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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 September 2016 - 07 November 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
22 January 2001
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: breeder: Janvier, le Genest-Saint-Isle, France.
- Age at the beginning of the treatment period: approximately 10-11 weeks old
- Mean body weight at the beginning of the treatment period: 305 g (range: 261 g to 353 g)
- Fasting period before study: no
- Housing: the animals were individually housed in polycarbonate cages (Tecniplast 2154, 940 cm2) with stainless steel lids and 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: 10 October 2016 to 07 November 2016.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on exposure:
PREPARATION OF DOSING FORMULATIONS:
- Emulsion in the vehicle
- Concentration in vehicle: 36, 72 and 144 mg/mL
- Amount of vehicle: 5 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 the acceptable range of variations (± 15%) when compared to the nominal values.
Homogeneity: the dose formulations containing the test item prepared at 1 and 200 mg/mL were found to be homogeneous.
Dose formulations ranging from 1 mg/mL to 200 mg/mL are therefore considered to be suitable for routine administration in GLP Toxicological studies.
Stability: diluted analytical samples prepared from 0.8 mg/mL and 240 mg/mL dose formulations in olive oil were found to be stable for 4 and 5 days, respectively.
Details on mating procedure:
The females were mated at the breeder's facility. The day of confirmed mating (detection of a vaginal plug) was designated as Day 0 p.c.
Duration of treatment / exposure:
The dose formulations were administered daily from Day 6 to Day 20 p.c., inclusive.
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
180 mg/kg bw/day (actual dose received)
Dose / conc.:
360 mg/kg bw/day (actual dose received)
Dose / conc.:
720 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:
- Rationale for dose selection:
The dose levels were selected in agreement with the Sponsor, based on the results of:
- a previous 2-week dose-range finding study by the oral route (gavage) performed in the male and female rats species. In this study, three groups of six Sprague Dawley rats were treated with the test item at dose levels of 120, 500 or 1000 mg/kg/day. Clinical signs were observed in all animals given 1000 mg/kg/day in both sexes such as hunched posture, piloerection, thin appearance and ptyalism during the whole treatment period. One male treated at 1000 mg/kg/day was prematurely killed due to poor clinical condition. All animals given 500 mg/kg/day had ptyalism until the end of the study and 1/3 females showed hunched posture, piloerection and thin appearance during the first 10 days. Dose-related lower body weight, body weight change and food consumption were recorded at 500 mg/kg/day in males (-10% and -28% for body weight and body weight change on Day 14, respectively) and at 1000 mg/kg/day in both sexes (-22% and -14%, -53% and -72% for body weight and body weight change in males and females, respectively),
- a combined repeated dose toxicity study with the reproduction/developmental toxicity Screening Test (OECD 422, Ref 06-119), conducted with grade BPA 5PO, showing developmental findings with a decrease of pups weights observed at 500 mg/kg/day (the females showed no changes in body weight during premating, pregnancy or the lactation period and thereby no maternal toxicity was seen in any of the dose groups),
- an ongoing OECD 408 study in rats conducted at 30, 120 and 500 mg/kg/day. There are no findings in females over the 3 first weeks of the treatment period.

Therefore, 720 mg/kg/day was selected as the high-dose level. The low-dose and mid dose were selected using a ratio representing approximately a 2-fold interval (i.e. 180 and 360 mg/kg/day).

- Rationale for animal assignment: computerized stratification procedure.

Examinations

Maternal examinations:
MORTALITY/MORBIDITY:
- Time schedule: animals were examined twice daily for mortality and morbidity.

CLINICAL OBSERVATIONS:
- 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, 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., and prior to premature euthanasia.

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 post-coitum.
- Examined: principal thoracic and abdominal organs.
Ovaries and uterine content:
The ovaries and uterus of the females were examined to determine:
- gravid uterus weight,
- 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,
- gross evaluation of placentas.
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
- Other: number dead and live, body weight, sex.
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 Fisher’s 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, treatment-related
Description (incidence and severity):
See table 1.
At 720 mg/kg/day, most of clinical signs were also observed in prematurely sacrificed and/or found dead animals. They were considered to be test item treatment-related and adverse.
There were no test item treatment-related clinical signs in the 360 and 180 mg/kg/day groups.

