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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 July 2016 - 09 February 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
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:
N-(3-aminopropyl)iminodiethanol
EC Number:
225-642-0
EC Name:
N-(3-aminopropyl)iminodiethanol
Cas Number:
4985-85-7
Molecular formula:
C7H18N2O2
IUPAC Name:
2-[(3-aminopropyl)(2-hydroxyethyl)amino]ethan-1-ol
Test material form:
liquid
Details on test material:
green liquid
storage conditions: at room temperature and protected from light under nitrogen atmosphere
Specific details on test material used for the study:
Name: N-(3-aminopropyl)iminodiethanol
Synonyms:
- Aminopropyldiethanolamine
- APDEA
- N-(3-Aminopropyl)diethanolamine
CAS No.: 4985-85-7
Batch No.: A290X2010101
Description: green liquid
Storage conditions: at room temperature and protected from humidity under nitrogen atmosphere
Purity: 80.83%
Expiry date: 01 September 2017

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 on the beginning of the treatment period
- Mean body weight at study initiation: a mean body weight of 305 g (range: 239 g to 358 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): approximately 8 to 15 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h

IN-LIFE DATES: 16 January 2017 to 09 February 2017

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Drinking water treated by reverse osmosis
Details on exposure:
PREPARATION OF DOSING FORMULATIONS:
- Solution in the vehicle
- Concentration in vehicle: 16, 50 and 150 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg/day
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Type of method: High Performance Liquid Chromatography with tandem Mass Spectrometry detection (LC/MS-MS)
Test item concentrations: remained within an acceptable range of variations (+2.2% to +9.5%) when compared to the nominal values (± 10% of the nominal concentrations).
Stability: The dose formulations containing the test item and prepared as a solution at 2 mg/mL and 200 mg/mL in drinking water treated by reverse osmosis were found to be stable after 5 days of storage at room temperature.
Duration of treatment / exposure:
Day 6 to Day 20 p.c.
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
80 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
750 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 level selection:
The dose-levels were selected in agreement with the Sponsor, based on the results of a previous OECD 422 study. The test item was administered by daily oral gavage to male and female Wistar Han rats at dose-levels of 100, 300 and 1000 mg/kg/day.
At 1000 mg/kg/day, parental toxicity consisted of reduced locomotor activity (total movements) with a normal habituation pattern, reduced body weight gain for male animals, affected hematological parameters, changes in blood biochemistry parameters, and organ weight changes (increased liver, kidney and adrenal weights and decreased thymus weights) without accompanying microscopic findings. The only apparent treatment-related macroscopic and microscopic changes were in the stomach. This was probably related to irritancy of the test substance. There were several dark red or reddish foci on the glandular mucosa of the stomach at macroscopic examination, and microscopic stomach abnormalities consisting of acute inflammation, hyperplasia, degenerative vacuolation, necrosis and hemorrhage.
Up to 1000 mg/kg/day, no toxicologically relevant changes in body weight, body weight gain or food consumption were noted for the pregnant females. Mating, fertility and conception indices, pre-coital time, numbers of corpora lutea and implantation sites, gestation index, duration of gestation, and pup body weights were unaffected by treatment.
No treatment-related findings were observed at the low and mid dose-levels.
Based on these results and considering the microscopic changes in the stomach observed at 1000 mg/kg/day, 750 mg/kg/day was selected as the high dose-level. The low and intermediate dose levels were selected using a ratio representing an approximately 3-fold interval (i.e. 250 and 80 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.

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.

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

PRESERVATION OF TISSUES:
See Table 1
- For all study animals, samples of the stomach were collected and preserved in 10% buffered formalin.
- Samples of the macroscopic lesions observed in females (including those from the females euthanized prematurely) were preserved in 10% buffered formalin (or in another appropriate fixative). As remarkable macroscopic lesions were observed in test item-treated animals, samples of the corresponding tissues (kidneys, adipose tissue and adrenals) from five control animals were collected and preserved.
Ovaries and uterine content:
The ovaries and uterine content were examined after termination, including::
- 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,
- evaluation of placentas.
Fetal examinations:
- External examinations: Yes: all fetuses per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
- Other : 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 2.
Test item treatment-related clinical signs, also noted in prematurely sacrificed females, consisted of round back, piloerection and loud breathing in 1/22 surviving females given 750 mg/kg/day.

