Registration Dossier

Administrative data

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

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
2016-11-02
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: Charles River Laboratories Italia, Calco, Italy.
- 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 272 g (range: 227 g to 309 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 (SICSA, Alfortville, France). Individual housing was chosen in order to not jeopardize gestations.
Each cage contained nylabone and rat hut for the environmental enrichment of the animals.
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. 799891 (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 study conditions for a period of 4 or 5 days before the beginning of the treatment period (arrival of the females on Day 1 or 2 p.c.).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 30% - 70%
- 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: From 2016-11-17 to 2016-12-15

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was administered as an emulsion in the vehicle. The required quantities were mixed progressively with the vehicle in order to obtain the desired concentration. After addition of the whole quantity of vehicle, the dose formulations were kept under magnetic stirring for at least 30 minutes to ensure effective solubilization of the test item. Based on test item dose formulation stability CiToxLAB France/Study No. 40029 AHS, the test item dose formulations were prepared on each day of treatment, stored and delivered at room temperature and protected from light. Control dose formulations were stored and delivered each day at room temperature.


VEHICLE
- Concentration in vehicle: 0, 20, 80 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/ kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Principle of the analytical method
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
(0.1 mg/mL). The diluted samples were analyzed by Ion Chromatography (IC) with conductivity detection,
bracketed by Quality Control samples and quantified using a calibration curve. The quantification was
based on the analysis of one component of the test item considered as a marker. The peak area of this
marker was used for quantification.

Solvent and reagent
Milli-Q water (CiToxLAB France)
Sodium Hydroxide S5881-500g (Sigma-Aldrich)
Sulfuric acid 320501-1L (Sigma-Aldrich)
Diluent: 0.01M sodium hydroxide.

Equipment
High Performance Liquid Chromatography Ionic system with conductivity detection (ICS5000) (Thermo)
Micro-balance; Balance (Mettler-Toledo)
Automatic pipettes (Biohit)
equipments for agitating (i.e. magnetic stirring, vortex…),
class A volumetric flasks.

Software
Empower 2 (Waters).

Samples preparation
For the determination of nominal concentrations, the dilution volumes used were calculated by measuring the weight of each sampling and assuming the density of the dose formulation sampled (d = 1.13).
Dilution factor = (V2 x V1) / (v1 x v2)
With:
V1 = total volume for the first dilution
V2 = total volume for the second dilution
v1 = volume taken for each sample (with v1 = weight of sample / density of the formulation)
v2 = volume taken for the second dilution

Composition of the analytical sequence
Analytical sequences are composed of at least:
. a blank sample (diluent) was checked for the absence of chromatographic interferences, . vehicle sample (when requested),
. a LOQ solution
. a calibration curve using five standard solutions at increasing concentrations within the range defined during validation
. at least two Quality Control (QC) samples at one concentration level prepared using independent standard solutions (different to those used for the calibration curve)
. study samples prepared from aliquots of the dose formulations bracketed by the QC samples.

Analytical note: for the determination of content of administered dose formulations analyzed in Week 3 (07 December 2016), the LOQ solution prepared at 2 μg/mL was not detected. The LOQ solution provided direct estimations of potential contamination in the group control. No interfering peaks was detected in the group control on 07 December 2016, thus this deviation to internal SOP has no impact on the results.

Quantification
The quantification was based on the analysis of one component of the test item, considered as a marker. Peak areas of this marker were used for the quantification.
Peak areas were determined for standard solutions ranging from 0.05 to 0.15 mg/mL of Reaction mass of diisobutyl hydrogen phosphate and isobutyl dihydrogen phosphate (five levels). A calibration curve was obtained by linear regression analysis of peak areas against concentrations and the regression analysis of the calibration data gave an equation as follows:
Y = a X + b
where:
Y = peak area of Reaction mass of diisobutyl hydrogen phosphate and isobutyl dihydrogen phosphate (μV.s)
X = concentration of Reaction mass of diisobutyl hydrogen phosphate and isobutyl dihydrogen phosphate (mg/mL)
a = slope value
b = intercept