Dacryorrhea, chromodacryorrhea and/or areas of hair loss on forelimbs were observed in only one animal in each low- and mid-dose group. They are common findings in this species and strain.
Mortality:
mortality observed, treatment-related
Description (incidence):
In the 720 mg/kg/day group, there were eight unscheduled deaths (all being pregnant females). Taking into account the similarity of the findings (clinical signs, effects on body weight/food consumption and macroscopic examination at necropsy), all these deaths were considered to be related to the test item treatment:
- one female (pregnant, 15 implantation sites) was prematurely sacrificed on Day 16 p.c. Prior to death, this animal progressively showed from Day 14 p.c., emaciated appearance, reddish vaginal discharge, soiled urogenital region and/or round back (in addition to ptyalism from Day 9 p.c.). This female lost 74 g of body weight on Days 6-15 p.c. (-23% vs. Day 6 p.c.) and had a markedly reduced food consumption on Days 12-15 p.c. (6 g/day). At necropsy, thymus reduced in size, forestomach with several thick and whitish colored areas, kidneys with irregular color, an enlarged adrenal and, vagina with thick and reddish content were observed,
- one female (pregnant, 13 implantation sites) was prematurely sacrificed on Day 19 p.c. Prior to death, this animal progressively showed from Day 13 p.c., piloerection, emaciated appearance, round back, hypoactivity, sedation, abdominal breathing, eyes half-closed and/or soiled urogenital area (in addition to ptyalism from Day 8 p.c.). This female lost 58 g of body weight on Days 6-18 (-20% vs. Day 6 p.c.) and had merely no food consumption on Days 15-18 p.c. At necropsy, thymus and spleen reduced in size were observed,
- one female (pregnant, 17 implantation sites) was prematurely sacrificed on Day 18 p.c. Prior to death, this animal progressively showed from Day 14 p.c., piloerection, emaciated appearance, round back, hypoactivity, loss of balance and/or abdominal breathing. This female had also half closed eyes and ptyalism on Days 8 to 17 p.c. This female lost 71 g of body weight on Days 6-18 (-24% vs. Day 6 p.c.) and had merely no food consumption on Days 15-18 p.c. At necropsy, forestomach with thickened mucosa, whitish colored areas and several brownish colored deposits were observed (this female had fetuses with reddish colored amniotic fluid),
- one female (pregnant, 16 implantation sites) was prematurely sacrificed on Day 20 p.c. Prior to death, this animal progressively showed from Day 14 p.c., piloerection, emaciated appearance, round back, soiled urogenital area and/or eyes half-closed (in addition to ptyalism from Day 13 p.c.). This female lost 41 g of body weight on Days 6-18 (-13% vs. Day 6 p.c.) and had merely no food consumption on Days 15-18 p.c. At necropsy, thymus and spleen reduced in size and, enlarged adrenals were observed,
- one female (pregnant, 14 implantation sites) was prematurely sacrificed on Day 20 p.c. Prior to death, this animal progressively showed from Day 14 p.c., piloerection, emaciated appearance, round back, hypoactivity, soiled urogenital area, brownish vaginal discharge and/or eyes half-closed (in addition to ptyalism from Day 7 p.c.). This female lost 34 g of body weight on Days 6-18 (-12% vs. Day 6 p.c.) and had merely no food consumption on Days 15 18 p.c. At necropsy, thymus and spleen reduced in size, enlarged adrenals and uterine horns with blackish colored content were observed,
- one female (pregnant, 14 implantation sites) was found dead on Day 18 p.c. Prior to death, this animal progressively showed from Day 14 p.c., piloerection, emaciated appearance, round back, hypoactivity, abdominal breathing, soiled urogenital area, reddish vaginal discharge and/or eyes half closed (in addition to ptyalism from Day 7 p.c.). This female lost 24 g of body weight on Days 6-15 (-8% vs. Day 6 p.c.) and had a low food consumption (9 g/day) on Days 12-15 p.c. At necropsy, thymus and spleen reduced in size, stomach with yellowish colored liquid count and enlarged adrenals were observed,
- one female (pregnant, 15 implantation sites) was found dead on Day 17 p.c. Prior to death, this animal progressively showed from Day 12 p.c., piloerection, emaciated appearance, round back, hypoactivity, chromorhinorrhea and/or eyes half-closed (in addition to ptyalism on Days 7-16 p.c.). This female lost 45 g of body weight on Days 6-15 (-14% vs. Day 6 p.c.) and had a low food consumption (5 g/day) on Days 12-15 p.c. At necropsy, thymus and spleen reduced in size, forestomach with several raised whitish colored foci, enlarged adrenals and uterine horns with a blackish colored content were observed,
- one female (pregnant, 16 implantation sites) was prematurely sacrificed on Day 19 p.c. Prior to death, this animal progressively showed from Day 12 p.c., piloerection, emaciated appearance, round back, cold to the touch, hypoactivity, lateral decubitus, dyspnea, brownish vaginal discharge and/or eyes half-closed (in addition to ptyalism from Day 8 p.c.). This female lost 65 g of body weight on Days 6-18 (-20% vs. Day 6 p.c.) and had merely no food consumption (1 or 0 g/day) on Days 12-18 p.c. At necropsy, thymus and spleen reduced in size, skin with reddish foci mainly on the tail region, caecum with thickened mucosa, forestomach and stomach with several brownish colored foci (depressed or not), ileum with blackish colored content and enlarged adrenals were observed.

There were no unscheduled deaths in the control, 180 and 360 mg/kg/day groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
See table 2.
At 720 mg/kg/day, there were mean body weight losses on Days 9 to 15 p.c. (-1 to -19 g, p < 0.001). When compared with controls, these resulted in decreases in mean body weights (down to -24% on Day 21 p.c., p < 0.001). These findings were considered to be test item-related and adverse.

At 360 mg/kg/day and when compared with controls, there were a few transient statistically significant differences in mean body weight (-5% vs. controls on Day 12 p.c., p < 0.05) and/or mean body weight changes (+6 g vs. +14 g on Days 6-9 p.c., p < 0.001 and +17 g vs. +23 g on Days 9-12 p.c., p < 0.05) which were considered to be test item treatment-related and adverse.

At 180 mg/kg/day and when compared with controls, there was a statistically significant difference in mean body weight change (+5 g vs. +14 g in controls on Days 6-9 p.c., p < 0.001) which was considered to be test item treatment-related but of limited toxicological significance (transient observation with no effect on mean body weight).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
See table 3.
At 720 mg/kg/day, there were decreases in mean food consumption (down to -60% vs. controls on Days 15-18 p.c., p < 0.001). These findings were considered to be test item-related and adverse.

At 360 mg/kg/day and when compared with controls, there were transient decreases in mean food consumption (down to -18% vs. controls on Days 6-9 p.c., p < 0.001) with a return to control values from Days 12-15 p.c. These findings were considered to be test item-related and adverse but of minor toxicological concern (less than 20% difference vs. controls).

At 180 mg/kg/day and when compared with controls, there was a transient decrease in mean food consumption (-14% vs. controls, p < 0.01) on Days 6-9 p.c. with a return to control values thereafter. This finding was considered to be test item-related but of minor toxicological concern.
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
See table 4.
At 720 mg/kg/day, there was a series of macroscopic findings affecting the thymus, spleen, stomach/forestomach, intestines and adrenals which were considered to be test item-related (also observed in almost all prematurely or found dead animals).

At 360 and 180 mg/kg/day, there were no test item treatment-related findings. The few findings recorded were also observed in the control group (enlarged lymph nodes) or are common observations in this species and strain (pancreas with edema and placenta with colored deposit).
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Net body weight change
See table 5.
At 720 mg/kg/day and when compared with controls, there were lower mean gravid uterus weight (-26%, p < 0.001), carcass weight (-24%, p < 0.001) and net body weight change (-28.4 g vs. +54.1 g, p < 0.001). These adverse findings were considered to be consecutive to the effects recorded on mean body weight and food consumption at this dose level and to the lower number of live fetuses.

At 360 mg/kg/day and when compared with controls, there was a decreased carcass weight (-6%, p < 0.05) resulting in a lower net body weight change (+33.4 g vs. +54.1 g, p < 0.05). These findings were considered to be test item treatment-related but of minor toxicological concern taking into account the amplitude of the differences.