Ptyalism, reddish vaginal discharge, dacryhorrhea, area of hair loss on forelimb, nodosities, abscess, cutaneous lesions and/or scabs on the neck were considered to be unrelated to the test item treatment as they were reported on a limited number of occasions and/or without any dose-relationship.
Mortality:
mortality observed, treatment-related
Description (incidence):
There were no test item treatment-related deaths in control animals or in the 80 and 250 mg/kg/day treated groups.

At 750 mg/kg/day, on Day 12 p.c., two pregnant females were prematurely sacrificed. Prior to death, these females showed signs of poor clinical condition (i.e. piloerection, emaciated appearance, abdominal and/or loud breathing, half-closed eyes and/or soft feces). Severe body weight loss was noted (between -43 to -64 g) over the last interval, together with reduced food consumption (1 to 8 g/animal/day).
At macroscopic post-mortem examination, the adrenal glands were enlarged (2/2 females) and brownish in color (1/2 females), the stomach mucosa showed several reddish discolored areas (2/2 females) and the cecum was dilated with gas (1/2 females). The right ureter, right kidney and right uterine horn were absent in one female.
These unscheduled deaths were considered to be test item treatment-related.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
See table 3.
At 750 mg/kg/day and when compared with controls, moderately lower mean body weight gain was noted on Days 9-12 p.c. (+11 g vs. +20 g in controls, p<0.05). This effect did not affect the mean body weight and did not correlate with any reduction in mean food intake.

At 80 or 250 mg/kg/day, there were no effects on mean body weight or mean body weight change.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
At 80, 250 or 750 mg/kg/day, there were no effects of treatment with the test item.
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 examined
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):
At 750 mg/kg/day, 1/22 females showed reddish colored foci on the forestomach. Since this finding was also observed in prematurely sacrificed females, it was considered to be test item treatment-related.

Other macroscopic findings (i.e. nodule in the right ovarian area, scabs and thickened subcutaneous tissue in the neck, unilateral absence of kidney and ureter together with unilateral kidney enlargement, unilateral atresia of uterine horn and/or enlarged placentae) were of isolated occurrence, not dose-related and/or due to spontaneous morphological malformations. They were, therefore, considered to be incidental.
Neuropathological findings:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
See table 5.
Net body weight change:
There were no dose-related effects on mean gravid uterus weight, carcass weight or net body weight change.

At 750 mg/kg/day and when compared with controls, females had a slightly lower mean net body weight change (+43 g vs. +50 g in controls); this was considered to be of no toxicological importance as this difference was minimal and did not correlate with any other finding.

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
See table 4.
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
See table 6.
There were no test item treatment-related effects.

At 750 mg/kg/day and when compared with controls, there was a slightly higher mean post-implantation loss (11.8% vs. 6.0%), along with a slightly higher mean number of early resorptions (1.2 vs. 0.8). This was mainly due to the contribution of two females, which had 80.0% and 64.3% post implantation losses, respectively. As these differences were not statistically significant, were close to the upper limit of the Historical Control Data and did not impact the mean number of live fetuses, a test item treatment relationship was considered to be unlikely.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
See table 6.
Early or late resorptions:
effects observed, non-treatment-related
Description (incidence and severity):
See table 6.
At 750 mg/kg/day and when compared with controls, there was a slightly higher mean post-implantation loss (11.8% vs. 6.0%), along with a slightly higher mean number of early resorptions (1.2 vs. 0.8). This was mainly due to the contribution of two females, which had 80.0% and 64.3% post implantation losses, respectively. As these differences were not statistically significant, were close to the upper limit of the Historical Control Data and did not impact the mean number of live fetuses, a test item treatment relationship was considered to be unlikely.
Dead fetuses:
no effects observed
Description (incidence and severity):
See table 6.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
See table 4.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
clinical signs
mortality

Maternal abnormalities

Abnormalities:
effects observed, non-treatment-related

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
See table 7.
There were no effects on mean fetal body weight at any dose level.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
See table 7.
There were no effects on sex-ratio (percentage of male fetuses) at any dose level.
External malformations:
effects observed, treatment-related
Description (incidence and severity):
See table 8.
Variations
There were no external variations at external fetal examination.

Malformations
In the 750 mg/kg/day group, two fetuses from two different litters had external malformations:
- one fetus (fetal body weight: 4.42 g): anasarca, cleft palate, mandibular micrognathia, short digits, micromelia, short trunk [split supraoccipital, split palate and absent hindpaw phalanx (at skeletal examination], bent tail and gastroschisis,
- one fetus (fetal body weight: 5.30 g): omphalocele.

