Bis(piperidinothiocarbonyl) hexasulphide

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

A screening study is available to evaluate the reprotoxic potential of DPTH (OECD 421). No adverse effect was observed in this study on parentals rats, on pups and on reproduction performance at 1000 mg/kg/day (the high-dose used).

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

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.

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

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)

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)

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: 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

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.

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

Critical effects observed:

no

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

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

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.

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

Critical effects observed:

no

Reproductive effects observed:

no

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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

This screening test is reliable, performing according to the OECD guideline n°421 (klimisch score of 1).

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

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. 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 5p.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 5p.p.and submitted for a macroscopic post-mortem examination of the principal thoracic and abdominal organs.

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 5p.p., pup sex ratio, pup macroscopicpost-mortemfindings and pathological findings in F0 animals. 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 4p.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.

Effects on developmental toxicity

Description of key information

A developmental toxicity study in rats was performed with DPTH. No adverse effects were observed in the female and in the foetus at the highest tested dose (1000 mg/kg bw/day).

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 May 2016 - 22 June 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

22 January 2001.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: breeder: Janvier, le Genest-Saint-Isle, France
- Age at study initiation: approximately 10-11 weeks old at the beginning of the treatment period
- Mean body weight at the beginning of the treatment period: 296 g (range: 254 g to 367 g)
- Fasting period before study: no
- Housing: the animals were individually housed in polycarbonate cages (Tecniplast 2154; 940 cm2; with stainless steel lids) containing autoclaved sawdust
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: for a period of 4 or 5 days before the beginning of the treatment period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): about 8 to 15 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h.

IN-LIFE DATES: 25 May 2016 to 22 June 2016.

Route of administration:

oral: gavage

Vehicle:

other: 0.5% methylcellulose aqueous solution

Details on exposure:

PREPARATION OF DOSING FORMULATIONS:
- Suspension in the vehicle
- Concentration in vehicle: 10, 30 and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg/day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Type of method: HPLC-UV (High Performance Liquid Chromatography with UV detection)
Test item concentrations: remained within an acceptable range of -14.0% to -1.3 % when compared to the nominal values (± 15% of the nominal concentrations).
Homogeneity/Stability: The dose formulations containing the test item in 0.5% methylcellulose (MC) in drinking water treated by reverse osmosis at 4 mg/mL and 100 mg/mL were found to be homogeneous and stable after 9 days at room temperature and protected from light.

Duration of treatment / exposure:

Days 6 to 20 p.c. (post-coitum)

Frequency of treatment:

Daily

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

24 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose-levels were selected in agreement with the Sponsor, on the basis of the results of an OECD 421 study where 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 Day 4 p.p., at dose-levels of 100, 300 or 1000 mg/kg/day. The No Observed Effect Level (NOEL) for parental toxicity, reproductive performance and toxic effects on progeny was considered to be 1000 mg/kg/day.

Therefore, 1000 mg/kg/day was selected as the high dose-level in the present study. The low-dose and mid dose were selected using a ratio representing approximately a 3-fold interval (i.e. 100 and 300 mg/kg/day).

- Rationale for animal assignment: computerized stratification procedure

Maternal examinations:

MORTALITY/MORBIDITY:
- Time schedule: each animal was checked for mortality and morbidity once a day before the treatment period and at least twice a day during the treatment period, including weekends.

CLINICAL SIGNS:
- Time schedule: From arrival, each animal was observed once a day as part of the routine examinations.
From the start of the treatment period, each animal was observed once a day, at approximately the same time of day, for the recording of clinical signs.

BODY WEIGHT:
- Time schedule: the body weight of each female was recorded on Days 2, 4, 6, 9, 12, 15, 18 and 21 p.c.

FOOD CONSUMPTION:
- Time schedule: the quantity of food consumed by each female was recorded for the following intervals: Days 2-4, 4-6, 6-9, 9-12, 12-15, 15-18 and 18-21 p.c.

POST-MORTEM MACROSCOPIC EXAMINATION:
- Sacrifice on Day 21 p.c.
- Examined: principal thoracic and abdominal organs

Ovaries and uterine content:

The ovaries and uterine content were examined to determine:
- number of corpora lutea,
- number and distribution of dead and live fetuses,
- number and distribution of early and late resorptions,
- number and distribution of uterine scars,
- number and distribution of implantation sites.