At 180 mg/kg/day, there were no test item treatment-related effects on mean gravid uterus weight, carcass weight and net body weight change.

Maternal developmental toxicity

Number of abortions:
not examined
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
See table 6.
At 720 mg/kg/day and when compared with control, there was a lower number of live fetuses per animal (9.3 vs. 12.1, p < 0.05) as a consequence of increased resorptions (early and scars) and dead fetuses (5.5 vs. 0.0) which resulted in an increased incidence of mean percent of post-implantation loss (26.8% vs. 5.3%, p < 0.01). All these findings were outside the range of the HCD and, considered to be test item treatment-related and adverse.

When compared with controls, there were no test item treatment-related effects on hysterectomy data in the 360 and 180 mg/kg/day groups.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
effects observed, treatment-related
Description (incidence and severity):
See table 6.
At 720 mg/kg/day and when compared with control, there was a lower number of live fetuses per animal (9.3 vs. 12.1, p < 0.05) as a consequence of increased resorptions (early and scars) and dead fetuses (5.5 vs. 0.0) which resulted in an increased incidence of mean percent of post-implantation loss (26.8% vs. 5.3%, p < 0.01). All these findings were outside the range of the HCD and, considered to be test item treatment-related and adverse.

When compared with controls, there were no test item treatment-related effects on hysterectomy data in the 360 and 180 mg/kg/day groups.
Dead fetuses:
effects observed, treatment-related
Description (incidence and severity):
See table 6.
At 720 mg/kg/day and when compared with control, there was a lower number of live fetuses per animal (9.3 vs. 12.1, p < 0.05) as a consequence of increased resorptions (early and scars) and dead fetuses (5.5 vs. 0.0) which resulted in an increased incidence of mean percent of post-implantation loss (26.8% vs. 5.3%, p < 0.01). All these findings were outside the range of the HCD and, considered to be test item treatment-related and adverse.

When compared with controls, there were no test item treatment-related effects on hysterectomy data in the 360 and 180 mg/kg/day groups.
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
effects observed, treatment-related
Description (incidence and severity):
See table 15.
All females were pregnant with the exception of two in the low-dose group.

At hysterectomy on Day 21 p.c., 24/24, 22/24, 24/24 and 14/16 surviving females were pregnant with live fetuses in the groups treated at 0, 180, 360 and 720 mg/kg/day, respectively.

In the 720 mg/kg/day group, two females had only implantation scars, two other pregnant females were found dead on Days 17 or 18 p.c., and six pregnant females were prematurely sacrificed on Day 16, 18, 19 or 20 p.c. for ethical grounds.

In the 180 mg/kg/day group, two females had no corpora lutea and no implantation sites.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
180 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
mortality
body weight and weight gain
food consumption and compound intake

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
See table 7.
At 720 mg/kg/day and when compared with control, fetuses (males and females) had a lower mean body weight (-23% vs. controls, p < 0.001). At this dose level, mean fetal body weight was below the lower limit of the HCD. This finding was considered to be test item treatment-related and adverse.
When compared with controls, there were no effects on mean fetal body weight in the 360 and 180 mg/kg/day groups.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
See table 7.
There were no effects on mean percentage of male fetuses (sex-ratio) at any dose tested.
External malformations:
effects observed, treatment-related
Description (incidence and severity):
Malformations
See table 8.
At 720 mg/kg/day, one female had nine fetuses with anasarca (generalized edema) and another female had a fetus with a cleft lip. One female had also four different fetuses with local edema (variation). While limited to two litters, these findings were observed in the high-dose group only and at incidences above the upper limit of the HCD. Therefore a test item treatment relationship cannot be excluded.
At 360 and 180 mg/kg/day, there were no malformations at external examination of the fetuses.
In the control group, one fetus had a gastroschisis, a malformation commonly observed in this species and strain.

Variations
See table 9
At 720 mg/kg/day there were fetuses with local edema (14.3% of litters vs. none in controls, p < 0.001). This observation was also recorded at higher litter and fetal incidences when compared with HCD. Therefore, this finding was considered to be test item treatment-related but not adverse (variation).
There were no external variations in fetuses from control, 180 and 360 mg/kg/day groups.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
Cartilage
See table 12.
When compared with controls, at 360 and/or 720 mg/kg/day, there was a series of statistically significant increases of cartilage present (related to absence of ossification). In the 720 mg/kg/day group only and at some occasions (cartilages of caudal vertebrae, sternebrae, metacarpal bones, distal phalanx, metatarsal bones and pelvic girdle), the litters and/or fetal incidences were above the upper limit of HCD. These findings were considered to be test item treatment-related and not adverse (the bone concerned being present).

In the 180 mg/kg/day group, one fetus had absent cartilage of cervical vertebrae (associated to absent hemicentrum). This finding was isolated, not dose-related or associated with any other findings. Therefore a test item treatment-related effect was considered unlikely.

Variations
See table 13.
From 360 mg/kg/day and when compared with controls, there were dose-related increases in the litter and/or fetal incidences of unossified bones or bones with incomplete ossification . In the 720 mg/kg/day group only, the litter and/or fetal incidences were above the upper limit of HCD. These findings were considered to be test item treatment-related and not adverse (variations of ossification degree, the cartilages being present).
At 180 mg/kg/day and when compared with controls, there were no test item treatment-related findings.

Malformations
See table 14.
At 720 mg/kg/day, there were increases in the litter and fetal incidences of split supraoccipital. This finding affected four fetuses from the same litter. While limited to one litter, this finding was observed in the high-dose group only and not recorded in the HCD. Therefore a test item treatment relationship cannot be excluded.

At 360 mg/kg/day, there were no skeletal malformations.

At 180 mg/kg/day, one fetus had a cervical vertebra with absent hemicentrum. This observation was isolated, not dose-related or associated with any other findings. Therefore a test item treatment-related effect was considered unlikely.