Acceptance criteria of an analytical sequence
Acceptance criteria are defined in CiToxLAB France Standard Operating Procedures (SOPs).
The acceptance criteria included:
. coefficient of determination: r2 ≥ 0.98,
. deviation calculated between the theoretical and measured concentrations (%) for calibration points
and Quality Controls: between -7% and +7%.
%DEV = (Cm-Ct)Ct x 100
With: Cm: measured concentration
Ct: theoretical concentration

Dose formulation analysis
Assay
Diluted samples of dose formulations were analyzed in single by Ion Chromatography (IC) with conductivity detection.
The test item peak area was determined for each sample and the corresponding concentration was calculated using the equation obtained from the calibration curve.
All the results are expressed as mg/mL of Reaction mass of diisobutyl hydrogen phosphate and isobutyl dihydrogen phosphate.

Acceptance criterion
Deviation calculated between measured concentration and theoretical concentration should be within
± 15%.
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 daily
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
400 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
24 animals per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose-levels were selected in agreement, on the basis of the results of a previous OECD 422 study performed in the same species and strain (CiToxLAB France/Study No. 40032 RSR), in which four groups of 10 males and 10 females received 0 (PEG 400), 100, 400 or 1000 mg/kg/day, daily by gavage, under a constant dosage volume of 5 mL/kg/day.
There were no test item-related deaths, and clinical signs consisted of ptyalism that was noted in up to six animals per sex at 400 mg/kg/day and in all animals at 1000 mg/kg/day. There were no test item treatment-related effects on mean body weight, mean food consumption, functional observation battery, mean hematological and urinalysis parameters or mean organ weights. There were no test item treatment-related effects on mating, fertility and delivery data, or on mortality, clinical signs, sex ratio, mean body weight or macroscopic post-mortem findings in pups.
There were minor blood biochemistry findings (decrease in mean total bilirubin and protein levels) in males given 1000 mg/kg/day. There were non-adverse pathology findings (thickening of duodenum, jejunum and/or ileum in females from 400 mg/kg/day and microscopic findings in the forestomach, stomach, duodenum and jejunum) suggesting local irritant properties of the compound in males and females from 400 mg/kg/day.
Therefore, the same dose-levels were used for this study.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Each animal was checked for mortality and morbidity once a day before the treatment period and at least twice a day during the treatment period, including weekends.

BODY WEIGHT: Yes
- Time schedule for examinations: 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: Yes
-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.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21
- Organs examined: the principal thoracic and abdominal organs
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- number of corpora lutea
- number and distribution of dead and live fetuses
- number and distribution of early and late resorptions
- number and distribution of uterine scars
- number and distribution of implantation sites.
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: Yes
Statistics:
Data were compared by one-way analysis of variance and Dunnett test (mean values being considered as normally distributed and variances being considered as homogeneous) or by Fisher exact probability test (proportions).
Historical control data:
Yes

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Test item treatment-related clinical signs consisted of:
- round back and/or piloerection observed in the second or third week of treatment in females at all dose-levels,
- ptyalism, usually observed from the second week of treatment in females given 100 or 400 mg/kg/day or from the first week of treatment in females given 1000 mg/kg/day,
- emaciated appearance noted from the second week of treatment in females given 400 or 1000 mg/kg/day,
- loud and/or abdominal breathing, pallor of extremities and/or soft feces observed from the second or third week of treatment in females given 1000 mg/kg/day.
Breathing difficulties were considered to be endpoints and therefore to have exceeded the Maximal Tolerated Dose.