Fetal examinations:

- External examinations: Yes: all fetuses per litter
- Soft tissue examinations: Yes: half fetuses per litter
- Skeletal examinations: Yes: half fetuses per litter
- Head examinations: Yes: half fetuses per litter
- Others: body weight, sex

Statistics:

Data were compared by one-way analysis of variance and Dunnett test (mean values being considered as normally distributed and variances being considered as homogeneous) or by Fisher exact probability test (proportions).

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 fetuses) / Number of implantation sites

Historical control data:

Cf attached document

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

See table 1.
There were no test item-related clinical signs.

There was one female at 300 mg/kg/day with round back, pilorection and loud breathing on Days 14 to 16 p.c. but this was considered to be incidental (absence of dose-relationship, isolated incidence).
The other clinical signs were of common background in rats and in isolated occurrence.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no premature deaths.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

See table 2.
There were no effects on mean body weight and mean body weight gain at any dose-levels.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

See table 3.
There were no effects on mean food consumption at any dose-levels.

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:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no macroscopic post-mortem findings.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

See table 4.
There were no effects on mean gravid uterus and carcass weights and on mean net body weight change at any dose-levels.

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

See table 5.
The differences from controls in mean number of post-implantation loss of test item groups were slight, not dose-related, within historical control data and not statistically significant.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

See table 5.
There were no test item-related effects on mean hysterectomy data.

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

See table 5.
The differences from controls in mean number of resorptions of test item groups were slight, not dose-related, within historical control data and not statistically significant.

Dead fetuses:

no effects observed

Description (incidence and severity):

See table 5.
There were no test item-related effects on mean hysterectomy data.

Changes in pregnancy duration:

not examined

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item effects on the pregnancy status of the females.
At hysterectomy on Day 21 p.c., all females were pregnant and had live fetuses, with the exception of one non-pregnant female at 1000 mg/kg/day.

Other effects:

not specified

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Abnormalities:

effects observed, non-treatment-related

Fetal body weight changes:

no effects observed

Description (incidence and severity):

See table 6.
There were no effects on mean fetal body weight.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed

Reduction in number of live offspring:

not specified

Changes in sex ratio:

no effects observed

Description (incidence and severity):

See table 7.
There were no effects on mean sex ratio.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

See table 5.
There were no test item-related effects on mean hysterectomy data.

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Variations
There were no fetal external variations.

Malformations
See table 8.
At 100 mg/kg/day, there was one litter with two fetuses having face external malformations (associated with head/face visceral or skeletal malformations): one with mandibular micrognathia and short snout, the other with synotia.
At 300 mg/kg/day, one litter also had one fetus with the face malformation mandibular micrognathia (together with aglossia).
Taking into account the limited litter incidence in each group, the absence of similar malformations in the high-dose group, and the presence of most of these malformations in historical control data, a test item effect was considered unlikely.

There were no external malformations in fetuses at 1000 mg/kg/day and the narrowed tail at 300 mg/kg/day was considered incidental (isolated incidence at the mid-dose only).

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Cartilage
Cartilages of short, unossified or incomplete/bipartite ossification of bones were present, with the following exceptions:
- a bipartite centrum of one thoracic vertebra in one fetus at 100 mg/kg/day,
- bilateral short 13th ribs in one fetus at 100 mg/kg/day and one fetus at 1000 mg/kg/day.
These findings being isolated in the groups, they were not attributed to the test item treatment.

Variations
See table 10.
There were no skeletal variations considered to be test item-related. At 1000 mg/kg/day, the higher litter and fetal incidences in dumbbell ossification of thoracic vertebra centrum, extra sternebral ossification site and ossification point on 14th thoracic vertebra(e) compared with controls and historical control data were considered incidental (no statistical significance, slight difference from controls and/or within/comparable with historical control data).
All other skeletal variations were noted at comparable incidence than controls or histological control data, in isolated incidence and/or there was no dose-relationship or statistical significance; they were therefore not considered test item-related.

Malformations
See table 11.
As there were no other fetuses and litters with skeletal malformations in the same group or at higher doses, the findings were not attributed to the test item treatment of the dam.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Variations

There were no test item-related fetal visceral variations (incidence lower or comparable to controls or historical control data, and no dose-relationship).