Visceral malformations:
effects observed, treatment-related
Description (incidence and severity):
Variations
See table 10.
At 720 mg/kg/day, one female had four fetuses with visceral edema, another female had one fetus with fluid-filled thoracic cavity and, another female had four fetuses with fluid-filled thoracic cavity and abdomen. While limited to three litters, these findings were observed in the high-dose group only and at incidences above the upper limit of the HCD. Therefore a test item treatment relationship cannot be excluded.
When compared with controls, there were no test item treatment-related variations at soft tissue examination of the fetuses from the 360 and 180 mg/kg/day groups.

Malformations
See table 11.
There were no test item treatment-related malformations at soft tissue examination of the fetuses.
In the control group, one fetus had an absent kidney/ureter, a malformation commonly observed in this species and strain.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
360 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
external malformations
skeletal malformations
visceral malformations
other: embryo-fetal toxicity

Overall developmental toxicity

Developmental effects observed:
yes
Lowest effective dose / conc.:
720 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
not specified

Any other information on results incl. tables

Table 1: Clinical signs

Dose level (mg/kg/day)

0

      180

     360

     720

Ptyalism

 

       22

       24

       16

Soiled urogenital area

 

 

 

         4

Lateral decubitus

 

 

 

         1

Piloerection

 

 

 

       16

Emaciated appearance

 

 

 

       12

Round back

 

 

 

       14

Cold to the touch

 

 

 

         3

Hypoactivity

 

 

 

         6

Abdominal breathing

 

 

 

         3

Eyes half-closed

 

 

 

         9

Dacryorrhea

 

         1

 

 

Chromodacryorrhea

 

         1

 

           

Chromorhinorrhea

 

           

 

         1

Area of hair loss on forelimbs

 

 

         1

 

Number of affected animals

0/24

22/24

24/24

16/16

 

Table 2: Body weight

Dose level (mg/kg/day)

0

180

360

720

Body weight (g)

 

 

 

 

Day 2p.c.

281

282
(0)

281
(0)

280
(0)

Day 4p.c.

293

296
(+1)

295
(+1)

295
(+1)

Day 6p.c.

305

307
(+1)

304
(0)

303
(-1)

Day 9p.c.

320

312
(-3)

310
(-3)

310
(-3)

Day 12p.c.

343

332
(-3)

327*
(-5)

309#
(-10)

Day 15p.c.

364

352
(-3)

347
(-5)

290#
(-20)

Day 18p.c.

406

399
(-2)

391
(-4)

299#
(-26)

Day 21p.c.

459

454
(-1)

443
(-3)

347#
(-24)

Body weight change (g)

 

 

 

 

Days 2-4p.c.

+12

+14

+14

+14

Days 4-6p.c.

+13

+12

+9*

+9*

Days 6-9p.c.

+14

+5#

+6#

+6**

Days 9-12p.c.

+23

+20

+17*

-1#

Days 12-15p.c.

+21

+21

+20

-19#

Days 15-18p.c.

+43

+47

+44

+5#

Days 18-21p.c.

+53

+55

+52

+30#

Days 6-21p.c.

+154

+147

+138

+45#

( ): in brackets, percentage differencevs.controls (%).

Statistical significance: *: p < 0.05; **: p < 0.01; #: p < 0.001.

Table 3: Food consumption

Dose level (mg/kg/day)

            0

180

360

720

Days 6-9p.c.

          22

19**
(-14)

18#
(-18)

17#
(-23)

Days 9-12p.c.

          25

22
(-12)

21**
(-16)

17#
(-32)

Days 12-15p.c.

          26

26
(0)

25
(-4)

13#
(-50)

Days 15-18p.c.

          30

30
(0)

30
(0)

12#
(-60)

Days 18-21p.c.

          30

30
(0)

30
(0)

23#
(-23)

(): in brackets, percentage difference (%)vs. controls.

Statistical significance: **: p < 0.01; #: p < 0.001.

Table 4: Macroscopic examination

 

Dose level (mg/kg/day)

0

180

360

720

Lymph node(s): enlarged

1

      2

      2

      0

Pancreas: edema

0

      0

      1

      0

Thymus: reduced in size

0

      0

      0

      3

Spleen: reduced in size

0

      0

      0

      2

Stomach: colored deposit

0

      0

      0

      1

Stomach: colored focus

0

      0

      0

      2

Intestines: colored contents

0

      0

      0

      1

Intestines: colored focus

0

      0

      0

      1

Adrenal: enlarged

0

      0

      0

      3

Uterus: colored contents in uterine horn

0

      0

      0

      1

Vagina: colored contents

0

      0

      0

      1

Placenta: colored deposit

0

      0

      1

      0

Number of affected animals

1/24

2/24

3/24

4/16

 

Table 5: Net body weight change

Dose level (mg/kg/day)

0

180

360

720

Gravid uterus weight

99.9

104.6
(+5)

105.1
(+5)

73.6#
(-26)

Carcass weight

359.5

349.3
(-3)

337.6*
(-6)

273.3#
(-24)

Net body weight change (g) from Day 6p.c.

+54.1

+42.1

+33.4*

-28.4#

(): in brackets, percentage difference (%)vs. controls.

Statistical significance: *: p < 0.05; #: p < 0.001.

Table 6: Hysterectimy data

Dose level (mg/kg/day)

  0

180

360

 720

HCD

Number of pregnant females

 24

  22

  24

  16

388

Mean number ofcorpora luteaper animal

 14.5

  15.2

  15.5

  13.9

[13.8; 16.0]

Mean number of implantation sites

 12.8

  13.4

  14.0

  12.5

[12.5; 14.5]

Mean pre-implantation loss (%)

 11.6

  11.4

    9.3

  10.2

[6.2; 14.0]

Mean number of live fetuses per animal

 12.1

  12.8

  13.2

    9.3*

[11.6; 13.8]

Mean number of dead fetuses

  0.0

   0.0

    0.0

    5.5

[0.00; 0.50]

Mean number of resorptions + scars

  0.6

   0.6

    0.9

    2.4*

[0.50; 1.35]

Mean number of implantation scars

  0.0

   0.0

    0.0

    1.3*

/

Mean number of early resorptions

  0.5

   0.5

    0.8

    0.9

/

Mean number of late resorptions per animal

  0.1

   0.1

    0.0

    0.2

/

Mean post-implantation loss (%)

  5.3

   4.3

    6.2

  26.8**

[3.5; 11.1]

HCD: Historical Control Data (Sprague-Dawley rats, March 2014 - July 2016, n = 18 studies) [min.; max.].