These findings, mainly affecting the trunk (mid-line defects: gastroschisis or omphalocele), were observed in two malformed fetuses from two different litters, and one of these fetuses also had a body weight significantly lower than the other fetuses. A test item treatment-related effect cannot be ruled out taking into account a potential dose-relationship although the incidences of these malformations were similar to the Historical Control Data.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
See tables 11 to 12.
Cartilage
There were no test item treatment-related cartilage observations.
Cartilage findings [i.e. cartilage of cervical vertebra(e) present and cartilage of distal phalanx present] were observed with a higher litter incidence in the 250 mg/kg/day group and were considered to be unrelated to the test item treatment as they were not dose-related.

Variations
At 250 and 750 mg/kg/day and when compared with controls, there were higher litter and fetal incidences of fetuses with incomplete ossification of the interparietal bone for which the differences from controls were statistically significant at 750 mg/kg/day.

At 750 mg/kg/day and when compared with controls, there was a statistically significant increase in delayed ossification of the supraoccipital and parietal bones (incomplete ossification).

These variations, mainly affecting the head bones from 250 mg/kg/day, were considered to be associated with the test item treatment, although the incidences were below the maximum values in the Historical Control Data.

Other skeletal variations, (i.e. unossified distal phalanx of the forepaw was observed with a statistically significant higher litter incidence in the 250 mg/kg/day group) were considered to be unrelated to the test item treatment as they were not dose-related.

Malformations
In the 750 mg/kg/day group, one litter had a malformed fetus, with split palate, split supraoccipital and absent hindpaw phalanx (along with cleft palate, mandibular micrognathia, short digits and micromelia at external examination).

In the 250 mg/kg/day group, one litter had a malformed fetus (fetal body weight 2.97 g), with bent ulna and bent radius (along with misshapen cartilage of the radius and ulna).

In the 80 mg/kg/day group, no malformed fetuses were observed.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
See tables 9 and 10.
Variations
Soft tissue variations (i.e. dilated renal pelvis, short or absent innominate artery, malpositioned artery, dilated ureter, and/or reddish foci on the thymus) were not dose-related and/or were observed with a similar or minimally different incidence in control and/or test item treated groups. Therefore, they were considered to be unrelated to the test item treatment.

Malformations
There was no test item treatment-related increase in the frequency of soft tissue malformations.
In the 250 mg/kg/day group, one litter contained one malformed fetus with no left kidney and left ureter. As this isolated malformation was not dose-related and was within the Historical Control Data, it was considered to be of no toxicological importance.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
external malformations
skeletal malformations

Fetal abnormalities

Abnormalities:
effects observed, treatment-related
Localisation:
external: cranium
external: face
external: limb
external: paw
external: tail
external: trunk
external: thorax
external: umbilicus
skeletal: skull sutures
skeletal: hindlimb

Overall developmental toxicity

Developmental effects observed:
yes
Lowest effective dose / conc.:
250 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects occurring together with maternal toxicity effects, but not 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

 

TISSUE PROCEDURE TABLE

Table 1: Preservation of tissues 

Organs

Organ

weights

Preservation

of tissues

Microscopic examination

Macroscopic lesions

 

X

 

Stomach

 

X

 

 

Table 2: Clinical signs

 

Dose-level (mg/kg/day)

0

80

250

750

Round back

 

 

 

1
(on Day 15p.c.)

Piloerection

 

 

 

1
(on Day 15p.c.)

Loud breathing

 

 

 

1
(from Day 13 to

19p.c. )

Number of affected animals

0/24

0/24

0/24

1/22

( ): in brackets: Days p.c. of occurrences.

Table 3: Body weight

 

Dose-level (mg/kg/day)

0

80

250

750

Body weight (g)

 

 

 

 

Day 6 p.c.

302

305

305

306

 

-

(+1)

(+1)

(+1)

Day 9 p.c.

318

322

322

323

 

-

(+1)

(+1)

(+2)

Day 12 p.c.

338

344

344

334

 

-

(+2)

(+2)

(-1)

Day 15 p.c.

357

363

365

360

 

-

(+2)

(+2)

(+1)

Day 18 p.c.

398

404

409

397

 

-

(+2)

(+3)

(0)

Day 21 p.c.

