Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2014-02-27 to 2014-09-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study, OECD 414 compliant

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
2013-01-10
Limit test:
no

Test material

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

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Sprague-Dawley, RjHan: SD (Rats CD®).
- Source: Breeder: Janvier, le Genest-Saint-Isle, France
- Age at study initiation: at the beginning of the treatment period, the animals were 10-11 weeks old
- Weight at study initiation: mean body weight of 251 g (range: 196 g to 305 g). The females were sexually mature and primigravid.
- Fasting period before study:
- Housing: The animals were individually housed in polycarbonate cages (Tecniplast 2154, 940 cm2) with stainless steel lids and containing autoclaved sawdust (SICSA, Alfortville, France). Individual housing was chosen in order to not jeopardize the gestation. Each cage contained an object for the environmental enrichment of the animal (rat hut). The cages were placed in numerical order on the racks.
- Diet (e.g. ad libitum): All animals had free access to SSNIFF R/M-H pelleted maintenance diet, batch No. 6730746 (SSNIFF Spezialdiäten GmbH, Soest, Germany) which was distributed weekly.
- Water (e.g. ad libitum): The animals had free access to bottles containing tap water (filtered with a 0.22 μm filter).
- Acclimation period: the animals were acclimated to the conditions of the study for a period of 4 or 5 days before the beginning of the treatment period (arrival of the females on Days 1 or 2 p.c.).

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: The vehicle was pH 7.2 sodium phosphate buffer 0.5M prepared using: . sodium phosphate monobasic monohydrate, batch BCBK7914V, . NaOH 1N, batch Nos. 14020004 and 13090001, . drinking water treated by reverse osmosis using ELIX 5 (Millipore SA).
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Calculation of correction factor: In this study, the dose-levels of the test item were expressed in terms of active ingredient. To obtain the test item amount to be weighed for dose formulation preparation, the amount of test item expressed in active ingredient was multiplied by a correction factor of 2.54.

Dose formulation preparation
The test item was administered as a solution in the vehicle. The test item was mixed with the required quantity of vehicle.
The test item dose formulations were prepared daily and delivered to the study room at room temperature and protected from light.

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle:0, 20, 60 and 200 mg/mL (0, 100, 300 and 1000 mg/kg)
- Amount of vehicle (if gavage): 5 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentrations of the test item in samples of each control and test item dose formulation prepared for use on the first day and last week of treatment were determined.

The analytical method was developed at CiToxLAB France. It consisted of sampling 1 mL of dose formulation and diluting it appropriately with diluent to reach the nominal concentration of injection. The diluted samples were analyzed by Ion Chromatography (IC) with conductivity detection, bracketed by standard solutions and quantified by the mean response factors calculated for the standard solutions.

Results
Concentration of ITC288 in administered dose formulations
Group number Nominal concentrations (mg/mL) Measured concentrations Day 01 Measured concentrations End of treatment
(mg/mL) Bias (%) (mg/mL) Bias (%)
1 0 < 0,1 nc < 0,1 nc
2 20 20,2 1,1 20,4 2,0
3 60 59,2 -1,3 60,4 0,6
4 200 194 -3,0 204 1,9
Details on mating procedure:
The females were mated at the breeder's facilities. 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:
Once a day
Duration of test:
14 days
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300 and 1000 mg/kg
Basis:
actual ingested
active ingredient
No. of animals per sex per dose:
24 mated females
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 preliminary prenatal study performed in the same species and strain (CiToxLAB France/Study No. 39997 RSR). In this study, four groups of five time-mated females received the test item as a solution in the vehicle (pH 7.2 sodium phosphate buffer 0.5M) at dose-levels of 100, 300, 750 or 1000 mg/kg/day. There were no significant effects at any dose-level, except transient and non-adverse lower mean body weight gain and mean food consumption at the beginning of the treatment period.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
Morbidity and mortality: Twice a day
Clinical signs
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 at least once a day, at approximately the same time, for the recording of clinical signs.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each female was recorded on Days 3, 6, 9, 12, 15, 18 and 21 p.c.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- The quantity of food consumed by each female was recorded for the following intervals:
. Days 3-6, 6-9, 9-12, 12-15, 15-18 and 18-21 p.c..

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations:

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #21
- Organs examined:
examination of principal thoracic and abdominal organs.
A gross evaluation of placentas
Ovaries and uterine content:
The ovaries and uterus of the females 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.

The following classification was used to record:
. uterine scar: uterine implantation without implant,
. early resorption: evidence of implant without recognizable embryo,
. late resorption: dead embryo or fetus with external degenerative changes,
. dead fetus: non live fetus with discernible digits.