/: not reported in HCD.

Statistical significance:*: p < 0.05; **: p < 0.01.

 

Table 7: Fetal body weights and sex ratio

Dose level (mg/kg/day)

    0

180

360

720

HCD

Mean male fetal body weight (g)

    6.02

5.94
(-1)

5.88
(-4)

4.59#
(-24)

[5.7; 6.1]

Mean female fetal body weight, (g)

    5.75

5.69
(-1)

5.55
(-4)

4.53#
(-21)

[5.4;5.7]

Mean percentage of male fetuses (%)

   48.1

46.9

48.4

53.9

[44.0; 55.4]

HCD: Historical Control Data (Sprague-Dawley rats, March 2014 - July 2016, n = 18 studies) [min.; max.].

(): in brackets, percentage difference (%)vs.controls.

Statistical significance:#: p < 0.001.

Table 8: External malformations

 

Dose level (mg/kg/day)

        0

    180

     360

      720

HCD

Number of litters

      24

      22

       24

       14

          388

Number of fetuses

    291

    282

     316

      148

         5000

Anasarca, F (L)

0 (0)

0 (0)

0 (0)

6.1# (7.1)

0 (0)(a)

Cleft lip, F (L)

0 (0)

0 (0)

0 (0)

0.7 (7.1)

0.4 (5.0)(a)

Gastroschisis, F (L)

0.3 (4.2)

0 (0)

0 (0)

0 (0)

0.4 (5.6)(a)

Litters affected, n (%)

1 (4.2)

0 (0)

0 (0)

2 (14.3)

11 (2.8)(b)

Fetus affected, n (%)

1 (0.3)

0 (0)

0 (0)

10# (6.8)

13 (0.3)(b)

n: number; F: fetal incidence; L: litter incidence.

HCD: Historical Control Data (Sprague-Dawley rats, March 2014 - July 2016, n = 18 studies).

(a): max.(b): mean.Statistical significance: #: p < 0.001.


 

Table 9: External variations

Dose level (mg/kg/day)

        0

    180

  360

         720

HCD

Number of litters

      24

      22

    24

          14

       388

Number of fetuses

    291

    282

  316

         148

     5000

Local edema, F (L)

0 (0)

0 (0)

0 (0)

6.1# (14.3)

0.8 (9.5)(a)

Litters affected, n (%)

0 (0)

0 (0)

0 (0)

2 (14.3)

6 (1.5)(b)

Fetus affected, n (%)

0 (0)

0 (0)

0 (0)

9# (6.1)

7 (0.1)(b)

n: number; F: fetal incidence; L: litter incidence.

HCD: Historical Control Data (Sprague-Dawley rats, March 2014 - July 2016, n = 18 studies).

(a): max.(b): mean.Statistical significance: #: p < 0.001.

Table 10: Soft tissue variations

Dose level (mg/kg/day)

       0

   180

    360

     720

HCD

Number of litters

     24

     22

      24

       13

       387

Number of fetuses

   140

   134

    153

       71

     2404

Dilated renal pelvis, F (L)

2.9 (12.5)

0 (0)

4.6 (20.8)

2.8 (7.7)

9.5 (28.6)(a)

Small testis, F (L)

0.7 (4.2)

0 (0)

0 (0)

0 (0)

0 (0)(a)

Absent innominate artery, F (L)

1.4 (8.3)

1.5 (9.1)

1.3 (8.3)

4.2 (7.7)

5.1 (25.0)(a)

Short innominate artery, F (L)

1.4 (4.2)

0.7 (4.5)

0 (0)

0 (0)

3.7 (22.7)(a)

Dilated ureter, F (L)

2.9 (12.5)

2.2 (13.6)

2.6 (12.5)

0 (0)

7.1 (28.0)(a)

Thymus: reddish foci, F (L)

0 (0)

1.5 (9.1)

0 (0)

0 (0)

0 (0)(a)

Edema, F (L)

0 (0)

0 (0)

0 (0)

5.6* (7.7)

0 (0)(a)

Fluid-filled thoracic cavity, F (L)

0 (0)

0 (0)

0 (0)

7.0** (15.4)

0 (0)(a)

Fluid-filled abdomen, F (L)

0 (0)

0 (0)

0 (0)

5.6* (7.7)

0.9 (5.0)(a)

Litters affected, n (%)

7 (29.2)

6 (27.3)

7 (29.2)

4 (30.8)

86 (22.2)(b)

Fetus affected, n (%)

9 (6.4)

7 (5.2)

10 (6.5)

14** (19.7)

119 (5.0)(b)

n: number; F: fetal incidence, L: litter incidence.

HCD: Historical Control Data (Sprague-Dawley rats, March 2014 - July 2016, n = 18 studies).

(a): max.(b): mean.

Statistical significance: *: p < 0.05; **: p < 0.01.

 

Table 11: Soft tissue malformations

Dose level (mg/kg/day)

       0

   180

    360

     720

HCD

Number of litters

     24

     22

      24

       13

       387

Number of fetuses

   140

   134

    153

       71

     2404

Absent kidney, F (L)

0.7 (4.2)

0 (0)

0 (0)

0 (0)

0.7 (4.5)(a)

Absent ureter, F (L)

0.7 (4.2)

0 (0)

0 (0)

0 (0)

/

Litters affected, n (%)

1 (4.2)

0 (0)

0 (0)

0 (0)

7 (1.8)(b)

Fetus affected, n (%)

1 (0.7)

0 (0)

0 (0)

0 (0)

13 (0.5)(b)

n: number; F: fetal incidence, L: litter incidence.

HCD: Historical Control Data (Sprague-Dawley rats, March 2014 - July 2016, n = 18 studies);/: not reported in HCD.

(a): max.(b): mean.

