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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 July 2012 - 12 September 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 guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

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: on the first day of treatment, the males were approximately 10 weeks old and had a mean body weight of 400 g (range: 366 g to 429 g) and the females were approximately 9 weeks old had a mean body weight of 225 g (range: 199 g to 248 g)
- Fasting period before study: no
- Housing: The animals were individually housed, except during pairing and lactation, in polycarbonate cages (Tecniplast 2154, 940 cm²) with stainless steel lids and containing autoclaved sawdust (SICSA, Alfortville, France). Individual housing was chosen because of software limitations and since it is preferable for pregnant animals.
Toward the end of gestation and during lactation, autoclaved wood shavings (SICSA, Alfortville, France) were provided to females and their litter as nesting material.
Each cage contained an object (rat hut) for the enrichment of the environment of the rats. The cages were placed in numerical order on the racks.
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: the animals were acclimated to the study conditions for a period of 7 days before the beginning of the treatment period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C. The temperature recorded in the animal room was twice outside the target range specified in the study plan (up to +26°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: 10 July 2012 to 24 August 2012.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5% methylcellulose
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
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. After mixing of the test item with the vehicle, the dose formulations were magnetically stirred for at least 1 hour before repartition into daily flasks.
The test item dose formulations were prepared for up to 9 days and stored at room temperature in brown flasks prior to use and delivery.

VEHICLE
- Concentration in vehicle: 10, 30 and 100mg/mL
- Amount of vehicle (if gavage): 10 mL/kg/day.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation (mating period): until mating occurs
- Proof of pregnancy: vaginal plug or sperm in the morning vaginal lavage referred to as day 0 post-coitum
- After successful mating each pregnant female was caged individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Type of method: HPLC-UV.
Test item concentrations: remained within an acceptable range of variation compared to nominal values.
Homogeneity: homogenous
Stability: stable after 9 days at room temperature and protected from light
Duration of treatment / exposure:
In the males:
- 2 weeks before mating,
- during the mating period,
- until sacrifice (i.e. at least 5 weeks in total),

In the females:
- 2 weeks before mating,
- during the mating period,
- during pregnancy,
- during lactation until day 5 post-partum inclusive,
- until sacrifice for females which had not delivered.
Frequency of treatment:
Daily
Details on study schedule:
- No F1 parents (only one generation mated)
- Age at mating of the mated animals in the study: 11-12 weeks
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
10 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose-levels were selected in agreement with the Sponsor, following the results of a previous 4-week toxicity study (CiToxLAB France/Study No. 38024 TSR). In this study, Sprague-Dawley rats received the test item at dose-levels of 100, 300 or 1000 mg/kg/day by gavage in 0.5% methylcellulose aqueous solution. There were no premature deaths, no relevant clinical signs and no effects on mean food consumption and mean body weight at any dose-level. There were no toxicologically significant effects on FOB, hematology, blood biochemistry and urinalysis results. There was no relevant macroscopic finding or effects on mean organ weights.

- Rationale for animal assignment: computerized stratification procedure.
Positive control:
no (not required)

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS:
- Time schedule: at least twice a day for mortality and once a day for clinical signs during the treatment period.

BODY WEIGHT (GAIN):
- Time schedule: Males: on the first day of treatment, then once a week until sacrifice. Females: on the first day of treatment, then once a week until mating, on Days 0, 7, 14 and 20 post-coitum and days 1 and 5 post-partum.

FOOD CONSUMPTION:
- Time schedule: the quantity of food consumed by each male was measured once a week, over a 7-day period, from the first day of treatment until the start of the pairing period.
The quantity of food consumed by each female was measured once a week, over a 7-day period, from the first day of treatment until the start of the pairing period, during gestation for the intervals days 0-7, 7-14 and 14-20 p.c. (except for one female) and during lactation for the interval days 1-5 p.p..

During the pairing period, the food consumption was not measured for males or females.

Food intake per animal and per day was calculated by noting the difference between the food given and that in the food-hopper the next time.


REPRODUCTION (apart from indices):
- Pre-coital time and duration of gestation were recorded.
Oestrous cyclicity (parental animals):
fresh vaginal lavage (stained with methylene blue), each morning during the pairing period, until females are mated.
Sperm parameters (parental animals):
Parameters examined in males of parental generation:
- testis weight (all groups) + microscopic evaluation (control and high-dose groups)
- epididymis weight (all groups) + microscopic evaluation (control and high-dose groups)
- microscopic evaluation of stages of the spermatogenic cycle and testicular interstitial cells (control and high-dose groups).
Litter observations:
STANDARDISATION OF LITTERS: No

PARAMETERS EXAMINED:
- number and sex of pups,
- number of live, dead and cannibalized pups,
- presence of gross anomalies, weight gain, clinical signs.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: all animals after the end of the mating period
- Female animals: all surviving animals = day 5 post-partum or, for females which had not delivered yet, day 25 post-coitum.

GROSS NECROPSY
Macroscopic post-mortem examination.