Reflux at dosing (noted on one occasion in 1/24 females given 100 mg/kg/day) resulted from the gavage procedure. Reddish vaginal discharge observed in 1/24 control females and 2/24 females given 400 mg/kg/day was considered to be unrelated to the test item treatment, as it was reported on a limited number of occasions and without any dose relationship.
Mortality:
mortality observed, treatment-related
Description (incidence):
There were no test item treatment-related deaths in control animals or the 100 and 400 mg/kg/day treated groups.
At 1000 mg/kg/day, one pregnant female was found dead (female J21511) on Day 18 p.c., and on Day 13, 18, 19 or 20 p.c., seven pregnant females were prematurely sacrificed (females J21509, J21514, J21515, J21516, J21520, J21521 and J21522). Prior to death, these females showed signs of poor clinical condition (i.e. piloerection, round back, pallor of extremities, emaciated appearance, hypotonia, hypoactivity, abdominal and/or loud breathing, dyspnea, chromodacryorrhea, chromorhinorrhea, half-closed eyes, soft feces, and/or soiled head or urogenital area). Ptyalism was also noted in all females. Moderate to severe body weight loss was noted in all females (between -23 to -83 g) over the last intervals, together with reduced food consumption in 4/8 females (3 to 17 g/animal/day).
At macroscopic post-mortem examination, the adrenal glands were enlarged (2/8 females), the spleen was reduced in size (5/8 females), the stomach was dilated (7/8 females) and whitish colored content was present (1 female), and there was whitish colored mucosa (4 females), thickening of mucosa (2 females) and/or whitish or reddish colored foci (2 females). The intestines were dilated (8/8 females) and/or the lungs were dilated or with whitish liquid content in the thoracic cavity (1/8 females).
These unscheduled deaths were considered to be test item- related. Macroscopic observations are consistent with the hypothesis that the deaths are linked to corrosive properties of the test item. However, in absence of microscopic examination it cannot be confirmed.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg/day and when compared with controls, slightly to markedly lower mean body weight gain was noted on Days 9-12 p.c. (+18 g vs. +24 g in controls) and on Days 15-18 p.c. (+20 g vs. +42 g in controls, p < 0.001). These variations were mainly due to the contribution of the body weight loss recorded in moribund females before their death and in surviving females J21524 and J21526. These findings were considered to be adverse although the terminal mean body weight on Day 21 p.c. was not affected.
Effects on mean body weight changes correlated with reduced food consumption between Days 15 and 18 p.c.

At 100 or 400 mg/kg/day, there were no effects on mean body weight or mean body weight change.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg/day and, when compared with controls, lower mean food consumption (-10%, p < 0.05) was recorded between Days 15 and 18 p.c., with a return to control values thereafter. This variation was mainly due to the contribution of the body weight loss recorded in moribund females before their death and in surviving females J21524 and J21526. This change was considered to be adverse. This difference correlated with the lower mean body weight gain also observed at 1000 mg/kg/day.

At 100 or 400 mg/kg/day, there were no effects of treatment with the test item.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
At 400 and 1000 mg/kg/day, 1/24 (J21501) and 1/16 females (J21526), respectively, showed whitish colored stomach mucosa. Dilatation of intestines, associated with reduced thymus and enlargement of one adrenal gland, was also noted in another female (J21524) given the high-dose level. Since these findings were also observed in decedent females, they were considered to be test item treatment-related.
Like for the decedent females, these observations are consistent with the hypothesis that the deaths are linked to corrosive properties of the test item. However, in absence of microscopic examination it cannot be confirmed.
Other macroscopic findings (i.e. renal pelvis dilatation in 1/24 females given 400 mg/kg/day and enlargement of iliac lymph nodes in 1/16 females given 1000 mg/kg/day) were of isolated occurrence and/or not dose-related. They were, therefore, considered to be incidental.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
In the 1000 mg/kg/day group, one dead fetus was observed. As the incidence of this finding was isolated and within the range of the Historical Control Data, it was considered to be incidental.
Changes in pregnancy duration:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
See chapter general toxicity (Maternal animals)

Effect levels (maternal animals)

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

Results (fetuses)

Fetal body weight changes:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex-ratio (percentage of male fetuses) was not affected by the test item treatment at any dose-level.
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
In the 1000 mg/kg/day group, when compared with controls and the HCD, mean fetal body weight was slightly lower ( 5%, p < 0.05). This variation was mainly due to the contribution of females J21524 and J21526 (net weight change:4.46 g and 4.30 g, respectively).As this test item treatment-related difference was minimal and did not correlate with a lower mean gravid uterus weight, it was considered to be of minor toxicological importance.
There were no effects on mean fetal body weight in the 100 and 400 mg/kg/day groups.

Litter size was not affected.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
There were no external variations or malformations at external fetal examination.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
At 400 and 1000 mg/kg/day and when compared with controls and Historical Control Data, there were higher litter and fetal incidences of fetuses with unossified hyoid for which cartilage was present (statistically significant at 1000 mg/kg/day).