 

Table 12: Skeletal examination (cartilage)

Dose level (mg/kg/day)

           0

     180

       360

     720

HCD

Number of litters

         24

      22

         24

      14

       388

Number of fetuses

       151

     148

       163

      77

     2596

Cartilage of hyoid: present, F (L)

0 (0)

1.4 (4.5)

4.3* (16.7)

0 (0)

7.4 (30.4) (a)

Cartilage of cervical vertebra(e): present, F (L)

2.6 (12.5)

2.7 (18.2)

3.1 (8.3)

20.8# (42.9)

22.1 (58.3) (a)

Cartilage of cervical vertebra(e): absent, F (L)

0 (0)

0.7 (4.5)

0 (0)

0 (0)

0 (0) (a)

Cartilage of thoracic vertebra(e): present, F (L)

6.0 (25.0)

5.4 (22.7)

14.1* (45.8)

14.3* (50.0)

31.5 (87.5) (a)

Cartilage of lumbar vertebra(e): present, F (L)

0.7 (4.2)

0 (0)

0 (0)

0 (0)

0.7 (4.5) (a)

Cartilage of sacral vertebra(e): present, F (L)

0 (0)

0 (0)

0 (0)

3.9* (14.3)

0.7 (5.0) (a)

Cartilage of caudal vertebra(e): present, F (L)

0.7 (4.2)

0 (0)

0 (0)

22.1# (28.6)

2.2 (15.0) (a)

Cartilage of sternebra(e): present, F (L)

0 (0)

0.7 (4.5)

0.6 (4.2)

23.4# (35.7**)

3.8 (21.7) (a)

Cartilage of rib(s): present, F (L)

2.6 (16.7)

1.4 (9.1)

0 (0)

0 (0)

5.1 (26.1) (a)

Cartilage of rib(s): absent, F (L)

0.7 (4.2)

0 (0)

0 (0)

1.3 (7.1)

5.1 (21.7) (a)

Cartilage of metacarpal bone(s): present, F (L)

0.7 (4.2)

0.7 (4.5)

0.6 (4.2)

26.0# (42.9**)

5.0 (20.0) (a)

Forepaw: cartilage of proximal phalanx present, F (L)

24.5 (75.0)

18.9 (54.5)

26.4 (62.5)

45.5** (85.7)

50.0 (85.0) (a)

Forepaw: cartilage of distal phalanx present, F (L)

59.6 (79.2)

46.6 (68.2)

56.4 (83.3)

46.8 (78.6)

59.0 (87.5) (a)

Cartilage of metatarsal bone(s): present, F (L)

5.3 (25.0)

10.8 (36.4)

19.0# (66.7**)

66.2# (85.7#)

36.8 (72.7) (a)

Hindpaw: cartilage of distal phalanx: present, F (L)

41.7 (75.0)

31.1 (63.6)

35.6 (75.0)

32.5 (64.3)

43.5 (75.0) (a)

Cartilage of pelvic girdle: present, F (L)

0 (0)

0 (0)

0 (0)

2.6 (14.3)

0.7 (5.0) (a)

Litters affected, n (%)

24 (100.0)

20 (90.9)

23 (95.8)

13 (92.9)

236 (60.8) (b)

Fetus affected, n (%)

110 (72.8)

93 (62.8)

113 (69.3)

62 (80.5)

990 (38.1) (b)

n: number; F: fetal incidence, L: litter incidence.

HCD: Historical Control Data (Sprague-Dawley rats, March 2014 - July 2016, n = 18 studies).

(a): max.(b): mean.

Statistical significance: *: p < 0.05; **: p < 0.01; #: p < 0.001.

 

Table 13: Skeletal variations

Dose level (mg/kg/day)

        0

     180

       360

       720

HCD

Number of litters

      24

      22

         24

         14

       388

Number of fetuses

     151

     148

       163

         77

     2596

Parietal: incomplete ossification, F (L)

0.7 (4.2)

0.7 (4.5)

0 (0)

20.8# (28.6)

9.2 (35.0)(a)

Interparietal: incomplete ossification, F (L)

0.7 (4.2)

0.7 (4.5)

0 (0)

16.9# (28.6)

9.2 (45.0)(a)

Supraoccipital: incomplete ossification, F (L)

0.7 (4.2)

0 (0)

0 (0)

9.1** (28.6)

5.6 (25.0)(a)

Frontal: incomplete ossification, F (L)

0 (0)

0.7 (4.5)

0 (0)

18.2# (28.6*)

1.7 (11.1)(a)

Nasal: incomplete ossification, F (L)

0 (0)

0 (0)

0 (0)

14.3# (28.6*)

0.8 (5.6)(a)

Hyoid: incomplete ossification, F (L)

0 (0)

1.4 (4.5)

4.3* (16.7)

0 (0)

14.8 (55.0)(a)

Incisive bone: incomplete ossification, F (L)

0 (0)

0.7 (4.5)

0 (0)

14.3# (21.4*)

07 (5.0)(a)

Cervical vertebra(e): incomplete ossification of centrum, F (L)

2.6 (12.5)

2.0 (13.6)

2.5 (8.3)

9.1* (35.7)

18.3 (54.2)(a)

Cervical vertebra(e): unossified centrum, F (L)

0 (0)

0 (0)

0.6 (4.2)

11.7# (14.3)

4.9 (29.2)(a)

Cervical vertebra(e): incomplete ossification of arch, F (L)

0 (0)

0 (0)

0 (0)

3.9* (7.1)

0.8 (5.6)(a)

Thoracic vertebra(e): bipartite ossification of centrum, F (L)

0 (0)

1.4 (9.1)

1.8 (8.3)

6.5** (28.6*)

5.0 (16.7)(a)

Thoracic vertebra(e): dumbbell ossification of centrum, F (L)

0.7 (4.2)

0 (0)

2.5 (16.7)

5.2* (28.6)

6.5 (30.0)(a)

Sacral vertebra(e): incomplete ossification of arch, F (L)

0 (0)

0 (0)

0 (0)

3.9* (14.3)

0.7 (5.0)(a)

Caudal vertebra(e): unossified arch, F (L)

0 (0)

0 (0)

0 (0)

7.8** (21.4*)

1.5 (10.0)(a)

Caudal vertebra(e): unossified centrum, F (L)

0 (0)

0 (0)

0 (0)

18.2# (28.6*)

1.4 (5.6)(a)

Caudal vertebra(e): incomplete ossification of arch, F (L)

0.7 (4.2)

0 (0)

0 (0)

14.3# (21.4)

2.3 (15.0)(a)

Caudal vertebra(e): incomplete ossification of centrum, F (L)

0 (0)

0 (0)

0 (0)

6.5** (21.4*)