Malformations
See table 9.
There were three litters (one at 100 mg/kg/day and two at 300 mg/kg/day) with heart or heart great vessels malformations (mainly absent aortic arch). Two of the concerned fetuses were the same fetuses as mentioned in the previous paragraph (with facial malformations). Besides, the three malformations are known to occur sometimes in association (by pair or even all three together). Moreover, no similar malformations were noted in the high-dose group. Therefore, a relationship with the test item was considered to be unlikely.

At 100 and 300 mg/kg/day, the soft tissue face malformations were associated with the external malformations described in the previous paragraph and a test item effect was similarly considered unlikely. Besides, anophtalmia was also found in a control litter of another similar study.

At 300 mg/kg/day, the marked dilated ureter and renal pelvis were considered to be incidental (isolated occurrence at the mid-dose only, within historical control data).

At 1000 mg/kg/day, the findings were noted at a similar incidence as in controls or historical control data and/or at an isolated incidence. There were not considered to be test item-related.

Other effects:

not specified

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Abnormalities:

effects observed, non-treatment-related

Developmental effects observed:

no

Table 1: Clinical signs

 

Dose-level (mg/kg/day)	0	100	300	1000
Round back			1^a
Piloerection			1^a
Loud breathing			1^a
Chromodacryhorrea			1	1^b
Reflux at dosing			1	1^b
Scab then cutaneous lesion on back			1
Number of affected animals	0/24	0/24	4/24	1/23

a: same female; b: same female.

Table 2: Body weight

Dose-level (mg/kg/day)	0	100	300	1000
Body weight (g)
Day 6p.c.	298	296	296	295
		-1	-1	-1
Day 21p.c.	449	443	447	449
		-1	0	0
Body weight change (g)
Days 6 - 21p.c.	+151	+146	+151	+154

Differences from controls initalic(%).

Table 3: Food consumption

Dose-level (mg/kg/day)	0	100	300	1000
. Days 6 - 9p.c.	25	25	26	25
. Days 9 - 12p.c.	27	27	27	28
. Days 12 - 15p.c.	30	31	29	30
. Days 15 - 18p.c.	33	33	33	34
. Days 18 - 21p.c.	34	33	33	35

 

Table 4: Gravid uterus weight and net body weight change

Dose-level (mg/kg/day)	0	100	300	1000
Gravid uterus weight^a	106	98.1	105	107
Carcass weight^a	344	345	342	342
Net body weight change from Day 6p.c.	45.4	48.3	46.1	46.8

a: rounded to three significant digits.

Table 5: Hysterectomy data

 

Dose-level (mg/kg/day)	0	100	300	1000	HCD
Number of females with live fetuses on Day 21p.c.	24	24	24	23	388
Number ofcorpora lutea	15.3	14.5	14.9	15.7	[13.8-16.0]
Number of implantation sites	13.5	13.2	14.0	14.2	[12.5-14.5]
Pre-implantation loss (%)	11.1	8.7	6.6	8.9	[6.2-14.0]
Number of live fetuses	12.9	12.0	12.8	13.2	[11.6-13.8]
Number of dead fetuses	0.0	0.0	0.0	0.0	[0.00-0.50]
Number of early resorptions	0.5	0.9	1.0	0.9	Early+late resorptions: [0.50-1.35]
Number of late resorptions	0.0	0.3	0.2	0.1
Post-implantation loss (%)	4.2	9.1	8.3	7.3	[3.5-11.1]

HCD: historical control data (Sprague-Dawley rats from Janvier; December 2016): study means [min-max].

 

Table 6: Fetal body weight

 

Dose-level (mg/kg/day)	0	100	300	1000
Mean fetal body weight (g)	5.87	5.87	5.88	5.89

 

Table 7: Fetal sex ratio

 

Dose-level (mg/kg/day)	0	100	300	1000
Mean percentage of male fetuses (%)	49.4	48.6	51.6	50.4

 

Table 8: External malformations

 

Dose-level (mg/kg/day)	0	100	300	1000	HCD
Mandibular micrognathia, F(L)		0.3 (4.2)^a	0.3 (4.2)^c		0.4 (5.0)^e
Short snout, F(L)		0.3 (4.2)^a			0.4 (5.0)
Synotia , F(L)		0.3 (4.2)^b			-
Narrowed tail, F(L)			0.3 (4.2)^d		0.4 (5.0)

F: fetal incidence (%), L: litter incidence (%).

a: fetus G23817-5; b: fetus G23817-10; c: fetus G23839-8; d: fetus G23828-14, e: found as short mandible in HCD, mandibular micrognathia was also found in a control fetus inCiToxLAB France/Study No. 42367 RSRin 2015.