At 1000 mg/kg/day and when compared with controls, there was an increase in delayed ossification of the caudal vertebrae (unossified centrum and/or incomplete ossification of arch and/or centrum), sternebrae (unossified or incomplete ossification 6th sternebra(e)] and metatarsal bone (unossified 1st metatarsal bone). This was considered to be associated with the test item treatment but of minor importance (see § Cartilage), although the incidences were below the maximum values in the Historical Control Data in some cases.
These findings were consistent with the slightly lower mean fetal body weight recorded at this dose-level and were also mainly due to the contribution of fetuses from females J21524 and J21526.

Other skeletal variations, [i.e. incomplete ossification of cervical vertebra(e) centrum observed in the 100 mg/kg/day group or dumbbell ossification of thoracic vertebra(e) centrum in the 400 mg/kg/day group] were considered to be unrelated to the test item treatment as they were not dose-related.

In the 1000 mg/kg/day group, two litters contained one or three malformed fetuses without correlate with any signs of maternal toxicity with the exception of loud breathing recorded in female J21503 from Day 18 to Day 20 p.c.

In the 100 and 400 mg/kg/day group, no malformed fetuses were observed.

In the control group, one litter contained one malformed fetus with split interparietal bone.
Visceral malformations:
effects observed, treatment-related
Description (incidence and severity):
When compared with controls, there was a tendency towards increased fetal and litter incidences of dilated ureter at all dose-levels (from 6.7% to 7.6% and from 30.4% to 37.5%, fetal and litter incidences, respectively). Taking into account that incidences (fetal and litter) of this non-adverse finding were higher at the high-dose level than the maximum incidence in the Historical Control Data, it was considered to be test item treatment-related.

Other soft tissue variations (i.e. dilated renal pelvis, short or absent innominate artery, paleness of spleen, and/or reddish foci on thymus) observed with a similar or minimal incidence in control and/or test item treated groups were considered to be unrelated to the test item treatment.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
400 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: See 'remarks'
Remarks on result:
other: see 'remarks'
Remarks:
in a context of severe maternal toxicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Pregnancy status

Dose-level (mg/kg/day)

        0

  100

       400

   1000

Number of females

      24

    24

         24

       24

Non-pregnant females

        0

      1

          0

        0

Found dead or prematurely sacrificed females

        0

      0

          0

        8

Females with total resorption

        0

      0

          0

        0

Females with live fetuses at term

      24

    23

         24

       16

 

Clinical signs

Test item treatment-related clinical signs (surviving females) are summarized as follows:

Dose-level (mg/kg/day)

0

100

400

1000

Round back

 

1
(for 2 days)

1
(for 3 days)

3
(
from 1 to 4 days duration)

Piloerection

 

1
(for 2 days)

2
(
for 1 or 6 days)

5
(
from 2 to 6 days duration)

Ptyalism

 

3
(from 1 to 5 days duration)

13
(from 3 to 10 days duration)

16
(from 3 to 15 days duration)

Reflux at dosing

 

1
(Day 18 p.c.)

 

 

Emaciated appearance

 

 

1a
(
from Day 13 to 20 p.c.)

1b
(
from Day 12 to 15 p.c.)

Loud breathing

 

 

 

2
(for 1 or 3 days)

Abdominal breathing

 

 

 

3
(from 1 to 5 days duration)

Pallor of extremities

 

 

 

1
(for 1 day)

Soft feces

 

 

 

1
(for 3 days)

Reddish vaginal discharge

1
(for 3 days)

 

2
(for 3 or 6 days)

 

Number of affected animals

1/24

5/24

15/24

16/16

( )  : in brackets: Daysp.c.of occurrences or length of the period.

a    : female J21494: +133 g between Days 12 and 21p.c.

b    : female J21507: - 29 g between Days 9 and 12p.c.

Body weight and body weight change

Dose-level (mg/kg/day)

Body weight (g)

0

100

400

1000

Day 6p.c.

271

271

271

275

-

(0)

(0)

(+1)

Day 9p.c.

287

289

289

290

-

(+1)

(+1)

(+1)

Day 12p.c.

311

313

310

309

-

(+1)

(0)

(-1)

Day 15p.c.

331

332

331

334

-

(0)

(0)

(+1)

Day 18p.c.