1.4 (10.0) (a)

Incomplete ossification of 1st to 4th sternebrae, F (L)

0 (0)

0.7 (4.5)

0.6 (4.2)

7.8** (21.4*)

3.1 (15.0) (a)

Incomplete ossification of 6th sternebra, F (L)

0 (0)

0 (0)

0 (0)

19.5# (28.6*)

2.9 (16.7)(a)

Unossified 5thsternebra, F (L)

0 (0)

0 (0)

0 (0)

6.5** (14.3)

1.7 (11.1)(a)

Unossified 6thsternebra, F (L)

0 (0)

0 (0)

0 (0)

3.9* (14.3)

0.7 (5.0)(a)

Incomplete ossification of metacarpal(s), F (L)

0.7 (4.2)

0 (0)

0.6 (4.2)

13.0# (28.6)

2.2 (10.0)(a)

Unossified metacarpal(s), F (L)

0 (0)

0.7 (4.5)

0 (0)

16.9# (28.6*)

2.9 (15.0)(a)

Forepaw: unossified proximal phalanx, F (L)

24.5 (75.0)

18.9 (54.5)

26.4 (62.5)

45.5** (85.7)

50.0 (85.0)(a)

Unossified 1stmetatarsal, F (L)

5.3 (25.0)

11.5 (40.9)

19.0# (66.7**)

66.2# (85.7#)

36.8 (72.7)(a)

Litters affected, n (%)

24 (100.0)

22 (100.0)

23 (95.8)

13 (92.9)

345 (88.9)(b)

Fetus affected, n (%)

115 (76.2)

104 (70.3)

117 (71.8)

62 (80.5)

1267 (48.8)(b)

n: number; F: fetal incidence, L: litter incidence.

HCD: Historical Control Data (Sprague-Dawley rats, March 2014 - July 2016, n = 18 studies).

(a): max.(b): mean.

Statistical significance: *: p < 0.05; **: p < 0.01; #: p < 0.001.

Table 14: Skeletal malformations

Dose level (mg/kg/day)

      0

     180

   360

     720

HCD

Number of litters

    24

      22

    24

      14

      388

Number of fetuses

   151

     148

   163

      77

    2596

Supraoccipital: split, F (L)

0 (0)

0 (0)

0 (0)

5.2* (7.1)

0 (0)(a)

Cervical vertebra(e): absent hemicentrum, F (L)

0 (0)

0.7 (4.5)

0 (0)

0 (0)

0 (0)(a)

Litters affected, n (%)

0 (0)

1 (4.5)

0 (0)

1 (7.1)

15 (3.9)(b)

Fetus affected, n (%)

0 (0)

1 (0.7)

0 (0)

4* (5.2)

18 (0.7)(b)

n: number; F: fetal incidence; L: litter incidence.

HCD: Historical Control Data (Sprague-Dawley rats, March 2014 - July 2016, n = 18 studies).(a): max;(b): mean.

Statistical significance: *: p < 0.05.

Table 15: Pregnancy status

Dose level (mg/kg/day)

         0

   180

        360

      720

Number of females

       24

     24

          24

        24

Females found dead

         0

      0

           0

         2

Females prematurely sacrificed

         0

      0

           0

         6

Non-pregnant females

         0

      2

           0

         0

Females with live fetuses on Day 21p.c.

       24

     22

          24

        16

 

Applicant's summary and conclusion

Conclusions:
The test item was administered to time-mated female Sprague-Dawley rats, by gavage, once daily, from Days 6 to 20 p.c., at dose levels of 180, 360 or 720 mg/kg/day.

On the basis of the results obtained in this study:
- the No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 180 mg/kg/day, based on adverse effects on body weight, body weight change and food consumption from 360 mg/kg/day and mortality at 720 mg/kg/day,
- the No Observed Adverse Effect Level (NOAEL) for embryo-fetal development was considered to be 360 mg/kg/day, based on embryo/fetal toxicity associated with malformations in a context of excessive maternal toxicity at 720 mg/kg/day.
Executive summary:

The objective of this GLP study was to evaluate the potentialtoxic 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 [Day 6 to Day 20 post-coitum (p.c.) inclusive].

 

Methods

Three groups of 24 time-mated female Sprague‑Dawley rats received the test item, at 180, 360 or 720 mg/kg/day by oral route (gavage) once daily from Days 6 to 20 p.c. A constant dosage-volume of 5 mL/kg/day was used. Another group of 24 rats received the vehicle alone (olive oil) under the same experimental conditions, and acted as a control group.

 

Formulation concentrations were checked in the start and end of the treatment period.

 

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, uterine scars, implantations, early and late resorptions, and live and dead fetuses were recorded. The live fetuses were sexed, weighed and examined for external, soft tissues and skeletal (cartilages + bones) abnormalities.

 

Results

Chemical analysis of dose formulations:

. the test item concentrations (-5.2% to -1.8%) in the administered dose formulations analyzed in Weeks 1 and 4 remained within the acceptable range of variations (± 15%) when compared to the nominal values,

. no test item was observed in the control dose formulation.

 

Pregnancy status:

. at hysterectomy on Day 21 p.c., 24/24, 22/24, 24/24 and 14/16 surviving females were pregnant (with live fetuses) in the groups treated at 0, 180, 360 and 720 mg/kg/day, respectively.

 

Mortality:

. in the 720 mg/kg/day group, there were eight unscheduled deaths. Taking into account the similarity of the findings associated to these deaths (clinical signs, effects on body weight/food consumption and macroscopic examination at necropsy), this mortality was considered to be related to the test item treatment,

. there were no unscheduled deaths in the control group, 180 and 360 mg/kg/day groups.

 

Clinical signs:

. at 720 mg/kg/day in surviving females, most of clinical signs (soiled urogenital area, lateral decubitus, piloerection, emaciated appearance, round back, cold to the touch, hypoactivity, abdominal breathing and/or eyes half-closed) were similar to those recorded in prematurely sacrificed and/or found dead animals. They were considered to be test item treatment-related and adverse,

. there were no test item treatment-related clinical signs in the 360 and 180 mg/kg/day groups.