448

453

463

447

 

-

(+1)

(+3)

(0)

Body weight change (g)

 

 

 

 

Days 6 - 9 p.c.

+16

+17

+17

+17

Days 9 - 12 p.c.

+20

+22

+22

+11*

Days 12 - 15 p.c.

+18

+18

+21

+19

Days 15 - 18 p.c.

+42

+41

+44

+38

Days 18 - 21 p.c.

+50

+49

+53

+50

Days 6 - 21 p.c.

+146

+148

+158

+140

 

-

(+1)

(+8)

(-4)

Statistical significance;*: p<0.05.

p.c.        :post-coitum.

-             : not applicable.

( )           : in brackets, percentage differencevs.controls.

Table 4: Pregnancy status

 

Dose-level(mg/kg/day)

        0

    80

       250

     750

Number of females

      24

    24

         24

       24

Non-pregnant females

        0

      0

           1

         0

Prematurely sacrificed females

        0

      0

           0

         2

Females with total resorption

        0

      0

           0

         0

Females with live fetuses at term

      24

    24

         23

       22

 

Table 5: Mean carcass, net change and gravid uterus weights (a) (g)

 

Dose-level (mg/kg/day)

0

80

250

750

Gravid uterus weight

96

-

95

(-1)

105

(+9)

98

(+2)

Carcass weight

352

-

358

(+2)

357

(+1)

350

(-1)

Net body weight change from Day 6.p.c.

+50

+53

+53

+43

( ): in brackets, percentage differencevs.controls.

p.c.        :post-coitum.

(a): weights are rounded values.

-             : not applicable.

Table 6: Hysterectomy data

 

Dose-level (mg/kg/day)

       0

    80

    250

750

HCD

Number of pregnant females
at hysterectomy

     24

    24

      23

22

      407

Number of females with live fetuses
at termination

     24

    24

      23

22

      388

Number of females with total resorption

       0

      0

       0

     0

          0

Mean number ofcorpora lutea

     14.2

    14.9

      16.3

    15.0

[13.8; 16.0]

Mean number of implantation sites

     13.0

    12.6

      14.4

    13.3

[12.5; 14.5]

Mean pre-implantation loss (%)

     10.0

    14.9

      11.4

    10.5

[6.2; 14.0]

Mean number of live fetuses

     12.2

    11.7

      13.3

    12.0

[11.6; 13.8]

Dead fetuses (mean %)

       0.0

      0.0

       0.0

     0.0

[0.00; 0.50]

Mean number of implantation scars

       0.0

      0.0

       0.0

     0.0

/

Mean number of early resorptions

       0.8

      0.9

       1.0

     1.2

/

Mean number of late resorptions

       0.1

      0.0

       0.2

     0.1

/

Mean post-implantation loss (%)

       6.0

      7.7

       8.6

    11.8

[3.5; 11.1]

HCD      : Historical Control Data (control data collected from 18 studies covering a period ranging from March 2014 to July 2016), [min.; max.].

/              : not reported in HCD.

Table 7: Fetal examinations ( Body weight and sex)

 

Dose-level (mg/kg/day)

0

80

250

750

HCD

Mean fetal body weight (g)

5.80
-

5.88
(+1)

5.72
(-1)

5.91
(+2)

[5.5; 5.9]

Mean fetal body weight
of males (g)

5.87
-

6.06
(+3)

5.88
(0)

6.07
(+3)

[5.7; 6.1]

Mean fetal body weight
of females (g)

5.70
-

5.69
(0)

5.57
(-2)

5.74
(+1)

[5.4; 5.7]

Mean percentage
of male fetuses (%)

47.4

51.8

47.5

50.0

[44.0; 55.4]

( ): in brackets, percentage differencevs.controls.

HCD: Historical Control Data (control data collected from 18 studies covering a period ranging from March 2014 to July 2016), [min.; max.].

-             : not applicable.