HCD: historical control data (Sprague-Dawley rats from Janvier; December 2016): max study fetal incidence (max study litter incidence) (%); -: none in HCD.

Table 9: Soft tissue malformations

 

Dose-level (mg/kg/day)	0	100	300	1000	HCD
Aglossia, F(L)			0.7 (4.2)^b		-
Anophthalmia, F(L)		0.7 (4.2)^a			0.8 (5.0)
Dilated 4^thcerebral ventricle F(L)	0.7 (4.2)			0.7 (4.3)	0.7 (4.2)
Ventricular septum defect F(L)		0.7 (4.2)^a			-
Truncus arteriosus F(L)		0.7 (4.2)^a			-
Absent aortic arch, F(L)		0.7 (4.2)^a	1.3 (8.3)^b		-
Marked dilated renal pelvis, F(L)			0.7 (4.2)^c		2.4 (4.8)
Marked dilated ureter, F(L)			0.7 (4.2)^c		4.8 (4.8)
Absent kidney, F(L)				0.7 (4.3)	0.7 (4.5)

F: fetal incidence (%), L: litter incidence (%); a: fetus G23817-5; b: including fetus G23839-8: c: fetus G23840-7.

HCD: historical control data (Sprague-Dawley rats from Janvier; December 2016): max study fetal incidence (max study litter incidence) (%); -: none in HCD.

Table 10: Skeletal variations

 

Dose-level (mg/kg/day)	0	100	300	1000	HCD
Thoracic vertebra(e): dumbbell ossification of centrum, F(L)	1.9 (12.5)	0.7 (4.2)	0.6 (4.2)	2.5 (17.4)	6.5 (30.0)
Thickened rib(s) , F(L)	0.6 (4.2)	2.6 (16.7)	3.8 (16.7)		5.6 (25.0)
Forepaw: unossified proxical phalanx, F(L)	23.6 (62.5)	25.2 (58.3)	30.6 (75.0)	19.1 (69.6)	50.0 (85.0)
Unossified 1^stmetatarsal, F(L)	14.3 (37.5)	16.6 (41.7)	13.4 (45.8)	8.3 (34.8)	36.8 (72.7)
Extra sternebral ossification site, F(L)		0.7 (4.2)	0.6 (4.2)	1.9 (8.7)	1.7 (10.0)
Ossification point on 14^ththoracic vertebra(e), F(L)	0.6 (4.2)	0.7 (4.2)	0.6 (4.2)	3.8 (13.0)	11.3 (40.0)

F: fetal incidence (%), L: litter incidence (%); HCD: historical control data (Sprague-Dawley rats from Janvier; December 2016): max study fetal incidence (max study litter incidence) (%).

Table 11: Skeletal malformations

 

Dose-level (mg/kg/day)	0	100	300	1000	HCD
Supraoccipital, absent, F(L)		0.7 (4.2)^a			-
Nasal, absent ,F(L)		0.7 (4.2)^a			-
Maxilla, absent, F(L)		0.7 (4.2)^a			-
Mandible, misshapen, F(L)		0.7 (4.2)^a			-
Palate, absent, F(L)		0.7 (4.2)^a			-

F: fetal incidence (%), L: litter incidence (%); a: fetus G23817-10.

HCD: historical control data (Sprague-Dawley rats from Janvier; December 2016): -: none in HCD.

Conclusions:

The test item was administered by gavage, once daily, from Days 6 to 20 p.c. inclusive, to pregnant Sprague-Dawley rats at dosages of 100, 300 and 1000 mg/kg/day.

Under the experimental conditions and results of this study:
- the No Observed Effect Level (NOEL) for maternal parameters was considered to be 1000 mg/kg/day in absence of effects in dams,
- the NOEL for embryo-fetal development was considered to be 1000 mg/kg/day in absence of effects in litters.

Executive summary:

The objective of this GLP study was to evaluate the potential toxic effects of the test item, on the pregnant female and on embryonic and fetal development, following daily oral administration (gavage) to pregnant female rats from implantation to the day before scheduled hysterectomy [Days 6 to 20 post-coitum (p.c.) inclusive].

