Registration Dossier

Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
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

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
Test material form:
solid: particulate/powder

Test animals

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.

Administration / exposure

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
Doses / concentrationsopen allclose all
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

Examinations

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

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

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.

Maternal developmental toxicity

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

Effect levels (maternal animals)

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

Maternal abnormalities

Abnormalities:
effects observed, non-treatment-related

Results (fetuses)

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

Effect levels (fetuses)

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

Fetal abnormalities

Abnormalities:
effects observed, non-treatment-related

Overall developmental toxicity

Developmental effects observed:
no

Any other information on results incl. tables

Table 1: Clinical signs

 

Dose-level (mg/kg/day)

0

100

300

1000

Round back

 

 

1a

 

Piloerection

 

 

1a

 

Loud breathing

 

 

1a

 

Chromodacryhorrea

 

 

1

1b

Reflux at dosing

 

 

1

1b

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 weighta

106

98.1

105

107

Carcass weighta

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 4thcerebral 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 1stmetatarsal, 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 14ththoracic 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.

Applicant's summary and conclusion

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.