Table 8: Fetal (litter) incidences (%) of external malformations

 

Dose-level (mg/kg/day)

       0

     80

    250

    750

HCD

Dams with live fetuses

     24

     24

     23

     22

        388

Number of live fetuses

    292

    280

    305

    264

       5000

Litters affected, n (%)

0 (0.0)

0 (0.0)

0 (0.0)

2 (9.1)

11 (2.8)(b)

Fetuses affected, n (%)

0 (0.0)

0 (0.0)

0 (0.0)

2 (0.8)

13 (0.3)(b)

Anasarca, F (L)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

0.4 (4.5)

/

Cleft palate, F (L)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

0.4 (4.5)

0.4 (5.0)(a)

Mandibular micrognathia, F (L)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

0.4 (4.5)

0.4 (5.0)(a)

Short digit(s), F (L)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

0.4 (4.5)

0.4 (5.0)(a)

Micromelia, F (L)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

0.4 (4.5)

0.4 (5.0)(a)

Gastroschisis, F (L)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

0.4 (4.5)

0.4 (5.6)(a)

Short trunk, F (L)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

0.4 (4.5)

0.4 (5.0)(a)

Omphalocele, F (L)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

0.4 (4.5)

0.4 (5.3)(a)

Bent tail, F (L)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

0.4 (4.5)

0.4 (5.0)(a)

F            : fetal incidence.

L            : litter incidence.

HCD      : Historical Control Data (control data collected from 18 studies covering a period ranging from March 2014 to July 2016).

/              : not reported in HCD.

(a)           : maximum incidence.

(b)           : mean incidence.

Table 9: Fetal (litter) incidences (%) of soft tissue variations

 

Dose-level (mg/kg/day)

        0

      80

     250

     750

HCD

Dams with live fetuses

      24

      24

      23

      21

       387

Number of live fetuses

    139

     136

     145

     125

     2404

Litters affected, n (%)

10 (41.7)

8 (33.3)

6 (26.1)

7 (33.3)

86 (22.2) (b)

Fetuses affected, n (%)

23 (16.5)

9 (6.6)

12 (8.3)

14 (11.2)

119 (5.0) (b)

Dilated renal pelvis, F (L)

2.9 (12.5)

1.5 (8.3)

5.5 (21.7)

4.0 (9.5)

9.5 (28.6) (a)

Short innominate artery, F (L)

1.4 (8.3)

0.7 (4.2)

0.7 (4.3)

1.6 (9.5)

3.7 (22.7) (a)

Absent innominate artery, F (L)

2.9 (16.7)

0.7 (4.2)

0.0 (0.0)

2.4 (14.3)

5.1 (25.0) (a)

Malpositioned subclavian artery, F (L)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

0.8 (4.8)

/

Dilated ureter, F (L)

10.8 (20.8)

3.7 (20.8)

4.8 (8.7)

4.8 (14.3)

7.1 (28.0) (a)

Thymus: reddish foci, F (L)

0.0 (0.0)

0.7 (4.2)

0.0 (0.0)

0.0 (0.0)

/

F: fetal incidence.

L: litter incidence.

HCD    : Historical Control Data (control data collected from 18 studies covering a period ranging from March 2014 to July 2016).

/: not reported in Historical Control Data.

(a): maximum incidence.

(b): mean incidence.

Table 10: Fetal (litter) incidences (%) of soft tissue malformations

 

Dose-level (mg/kg/day)

       0

      80

    250

    750

HCD

Dams with live fetuses

     24

      24

      23

      21

       387

Number of live fetuses

    139

    136

    145

    125

     2404

Litters affected, n (%)

0 (0.0)

0 (0.0)

1 (4.3)

0 (0.0)

7 (1.8)(b)

Fetuses affected, n (%)

0 (0.0)

0 (0.0)

1 (0.7)

0 (0.0)

13 (0.5)(b)

Absent kidney, F (L)

0.0 (0.0)

0.0 (0.0)

0.7 (4.3)

0.0 (0.0)

0.7 (4.5)(a)

Absent ureter, F (L)

0.0 (0.0)

0.0 (0.0)

0.7 (4.3)

0.0 (0.0)

/

F: fetal incidence.

L: litter incidence.

HCD: Historical Control Data (control data collected from 18 studies covering a period ranging from March 2014 to July 2016).

/              : not reported in Historical Control Data.

(a)           : maximum incidence.

(b)           : mean incidence.

Table 11: Fetal (litter) incidences (%) of skeletal variations

 

Dose-level (mg/kg/day)

         0

       80

   250

     750

HCD

Dams with live fetuses

       24

       24

     23

       22

        388

Number of live fetuses

     153

     144

   160

     139

      2596

Supraoccipital: incomplete ossification, F (L)

0.0 (0.0)

0.7 (4.2)

0.0 (0.0)

3.6* (9.1)

5.6 (25.0)(a)

Parietal: incomplete ossification, F(L)

0.7 (4.2)

0.0 (0.0)

0.6 (4.3)

4.3*(22.7)

9.2 (35.0)(a)

Interparietal: incomplete ossification, F(L)

0.0 (0.0)

0.7 (4.2)

1.9 (13.0)

5.0** (18.2*)

9.2 (45.0)(a)

F: fetal incidence.