 

Methods

Three groups of 24 time-mated female Sprague-Dawley rats received the test item, at 100, 300 or 1000 mg/kg/day by oral route (gavage) once daily from Days 6 to 20 p.c. A constant dosage-volume of 10 mL/kg/day was used. Another group of 24 rats received the vehicle alone (0.5% methylcellulose in drinking water treated by reverse osmosis) under the same experimental conditions, and acted as a control group.

 

Formulation concentrations were checked in the first and last weeks of treatment in the study.

 

The animals were checked daily for mortality and clinical signs. Body weight and food consumption were recorded every 2 to 3 days. On Day 21 p.c., females were sacrificed and submitted to a macroscopic post-mortem examination. Hysterectomy was performed and the numbers of corpora lutea, implantations, early and late resorptions, and live and dead fetuses were recorded. The fetuses were sexed, weighed and examined for external, soft tissues and skeletal (cartilages + bones) abnormalities.

 

Results

The test item concentrations in the dose formulations analyzed were within an acceptable range of variations when compared to the nominal values and no test item was observed in the control dose formulations.

 

In F0 females, there were no premature deaths and no test item-related effects on pregnancy status, clinical condition, mean body weight, mean body weight gain, mean food consumption, necropsy observation, mean gravid uterus weight, mean carcass weight, mean net body weight change and mean hysterectomy data.

 

There were no test item-related effects on mean fetal body weight or mean sex ratio. There were no fetal external variations, and no test item-related visceral and skeletal variations, or external, visceral and skeletal malformations, or test item-related effects on cartilages.

 

Conclusion

The test item was administered by gavage, once daily, from Days 6 to 20 p.c. inclusive, to pregnant Sprague-Dawley rats at dosages of 100, 300 and 1000 mg/kg/day.

 

Under the experimental conditions and results of this study:

.            the No Observed Effect Level (NOEL) for maternal parameters was considered to be 1000 mg/kg/day in absence of effects in dams,

.            the NOEL for embryo-fetal development was considered to be 1000 mg/kg/day in absence of effects in litters.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The study is considered to be reliable with a klimisch score of 1.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

The objective of this GLP study was to evaluate the potential toxic effects of the test item, on the pregnant female and on embryonic and fetal development, following daily oral administration (gavage) to pregnant female rats from implantation to the day before scheduled hysterectomy [Days 6 to 20 post-coitum (p.c.) inclusive].

Three groups of 24 time-mated female Sprague-Dawley rats received the test item, at 100, 300 or 1000 mg/kg/day by oral route (gavage) once daily from Days 6 to 20p.c.A constant dosage-volume of 10 mL/kg/day was used. Another group of 24 rats received the vehicle alone (0.5% methylcellulose in drinking water treated by reverse osmosis) under the same experimental conditions, and acted as a control group.

The animals were checked daily for mortality and clinical signs. Body weight and food consumption were recorded every 2 to 3 days. On Day 21p.c., females were sacrificed and submitted to a macroscopicpost-mortemexamination. Hysterectomy was performed and the numbers of corpora lutea, implantations, early and late resorptions, and live and dead fetuses were recorded. The fetuses were sexed, weighed and examined for external, soft tissues and skeletal (cartilages + bones) abnormalities.

In F0 females, there were no premature deaths and no test item-related effects on pregnancy status, clinical condition, mean body weight, mean body weight gain, mean food consumption, necropsy observation, mean gravid uterus weight, mean carcass weight, mean net body weight change and mean hysterectomy data.

There were no test item-related effects on mean fetal body weight or mean sex ratio. There were no fetal external variations, and no test item-related visceral and skeletal variations, or external, visceral and skeletal malformations, or test item-related effects on cartilages.

 

Under the experimental conditions and results of this study:

.            the No Observed Effect Level (NOEL) for maternal parameters was considered to be 1000 mg/kg/day in absence of effects in dams,

.            the NOEL for embryo-fetal development was considered to be 1000 mg/kg/day in absence of effects in litters.

Justification for classification or non-classification

Based on the available data, no classification for reproduction toxicity is required for Dipentamethylenethiuram hexasulfide according to the Regulation EC n°1272/2008.