Body weight and body weight change:

. at 720 mg/kg/day, there were body weight losses on Days 9 to 15 p.c. (-1 to -19 g, p < 0.001). When compared with controls, these resulted in decreased mean body weights (down to -24% on Day 21 p.c., p < 0.001). These findings were considered to be test item-related and adverse,

. at 360 mg/kg/day and when compared with controls, there were a few transient statistically significant differences in mean body weight (-5% vs. controls on Day 12 p.c., p < 0.05) and/or mean body weight changes (+6 g vs. +14 g on Days 6-9 p.c., p < 0.001 and +17 g vs. +23 g on Days 9-12 p.c., p < 0.05) which were considered to be test item treatment-related and adverse,

. at 180 mg/kg/day and when compared with controls, there were no toxicologically significant effects on mean body weight or mean body weight changes. However, the transient statistically significant difference in mean body weight change (+5 g vs. +14 g in controls on Days 6 -9 p.c., p < 0.001) was considered to be test item treatment-related but of limited toxicological significance.

 

Food consumption:

. at 720 mg/kg/day, there were decreases in mean food consumption (down to -60% vs. controls on Days 15-18 p.c., p < 0.001). These findings were considered to be test item-related and adverse,

. at 360 mg/kg/day and when compared with controls, there were transient decreases in mean food consumption (down to -18% vs. controls on Days 6-9 p.c., p < 0.001) with a return to control values from Days 12-15 p.c. These findings were considered to be test item-related but of minor toxicological concern (less than 20% difference vs. controls),

. at 180 mg/kg/day and when compared with controls, there was a transient decrease in mean food consumption (-14% vs. controls, p < 0.01) on Days 6 -9 p.c. with a return to control values thereafter. This finding was considered to be test item-related but of minor toxicological concern.

 

Macroscopic post-mortem examination of the dams:

. at 720 mg/kg/day, there was a series of macroscopic findings affecting the thymus, spleen, stomach/forestomach, intestines and adrenals which were considered to be test item-related (also observed in almost all prematurely or found dead animals),

. at 360 and 180 mg/kg/day, there were no test item treatment-related findings.

 

Gravid uterus weight, carcass weight and net body weight change:

. at 720 mg/kg/day and when compared with controls, there were weight losses affecting the gravid uterus (-26%, p < 0.001), carcass (-24%, p < 0.001) and net body (-28.4 g vs. +54.1 g, p < 0.001). These adverse findings were considered to be consecutive to the effects recorded on mean body weight and food consumption at this dose level,

. at 360 mg/kg/day and when compared with controls, there was a decreased carcass weight (-6%, p < 0.05) resulting in a lower net body weight change (+33.4 g vs. +54.1 g, p < 0.05). These findings were considered to be test item treatment-related but of minor toxicological concern taking into account the amplitude of the differences,

. at 180 mg/kg/day, there were no test item treatment-related effects.

 

Hysterectomy data:

. at 720 mg/kg/day and when compared with control, there was a lower percentage of live fetuses per animal (73.2 vs. 94.7, p < 0.01) as a consequence of increased resorptions (late, early and scars) and dead fetuses (5.5% vs. 0.0%) which resulted in an increased incidence of mean percent of post‑implantation loss (26.8% vs. 5.3%, p < 0.01). All these findings were outside the range of the Historical Control Data and, considered to be test item treatment-related and adverse,

. there were no test item treatment-related effects on hysterectomy data in the 360 and 180 mg/kg/day groups.

Fetal examination:

Fetal weights:

. at 720 mg/kg/day and when compared with control, fetuses (males and females) had a lower mean body weight (-23% vs. controls, p < 0.001). At this dose level, mean fetal body weight was below the lower limit of the HCD. This finding was considered to be test item treatment-related and adverse,

. there were no effects on mean fetal body weight in the 360 and 180 mg/kg/day groups.

 

Sex ratio:

. there were no effects on mean percentage of male fetuses (sex-ratio) at any dose tested.

 

External examination:

. at 720 mg/kg/day there were fetuses with local edema (a variation affecting 14.3% of litters vs. none in controls, p < 0.001). One female had nine fetuses with anasarca (a term used to define a malformation corresponding to a generalized edema) and another one had a fetus with a cleft lip. These findings were observed in the high-dose group only and/or at incidences above the upper limit of the HCD. Therefore a test item treatment relationship was not excluded,

. at 360 and 180 mg/kg/day, there were no variations or malformations at external examination of the fetuses.

 

Soft tissue examination:

. at 720 mg/kg/day, three females had fetuses with visceral edema, fluid-filled thoracic cavity and/or, fluid-filled abdomen. While limited to 3 litters, these variations were observed in the high-dose group only and at incidences above the upper limit of the HCD. Therefore a test item treatment relationship cannot be excluded. There were no test item treatment-related variations at soft tissue examination of the fetuses from 360 and 180 mg/kg/day groups,

. there were no test item treatment-related malformations at soft tissue examination of the fetuses from any group.

 

Skeletal examination:

. from 360 mg/kg/day and when compared with controls, there were dose-related increases in the litter and/or fetal incidences of skeletal variations (unossified bones or bones with incomplete ossification). In the 720 mg/kg/day group only, the litter and/or fetal incidences were above the upper limit of HCD. These findings were considered to be test item treatment-related and not adverse. At 180 mg/kg/day, there were no test item treatment-related skeletal variations,

. at 720 mg/kg/day, there were increases in the litter and fetal incidences of split supraoccipital. This malformation affected four fetuses from the same litter. While limited to one litter, this finding was observed in the high-dose group only and not recorded in the HCD. Therefore a test item treatment relationship cannot be excluded,

. at 360 and 180 mg/kg/day, there were no test item treatment-related skeletal malformations.

 

Conclusion

The test item was administered to time-mated female Sprague-Dawley rats, by gavage, once daily, from Days 6 to 20 p.c., at dose levels of 180, 360 or 720 mg/kg/day. 

On the basis of the results obtained in this study:

. the No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 180 mg/kg/day, based on adverse effects on body weight, body weight change and food consumption from 360 mg/kg/day and mortality at 720 mg/kg/day,

. the No Observed Adverse Effect Level (NOAEL) for embryo-fetal development was considered to be 360 mg/kg/day, based on embryo/fetal toxicity associated with malformations in a context of excessive maternal toxicity at 720 mg/kg/day.