L: litter incidence.

Statistically significant; *: p<0.05 and **: p<0.01 for the number of fetuses or litters affected or for affected fetuses/litter (incomplete ossification of parietal).

HCD      : Historical Control Data (control data collected from 18 studies covering a period ranging from March 2014 to July 2016).

(a)           : maximum incidence.

Table 12: Fetal (litter) incidences (%) of fetal skeletal malformations

 

Dose-level (mg/kg/day)

       0

      80

   250

     750

HCD

Dams with live fetuses

     24

      24

     23

       22

        388

Number of live fetuses

    153

    144

   160

     139

      2596

Litters affected, n (%)

0 (0.0)

0 (0.0)

1 (4.3)

1 (4.5)

15 (3.9)(b)

Fetuses affected, n (%)

0 (0.0)

0 (0.0)

1 (0.6)

1 (0.7)

18 (0.7)(b)

Supraoccipital: split, F (L)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

0.7 (4.5)

/

Palate: split, F (L)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

0.7 (4.5)

0.7 (5.0)(a)

Ulna: bent, F(L)

0.0 (0.0)

0.0 (0.0)

0.6 (4.3)

0.0 (0.0)

/

Radius: bent, F(L)

0.0 (0.0)

0.0 (0.0)

0.6 (4.3)

0.0 (0.0)

/

Hindpaw: absent phalanx, F (L)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

0.7 (4.5)

/

F: fetal incidence.

L: litter incidence.

HCD: Historical Control Data (control data collected from 18 studies covering a period ranging from March 2014 to July 2016).

/              : not reported in Historical Control Data.

(a)           : maximum incidence.

(b)           : mean incidence.

Table 13:

Distribution of fetal malformations

 

Dose-level (mg/kg/day)

0

80

250

750

HCD

External

 

Litters affected, n (%)

0 (0.0)

0 (0.0)

0 (0.0)

2 (9.1)

11 (2.8)(a)

Fetuses affected, n (%)

0 (0.0)

0 (0.0)

0 (0.0)

2 (0.8)

13 (0.3)(a)

Soft tissue

 

 

 

Litters affected, n (%)

0 (0.0)

0 (0.0)

1 (4.3)

0 (0.0)

7 (1.8)(a)

Fetuses affected, n (%)

0 (0.0)

0 (0.0)

1 (0.7)

0 (0.0)

13 (0.5)(a)

Skeletal

 

Litters affected, n (%)

0 (0.0)

0 (0.0)

1 (4.3)

1 (4.5)

15 (3.9)(a)

Fetuses affected, n (%)

0 (0.0)

0 (0.0)

1 (0.6)

1 (0.7)

18 (0.7)(a)

Total

 

 

 

 

 

Litters affected / evaluated (%)

0/24 (0.0)

0/24 (0.0)

2/23 (8.7)

2/22 (9.1)

/

Fetuses affected / evaluated (%)

0/292 (0.0)

 0/280 (0.0)

2/305 (0.7)

2/264 (0.8)

/

HCD: Historical Control Data (control data collected from 18 studies covering a period ranging from March 2014 to July 2016).

(a): mean incidence.

/: not reported 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 80, 250 and 750 mg/kg/day. The control group received the vehicle, drinking water treated by reverse osmosis, under the same experimental conditions.

On the basis of the results obtained in this study:
- the No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 250 mg/kg/day (based on poor clinical condition leading to premature euthanasia at 750 mg/kg/day),
- the NOAEL for embryo-fetal development was considered to be 250 mg/kg/day (based on 2 fetuses with fetal malformations at 750 mg/kg/day) in a context of severe maternal toxicity.
Executive summary:

The objective of this 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 until the day before scheduled hysterectomy [Day 6 to Day 20 post-coitum (p.c.) inclusive].

This GLP study was carried out according to OECD test guideline No. 414 (22 January 2001).