373

372

375

354

-

(0)

(+1)

(-5)

Day 21p.c.

433

429

430

422

-

(-1)

(-1)

(-3)

 Body weight change (g)

Days 9 - 12p.c.

+24

+24

+21

+18

Days 12 - 15p.c.

+20

+19

+21

+23

Days 15 - 18p.c.

+42

+40

+44

+20#

Days 18 - 21p.c.

+60

+57

+55

+54

Days 6 - 21p.c.

+162

+158

+158

+146

 

-

(-2)

(-2)

(-10)

Statistical significance;#: p < 0.001.

p.c.        :post-coitum.

-    : not applicable.

( )  : in brackets, percentage differencevs.controls.

 

Food consumption 

Dose-level (mg/kg/day)

0

100

400

1000

Days 6 - 9p.c.

26

-

27

(+4)

26

(0)

25

(-4)

Days 9 - 12p.c.

27

-

29

(+7)

27

(0)

27

(0)

Days 12 - 15p.c.

29

-

29

(0)

29

(0)

30

(+3)

Days 15 - 18p.c.

31
-

31
(0)

32
(+3)

28*
(-10)

Days 18 - 21p.c.

32

-

31

(-3)

31

(-3)

29

(-9)

Statistical significance;*: p < 0.05.

p.c.  :post-coitum.

-       : not applicable.

( )     : in brackets, percentage differencevs. controls.

 

Maternal necropsy findings

Dose-level (mg/kg/day)

0

   100

     400

  1000

Stomach: whitish colored mucosa

0

      0

        1

       1

Intestines: dilated

0

      0

        0

       1

Adrenal glands: enlarged

0

      0

        0

       1

Thymus: reduced in size

0

      0

        0

       1

Number of affected animals

0/24

0/24

1/24

2/16

 

Mean carcass, net change and gravid uterus weights

Dose-level (mg/kg/day)

0

100

400

1000

Gravid uterus weight

116

-

115

(-1)

116

(0)

112

(-3)

Carcass weight

317

-

314

(-1)

314

(-1)

310

(-2)

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

+45

+43

+43

+35

( )     : in brackets, percentage differencevs.controls.

p.c.  :post-coitum.

(a)    : weights are rounded values.

-       : not applicable.

 

Hysterectomy data

Dose-level (mg/kg/day)

      0

  100

    400

1000

HCD

Number of pregnant females
at hysterectomy

     24

    23

     24

   16

      407

Number of females with live fetuses
at termination

     24

    23

     24

   16

      388

Number of females with total resorption

      0

      0

       0

     0

          0

Mean number ofcorpora lutea

     16.7

    17.2

     17.5

   18.0

[13.8; 16.0]

Mean number of implantation sites

     15.2

    15.4

     15.4

   15.6

[12.5; 14.5]

Mean pre-implantation loss (%)

      8.9

      9.3

     11.1

   11.5

[6.2; 14.0]

Mean number of live fetuses

     14.8

    14.8

     14.9

   15.3

[11.6; 13.8]

Dead fetuses (mean %)

      0.0

      0.0

       0.0

     0.40

[0.00; 0.50]

Mean number of implantation scars

      0.0

      0.0

       0.0

     0.0

/

Mean number of early resorptions

      0.3

      0.5

       0.5

     0.3

/

Mean number of late resorptions

      0.1

      0.1

       0.1

     0.1

/

Mean post-implantation loss (%)

      2.2

      3.8

       3.6

     2.3

[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.

 

Fetal body weight and sex

Dose-level (mg/kg/day)

0

100

400

1000

HCD

Mean fetal body weight (g)

5.74
-

5.73
(0)

5.77
(+1)

5.44*
(-5)

[5.5; 5.9]

Mean fetal body weight
of males (g)

5.91
-

5.88
(-1)

5.89
(0)

5.58*
(-6)

[5.7; 6.1]

Mean fetal body weight
of females (g)

5.57
-

5.58
(0)

5.63
(+1)

5.27*
(-5)

[5.4; 5.7]

Mean percentage
of male fetuses (%)

48.9

51.6

48.7

51.2

[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.].

Statistically significant;*: p < 0.05.

-       : not applicable.