HISTOPATHOLOGY
- ovaries or testes and epididymides of all control and high-dose animals.
- see above sperm parameters

ORGAN WEIGHTS: epididymides, ovaries and testes.
Postmortem examinations (offspring):
SACRIFICE: on day 5 post-partum

GROSS NECROPSY: on all pups (surviving and found dead)

HISTOPATHOLOGY: No

ORGAN WEIGTHS: No
Statistics:
Data were compared by one-way analysis of variances and Dunnett test (mean values being considered as normally distributed, variances being considered as homogenous) or by Fischer exact probability test (proportions). PathData software (version 6.2d2) was used to perform the statistical analysis of organ weight data (level of significance of 0.05 or 0.01).
Reproductive indices:
Pre-implantation loss = 100 * (Number of corpora lutea - Number of implantation sites) / Number of corpora lutea
Post-implantation loss = 100 * (Number of implantation sites - Number of live pups) / Number of implantations
Mating index = 100 * (Number of mated animals / Number of paired animals)
Fertility index = 100 * (Number of pregnant female partners / Number of mated pairs)
Gestation index = 100 * (Number of females with live born pups / Number of pregnant females)
Offspring viability indices:
Live birth index = 100 * (Number of live born pups / Number of delivered pups)
Viability index on day 4 p.p. = 100 * (Number of surviving pups on day 4 p.p. / Number of live born pups)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
(limited data)
Reproductive function: sperm measures:
not examined
Description (incidence and severity):
no in-vivo observation of sperm
Reproductive performance:
no effects observed

Details on results (P0)

MORTALITY:
There were no unscheduled deaths.

CLINICAL SIGNS:
There were no test item-related clinical signs.
Incidental findings in test item-treated groups included cutaneous lesions and areas of hair loss.

BODY WEIGHT (GAIN):
There were no effects on mean body weight and mean body weight gains.

FOOD CONSUMPTION:
There were no effects on mean food consumption.

REPRODUCTIVE PERFORMANCE:
Mating and fertility data
There were no test item-related effects on mating and fertility data.
The unique non pregnancy was noted at 300 mg/kg/day and considered to be fortuitous.

Delivery data
There were no test item-related effects on delivery data.

Organ weights of F0 animals
There were no changes in organ weights attributable to the test item administration. Differences in organ weights were marginal and reflected the usual range of individual variations.

Macroscopic post-mortem examination of F0 animals
The few macroscopic findings noted at necropsy had no histological correlates or correlated with common histological findings in control rats, and were considered to be incidental.

Microscopic examination of F0 animals
There were no microscopic findings in the testis, epididymis, ovary and oviduct.

Effect levels (P0)

Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: For parental toxicity and reproductive performance.
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Other effects:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

MORTALITY/VIABILITY/CLINICAL SIGNS:
There were no effects on pup viability and clinical signs by day 5 p.p.

BODY WEIGHT (GAIN):
There were no effects on mean pup body weights and mean pup body weight gains. The slight differences from controls seen at 100 and 300 mg/kg/day were considered to be related to the slight differences in the number of pups born.

POST-MORTEM EXAMINATIONS
Macroscopic post-mortem examination of pups
There were no test item-related macroscopic findings in pups.

Effect levels (F1)

Dose descriptor:
NOEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
Based on the experimental conditions of this study:
- the No Observed Effect Level (NOEL) for parental toxicity was considered to be 1000 mg/kg/day,
- the NOEL for reproductive performance (mating and fertility) was considered to be 1000 mg/kg/day,
- the NOEL for toxic effects on progeny was considered to be 1000 mg/kg/day.
Executive summary:

The objective of this study was to evaluate the potential toxic effects of the test item following daily oral administration (gavage) to male and female rats from before mating, through mating and, for females, through gestation until day 4 post-partum (p.p.), according to OECD No. 421, 27 July 1995.

This study provided initial information on male and female reproductive performance, such as gonadal function, mating behavior, conception, development of the conceptus and parturition.

 

Methods

Three groups of ten male and ten female Sprague-Dawley rats received the test item daily, by oral (gavage) administration, before pairing and through pairing and, for the females, through gestation until day 4 p.p. The test item was administered as a suspension in the vehicle, 0.5% methylcellulose aqueous solution, at dose-levels of 100, 300 or 1000 mg/kg/day.

Another group of ten males and ten females received the vehicle, alone, under the same experimental conditions and acted as a control group. A constant dosage-volume of 10 mL/kg/day was used.

 

The concentration of the dose formulation was checked prior to administration of the dose formulation in study weeks 1, 3 and 6.

 

The animals were checked at least twice daily during the dosing period for mortality and morbidity and at least once daily for clinical signs. Body weight and food consumption were recordedonce a week. The animals were paired for mating after 2 weeks of treatment and the dams were allowed to litter and rear their progeny until day 5 p.p. The total litter sizes and the sex of each pup were recorded after birth. The pups were observed daily for clinical signs, abnormal behaviour and external abnormalities and weighed on days 1 and 5 p.p.

The males were sacrificed after completion of the pairing period and the dams on day 5 p.p. Final body weights and selected organs weights (epididymes, testes, ovaries) were recorded and a complete macroscopic post-mortem examination was performed, with particular attention paid to the reproductive organs. Reproductive organs were preserved and a microscopic examination was performed on the epididymes and testes or ovaries from all animals of the control and high-dose groups, and on all macroscopic lesions.

The pups were sacrificed on day 5 p.p.and submitted for a macroscopic post-mortem examination of the principal thoracic and abdominal organs.

 

Results

The test item concentrations in the administered dose formulations analyzed in weeks 1, 3 and 6 were within the acceptance criteria (± 10% of the nominal concentrations).

 

There were no test item-related deaths, clinical signs or effects on mean body weight, food consumption, mating and fertility data, delivery data,pup viability and clinical signs by day 5 p.p., pup sex ratio, pup macroscopic post-mortem findings and pathological findings in F0 animals.


Conclusion

The test item was administered daily by oral gavage to male and female Sprague-Dawley rats, for 2 weeks before pairing, during pairing, gestation and until up to day 4 p.p., at dose-levels of 100, 300 or 1000 mg/kg/day.

 

Based on the experimental conditions of this study:

. the No Observed Effect Level (NOEL) for parental toxicity was considered to be 1000 mg/kg/day,

. the NOEL for reproductive performance (mating and fertility) was considered to be 1000 mg/kg/day,

. the NOEL for toxic effects on progeny was considered to be 1000 mg/kg/day.