For apparently non-pregnant females, the presence of implantation scars on the uterus was checked using the ammonium sulphide staining technique (Salewski, 1964).
A gross evaluation of placentas was also undertaken. The placenta weight was also recorded for each live fetus (except of placentas from fetuses No. C24486-5 to C24486-8 which were not found, their dam having started to deliver).

Fetal examinations:
The body weight of each fetus was recorded.

The sex of each fetus was determined at the time of hysterectomy by visual assessment of the anogenital distance and was confirmed by internal examination of sexual organs at detailed dissection of the soft tissues or at evisceration.

External examination
Each fetus was subjected to a detailed external examination, which included the observation of all visible structures, surfaces and orifices.

Soft tissue examination
As soon as possible after sacrifice, approximately half of the fetuses in each litter were subjected to a detailed dissection of the soft tissues, which included the observation of all organs and structures of the neck, thorax and abdomen. The fetuses were then eviscerated and fixed with Harrison's fluid for examination of the structures of the head.

Skeletal examination
The remaining fetuses per litter were eviscerated and fixed with ethyl alcohol. A detailed examination of the skeleton (bones and cartilages) was performed after staining with alizarin red S and alcian blue. This examination included the observation of all bones and cartilage structures of the skull, spine, rib cage, pelvis and limbs.

Photographs
Photographes of fetuses with rare malformations (not considered as raw data) were taken to document findings and kept with the study archives.
Statistics:
Mean values were compared by one-way analysis of variance and Dunnett test (mean values being considered as normally distributed and variances being considered as homogeneous).
Percentage values were compared by Fisher exact probability test (proportions).
Historical control data:
No

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
act. ingr.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
act. ingr.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Body weight (Figures 1 and 2, Tables 2 to 4, Appendices 6 to 8)

Dose-level (mg/kg/day)

0

100

300

1000

Body weight (g)

Day 6p.c.

251

250

252

252

Day 21p.c.

394

383

393

387

 

 

(-3)

(0)

(-2)

Body weight change (g)

Days 6 - 9p.c.

16

15

14

12*

Days 15 -18p.c.

378

32**

37

36

Days 6 - 21p.c.

+143

+133

+141

+135

 

 

(-7)

(-1)

(-6)

Statistically significantvs.controls: *: p<0.05, **: p<0.01; (): percentage differences from controls (%).

(): percentage differences from controls (%).

 

Mean carcass weights and mean net body weight changes

Dose-level (mg/kg/day)

0

100

300

1000

Carcass weight (g)

297.2

294.6

294.0

290.2

Net body weight change from day 6p.c.(g)21p.c.

46.2

44.5

42.4

39.3

 

 

(-4)

(-8)

(-15)

(): percentage differences from controls (%).

 

Gravid uterus weight

Dose-level (mg/kg/day)

0

100

300

1000

Gravid uterus weight (g)

297.2

294.6

294.0

290.2

 

 

 

 

 

 

 

Macroscopicpost-mortemexamination

There were no test item-related macroscopic changes at necropsy

 

Hysterectomy data

Dose-level (mg/kg/day)

0

100

300

1000

Number of females with live fetuses at termination

20

24

22

24

Mean number ofcorpora lutea

15.6

14.4

14.9

14.6

Mean number of implantations

13.3

12.9

13.5

13.4

Mean pre-implantation loss (%)

12.7

10.0

9.1

7.7

Mean number of fetuses

12.5

11.5

12.9

12.9

Percentage of dead fetuses (%)

0

0

0

0

Mean number of implantation scars

0

0

0

0

Mean number of early resorptions

0.8

1.3

0.6

0.5

Mean number of late resorptions

0.1

0.1

0.0

0.0

Mean number of late resorptions

6.2

11.3

4.4

4.3

Percentage of females affected

50.0

66.7

36.4

37.5

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

 

Fetal examinations

Dose-level (mg/kg/day)

0

100

300

1000

Mean percentage of male fetuses (%)

49.4

48.4

52.7

48.8

Mean fetal body weight (g), both sexes

5.73

5.59

5.58

5.45**

Variation fetal body weight from control (%)

 

(-2)

(-3)

(-5)

Mean fetal body weight (g), Males

5.84

5.76

5.71

5.55**

Mean fetal body weight (g), Females

5.61

5.44

5.44

5.35

Mean litter weighta(g)

71.6

64.2

71.9

69.9

Variation Mean litter weight from control (%)

 

(-10)

(0)

(-2)

Mean placenta weight (g)

0.58

0.63

0.56

0.60

Mean fetal/placenta weight ratioa

10.18

9.42

10.20

9.43

Statistically significantvs.controls: **: p<0.01; a: manually calculated, no statistics performed.