 

Fetal external variations

Dose-level (mg/kg/day)

       0

   100

   400

 1000

HCD

Dams with live fetuses

     24

     23

     24

     16

        388

Number of live fetuses

   356

   340

   357

   244

      5000

Litters affected, n (%)

0 (0)

0 (0)

0 (0)

0 (0)

6 (1.5)(b)

Fetuses affected, n (%)

0 (0)

0 (0)

0 (0)

0 (0)

7 (0.1)(b)

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).

 (b)    : mean incidence.

 

 

 

Fetal soft tissue variations

Dose-level (mg/kg/day)

        0

    100

    400

   1000

HCD

Dams with live fetuses

      24

      23

      24

      16

       387

Number of live fetuses

    171

    165

    175

    118

     2404

Litters affected, n (%)

6 (25.0)

9 (39.1)

7 (29.2)

8 (50.0)

86 (22.2)(b)

Fetuses affected, n (%)

11 (6.4)

15 (9.1)

16 (9.1)

12 (10.2)

119 (5.0)(b)

Dilated renal pelvis, F (L)

4.7 (12.5)

6.7 (34.8)

3.4 (16.7)

2.5 (18.8)

9.5 (28.6)(a)

Short innominate artery, F (L)

0 (0)

0 (0)

0 (0)

0.8 (6.3)

3.7 (22.7)(a)

Absent innominate artery, F (L)

1.2 (8.3)

1.2 (8.7)

1.1 (4.2)

0 (0)

5.1 (25.0)(a)

Dilated ureter, F (L)

4.7 (16.7)

6.7 (30.4)

6.9 (20.8)

7.6 (37.5)

7.1 (28.0)(a)

Spleen: paleness, F (L)

0 (0)

0.6 (4.3)

0 (0)

0 (0)

0.8 (5.0)(a)

Thymus: reddish foci, F (L)

0 (0)

0 (0)

0 (0)

0.8 (6.3)

0 (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).

(a)     : maximum incidence.

(b)     : mean incidence.

 

Fetal soft tissue malformations

Dose-level (mg/kg/day)

       0

    100

    400

  1000

HCD

Dams with live fetuses

     24

      23

      24

      16

       387

Number of live fetuses

   171

    165

    175

    118

     2404

Litters affected, n (%)

0 (0)

0 (0)

0 (0)

1 (6.3)

7 (1.8)(b)

Fetuses affected, n (%)

0 (0)

0 (0)

0 (0)

1 (0.8)

13 (0.5)(b)

Marked dilated ureter, F (L)

0 (0)

0 (0)

0 (0)

0.8 (6.3)

4.8 (4.8)(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).

(a)     : maximum incidence.

(b)     : mean incidence.

 

Fetal incidences of cartilage

Dose-level (mg/kg/day)

        0

     100

   400

    1000

HCD

Dams with live fetuses

       24

       23

     24

        16

        388

Number of live fetuses

     185

     175

   182

      126

      2596

Cartilage of metatarsal
bone present, F(L)

7.6 (37.5)

6.3 (34.8)

10.4 (41.7)

25.4#(50.0)

36.8 (72.7)(a)

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

3.2 (16.7)

2.9 (21.7)

1.1 (8.3)

18.3#(31.3)

2.2 (15.0)(a)

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

1.6 (12.5)

2.3 (8.7)

0.5 (4.2)

11.9#(25.0)

3.8 (21.7)(a)

F      : fetal incidence.

L      : litter incidence.

Statistically significant;#: p < 0.001 for the number of fetuses affected.

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

(a)     : maximum incidence.

(b)     : mean incidence.