 

Methods

Three groups of 24 time-mated female Sprague-Dawley rats received the test item by the oral route (gavage), at 80, 250 or 750 mg/kg/day, once daily from Day 6 to Day 20 p.c. inclusive. Another group of 24 time-mated rats received the vehicle only, drinking water treated by reverse osmosis, under the same experimental conditions, and acted as a control group. A constant dosage volume of 5 mL/kg/day was used. The test item concentrations were determined in the dose formulations.The animals were checked daily for mortality and clinical signs. Body weight and food consumption were recorded over designated intervals. On Day 21 p.c., the females were sacrificed and a macroscopic post-mortem examination was performed. Hysterectomy was performed and the numbers of corpora lutea, implantation sites, uterine scars, early and late resorptions, and live and dead fetuses were recorded. The fetuses were weighed, sexed and examined for external, soft tissue and skeletal abnormalities (including cartilage).

Samples of stomach and macroscopic lesions were collected from all dams and preserved in 10% buffered formalin.

 

Results

Chemical analyses

The concentrations of the test item in the dose formulations remained within acceptable ranges of variations (+2.2% to +9.5%) when compared with the nominal concentrations (± 10%).

The test item concentration was below the Limit Of Quantification (0.01 mg /mL) in control dose formulations.

 

Pregnancy status

There were 24, 24, 23 and 22 females with live fetuses in the groups given 0, 80, 250 and 750 mg/kg/day, respectively.

 

Mortality

At 750 mg/kg/day, two pregnant females were prematurely sacrificed due to signs of poor clinical condition (piloerection, emaciated appearance, abdominal and/or loud breathing, half-closed eyes and/or soft feces, and severe body weight loss together with reduce food intake).At necropsy, the adrenal glands were enlarged and/or brownish in color, the stomach mucosa showed several reddish discolored areas and/or the cecum was dilated with gas.These findings were considered to be test item treatment-related.

No other death occurred in the low and mid dose groups.

 

Clinical signs

Test item treatment-related clinical signs were transient and consisted of round back, piloerection and loud breathing in 1/22 females given 750 mg/kg/day.


Body weight

There was no effect on mean body weight at any dose-level.

At the high dose-level of 750 mg/kg/day, mean body weight gain was significantly lower than controls

(-45%) on Days 9-12 p.c. (+11 g vs. +20 g, p<0.05).

 

Food consumption

There were no test item treatment-related effects at any dose-level.

 

Necropsy and macroscopic post-mortem examination

Reddish colored foci on the forestomach mucosa was observed in 1/22 females given 750 mg/kg/day and sacrificed as scheduled. No test item-related findings were observed in the low and mid dose-level groups.

 

Net body weight change

There were no dose-related effects on mean gravid uterus weight, carcass weight or net body weight change at any dose-level.

 

Hysterectomy data

There were no test item treatment-related effects.

Fetal body weight and percentage of male fetuses

There were no test item treatment-related effects.

 

Fetal examinations

External examination

There were no test item-related external or oral cavity variations at examination of the fetuses at any dose-level. At 750 mg/kg/day, two fetuses, from two different litters had anasarca, cleft palate, mandibular micrognathia, short digits, micromelia, short trunk, bent tail, gastroschisis and/or omphalocele for which a relationship to the test item could not be ruled out.

 

Soft tissue examination

There were no test item-related variations or malformations at soft tissue examinationat any dose-level.

 

Cartilage and skeletal examinations

At 250 and 750 mg/kg/day and when compared with controls, there were higher litter and fetal incidences of fetuses with delayed ossification: incomplete ossification of the interparietal bone (statistically significant at 750 mg/kg/day).

 

At 750 mg/kg/day and when compared with controls, there were statistically significant increases in litter and fetal incidences of fetuses with skeletal abnormalities mainly involving the head bones, such as incomplete ossification of the supraoccipital and parietal bones (variations), split palate and split supraoccipital (malformations observed in one fetus also showed an absence of the hindpaw phalanx).


Conclusion

The test item was administered by gavage, once daily, from Days 6 to 20 p.c., inclusive, to pregnant Sprague-Dawley rats at 80, 250 and 750 mg/kg/day. The control group received the vehicle, drinking water treated by reverse osmosis, under the same experimental conditions.

 

On the basis of the results obtained in this study:

.         the No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 250 mg/kg/day (based on poor clinical condition leading to premature euthanasia at 750 mg/kg/day),

.         the NOAEL for embryo-fetal development was considered to be 250 mg/kg/day (based on 2 fetuses with fetal malformations comparable to HCD at 750 mg/kg/day) in a context of maternal toxicity.

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