(): percentage differences from controls (%).

There were no effects on mean percentage of male fetuses (sex ratio).

There were no effects on mean placenta weight and on mean fetal/placental weight ratio at any dose-level.

External examination

There were no external variations.

Soft tissue examination

There were no soft tissue malformations.

 

Repartition of fetuses and litters with soft tissue variations

 

Dose-level (mg/kg/day)

0

100

300

1000

Number of litters

20

24

22

24

Number of fetuses

119

132

134

145

Dilated renal pelvis, L(F)

3 (3)

2 (3)

4 (4)

2 (3)

Dilated ureter, L(F)

 

1 (1)

1 (1)

1 (1)

Absent innominate artery, L(F)

1 (1)

 

 

 

Short innominate artery, L(F)

 

 

2 (2)

 

Liver: colored focus, L(F)

1 (1)

 

 

 

L (F): Litter (Fetal) incidences.

There were no test item treatment-related soft tissue variations.

 

Skeletal malformations in litters (fetuses)

 

Dose-level (mg/kg/day)

0

100

300

1000

Number of litters

20

24

22

24

Number of fetuses

131

144

149

164

Thoracic vertebra(e): absent

hemicentrum (no cartilage)

+ fused sternebrae + fused ribs, L(F)

 

1 (1)

(C24423-14)

 

 

Lumbar vertebra(e): absent, unossified

arches + Sacral vertebrae: absent (no

cartilage for the absent vertebrae), L (F)

 

 

1 (1)

(C24460-5:

had also

constricted tail at

external

observation)

 

Sternoschisis + fused sternebrae, L (F)

 

 

1 (1)

(C24450-9)

 

Total fetal skeletal malformations L(F)

0

1 (1)

2 (2)

0

Variations

Total fetal skeletal variations, L (F)

19 (64)

23 (74)

19 (76)

19 (56)

L(F): Litter (Fetal) incidences.

One (100 mg/kg/day) and two fetuses (300 mg/kg/day) had multiple variations and malformation of the axial skeleton.

In the absence of dose-related effects and/or similar findings in other groups, a test item treatment-related effect was considered unlikely.

Applicant's summary and conclusion

Conclusions:
The test item, ITC288 (batch No. B288P22E1), was administered by gavage, once daily, from Days 6 to 20 p.c., inclusive, to time-mated female Sprague-Dawley rats at dosages of 100, 300 and 1000 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 1000 mg/kg/day, based on the absence of adverse effects on the dams at the dose-level.
. The NOAEL for embryo-fetal development was considered to be 1000 mg/kg/day, based on the absence of adverse effects on the offspring development at the dose-level.
Executive summary:

Objectives

The objective of this study was to evaluate the potential toxic effects of the substance, ITC288, on the pregnant female rat and on embryonic and fetal development, following daily oral administration (gavage) to pregnant female rats from implantation to the day prior to the scheduled hysterectomy (Day 6 to Day 20 post-coitum (p.c.) inclusive) acording to the OECD 414 guideline.

The test item concentrations (100, 300 and 1000 mg/kg expressed as active ingredient) in the administered dose formulations analyzed were within the acceptance criteria.

Dams

At termination on Day 21 p.c., there were 20, 24, 22 and 24 dams with live fetuses at 0, 100, 300 and 1000 mg/kg/day, respectively.

  • There were no premature deaths and no test item treatment-related clinical signs.
  • There were no test item treatment-related effects on mean food consumption, mean body weight, mean body weight gain, mean gravid uterus weight and mean hysterectomy data.
  • There were no toxicologically relevant effects on mean carcass weight and mean net body weight change. There were no test item treatment-related macroscopic findings at necropsy.

Fetuses

  • There were no effects on sex ratio, mean placenta weight and mean fetal/placenta weights ratio. There were no toxicologically significant effects on mean fetal weight.
  • There were no test item treatment-related variations or malformations at external, soft tissues and skeletal examinations.

Conclusion

The test item, ITC288 (batch No. B288P22E1), was administered by gavage, once daily, from Days 6 to 20 p.c., inclusive, to time-mated female Sprague-Dawley rats at dosages of 100, 300 and 1000 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 1000 mg/kg/day, based on the absence of adverse effects on the dams at the dose-level.
  • The NOAEL for embryo-fetal development was considered to be 1000 mg/kg/day, based on the absence of adverse effects on the offspring development at the dose-level.