 

Fetal incidences of skeletal

Dose-level (mg/kg/day)

        0

     100

   400

   1000

HCD

Dams with live fetuses

       24

       23

     24

       16

        388

Number of live fetuses

     185

     175

   182

     126

      2596

Hyoïd: unossified, F (L)

0.5 (4.2)

0.6 (4.3)

1.6 (8.3)

4.0* (12.5)

1.3 (5.0)(a)

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

1.1 (8.3)

1.1 (8.7)

0 (0)

7.9**(25.0)

2.3 (15.0)(a)

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

1.1 (8.3)

2.3 (17.4)

0.5 (4.2)

8.7** (18.8)

1.4 (10.0)(a)

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

0 (0)

0 (0)

0 (0)

7.1#(18.8)

1.4 (5.6)(a)

6thsternebra: unossified, F (L)

0 (0)

0 (0)

0 (0)

4.0* (12.5)

0.7 (5.0)(a)

6thsternebra: incomplete ossification, F (L)

0 (0)

1.7 (4.3)

0 (0)

7.1#(12.5)

2.9 (16.7)(a)

Metatarsal bone: unossified

first metatarsal, F(L)

7.6 (37.5)

6.3 (34.8)

10.4(41.7)

23.0#(43.8)

36.8 (72.7)(a)

F      : fetal incidence.

L      : litter incidence.

Statistically significant; *: p < 0.05, **: p < 0.01 and #: p < 0.001 for the number of fetuses affected.

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

(a)     : maximum incidence.

(b)     : mean incidence.

 

Fetal skeletal malformations

Dose-level (mg/kg/day)

       0

    100

   400

   1000

HCD

Dams with live fetuses

     24

      23

     24

       16

        388

Number of live fetuses

   185

    175

   182

     126

      2596

Litters affected, n (%)

1 (4.2)

0 (0)

0 (0)

2 (12.5)

15 (3.9)(b)

Fetuses affected, n (%)

1 (0.5)

0 (0)

0 (0)

4 (3.2)

18 (0.7)(b)

Interparietal: split, F(L)

0.5 (4.2)

0 (0)

0 (0)

0 (0)

/

Lumbar vertebra(e): absent, F(L)

0 (0)

0 (0)

0 (0)

2.4 (6.3)

1.3 (8.7)(a)

Fused sternebra(e), F(L)

0 (0)

0 (0)

0 (0)

0.8 (6.3)

1.3 (8.7)(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 Historical Control Data.

(a)     : maximum incidence.

(b)     : mean incidence.

 

Distribution of fetal malformations

Dose-level (mg/kg/day)

0

100

400

1000

HCD

External

Litters affected, n (%)

0 (0)

0 (0)

0 (0)

0 (0)

11 (2.8)(a)

Fetuses affected, n (%)

0 (0)

0 (0)

0 (0)

0 (0)

13 (0.3)(a)

Soft tissue

Litters affected, n (%)

0 (0)

0 (0)

0 (0)

1 (6.3)

7 (1.8)(a)

Fetuses affected, n (%)

0 (0)

0 (0)

0 (0)

1 (0.8)

13 (0.5)(a)

Skeletal

Litters affected, n (%)

1 (4.2)

0 (0)

0 (0)

2 (12.5)

15 (3.9)(a)

Fetuses affected, n (%)

1 (0.5)

0 (0)

0 (0)

4 (3.2)

18 (0.7)(a)

Total

Litters affected / evaluated (%)

1/24 (4.2)

0/23 (0)

0/24 (0)

2/16 (12.5)

/

Fetuses affected / evaluated (%)

1/356 (0.3)

 0/340 (0)

0/357 (0)

5/244 (2.0)

/

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, Reaction mass of diisobutyl hydrogen phosphate and isobutyl dihydrogen phosphate (batch No. 1629110) was administered by gavage, once daily, from Days 6 to 20 p.c., inclusive, to pregnant Sprague-Dawley rats at the dose-levels of 100, 400 and 1000 mg/kg/day. The control group received the vehicle, Polyethylene Glycol 400 (PEG 400), 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 400 mg/kg/day (based on clinical signs, food consumption, body weight gain and mortality at 1000 mg/kg/day),
- the NOAEL for embryo-fetal development was considered to be 400 mg/kg/day (based on fetal dysmorphogenesis at 1000 mg/kg/day) in a context of severe maternal toxicity.
Executive summary:

SUMMARY

The objective of this study was to evaluate the potential toxic effects of the test item, Reaction mass of diisobutyl hydrogen phosphate and isobutyl dihydrogen phosphate, on the pregnant female and on embryonic and fetal development following daily oral administration (gavage) to pregnant female ratsfrom 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,Reaction mass of diisobutyl hydrogen phosphate and isobutyl dihydrogen phosphate(batch No. 1629110), by the oral route (gavage), at a dose-level of 100, 400 or 1000 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,Polyethylene Glycol 400 (PEG 400),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 recordedat designated intervals. On Day 21p.c.,the females were sacrificed and a macroscopicpost-mortemexamination 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 the macroscopic lesions were collected and preserved in 10% buffered formalin.

 

Results

Chemical analyses

The concentrations of the test item in the dose formulations (-9.1% to -2.8%) remained within an acceptable range of variations (± 15%) when compared to the nominal concentrations.

No test item was observed in the control dose formulation.

 

Pregnancy status

At study termination on Day 21p.c.,there were 24, 23, 24 and 16 females with live fetuses in the groups given 0, 100, 400 and 1000 mg/kg/day, respectively.

 

Mortality

At 1000 mg/kg/day, one pregnant female was found dead and seven pregnant females were prematurely sacrificed.These unscheduled deaths were considered to be test item treatment-related.

 

Clinical signs

The following test item treatment-related findings were observed:

.          from 100 mg/kg/day: ptyalism, round back and/or piloerection,

.          from 400 mg/kg/day: emaciated appearance in isolated animals,

.          at 1000 mg/kg/day: loud and/or abdominal breathing defined as endpoints, pallor of extremities and/or soft feces.

 


Body weight

At 1000 mg/kg/day and when compared with controls, lower mean body weight gain was observed on Days 9-12 p.c. (+18 g vs. +24 g in controls) and Days 15-18 p.c. (+20 g vs. + 42 g in controls, p < 0.001). These findings were considered to be test item treatment-related and adverse.

The terminal mean body weight was not affected on Day 21 p.c.

 

Food consumption

At 1000 mg/kg/day and when compared with controls, there was lower mean food consumption (-10%, p < 0.05) that correlated with lower mean body weight gain on Days 15-18 p.c. This finding was considered to be test-item treatment-related and adverse.

 

Necropsy and macroscopicpost-mortemexamination

Whitish coloration of stomach mucosa, dilatation of intestines, small thymus and/or enlargement of adrenals were observed in 1/24 females given 400 mg/kg/day and 2/16 females given 1000 mg/kg/day; these were considered to be test item treatment-related.

 

Net body weight change

At 1000 mg/kg/day and when compared with controls, a lower mean net body weight change (+35 g vs. +45 g in controls, not statistically significant) was noted.

 

Hysterectomy data

There were no test item treatment-related effects.

Fetal body weight and percentage of male fetuses

There was no toxicologically important effect on mean fetal body weight.

The sex ratio was not affected by the test item treatment at any dose-level.

 

Fetal examinations

External examination

There were no test item-related variations or malformations at external examination.

 

Soft tissue examination

At all dose-levels and when compared with controls, there were higher litter and fetal incidences of dilated ureter (considered to be a non-adverse finding).

At 1000 mg/kg/day, one malformed fetus had marked dilatation of the ureter, for which a relationship to the test item treatment cannot be ruled out.

 

Cartilage and skeletal examinations

At 1000 mg/kg/day and when compared with controls, there were higher litter and fetal incidences of fetuses with delayed ossification: hyoid (unossified), caudal vertebra(e)(unossified centrum and/or incomplete ossification of arch and/or centrum), 6th sternebra (unossified or incomplete ossification) and 1st metatarsal bone (unossified).

One litter contained one malformed fetus (fused sternebrae) and another one contained three malformed fetuses[absent lumbar vertebra(e)],for which a test item treatment-related effect cannot be excluded.


Conclusion 

The test item, Reaction mass of diisobutyl hydrogen phosphate and isobutyl dihydrogen phosphate (batch No. 1629110) was administered by gavage, once daily, from Days 6 to 20 p.c.,inclusive, to pregnant Sprague-Dawley rats at the dose-levels of 100, 400 and 1000 mg/kg/day.The control group received the vehicle, Polyethylene Glycol 400 (PEG 400), 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 400 mg/kg/day (based on clinical signs, food consumption, body weight gain and mortality at 1000 mg/kg/day),

.          the NOAEL for embryo-fetal development was considered to be 400 mg/kg/day (based on fetal dysmorphogenesis at 1000 mg/kg/day)in a context of severe maternal toxicity.