3-methyl-1,5-pentanediyl diacrylate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 March 2018 - 16 May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

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

Test material information

Constituent 1

Reference substance name:

3-methyl-1,5-pentanediyl diacrylate

EC Number:

264-727-7

EC Name:

3-methyl-1,5-pentanediyl diacrylate

Cas Number:

64194-22-5

Molecular formula:

C12H18O4

IUPAC Name:

3-methyl-5-(prop-2-enoyloxy)pentyl prop-2-enoate

Test material form:

liquid

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 the beginning of the treatment period: the females were 10-11 weeks old
- Mean at the beginning of the treatment period: the females had a mean body weight of 296 g (range: 254 g to 337 g)
- Fasting period before study: no
- Housing: individually
- 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: 26 March 2018 to 16 May 2018

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING FORMULATIONS:
- Solution in the vehicle
- Justification for use and choice of vehicle (as other than water): suitable formulation in the selected vehicle
- Concentration in vehicle: 20, 60 and 200 mg/mL
- Amount of vehicle: 5 mL/kg/day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Type of method: Gas chromatography with FID detection (GC-FID)
Test item concentrations: remained within an acceptable range of variations (-0.6% to +8.2%) when compared with the nominal values (± 10% of the nominal concentrations).
Homogeneity: not assessed, dose formulations were solutions
Stability: stableformulation at 2 mg/mL and 200 mg/mL, at room temperature for 12 days.

Details on mating procedure:

- Impregnation procedure: purchased time pregnant
- Proof of pregnancy: vaginal plug (at the breeder's facility); referred to as Day 0 post coitum

Duration of treatment / exposure:

Days 6 to 20 post coitum inclusive

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

24 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Rationale for dose selection:
The dose levels were selected by the Sponsor based on a preliminary study for effects on embryo-fetal development by the oral route in rats.
In this study, three groups of eight pregnant female Sprague-Dawley rats received the test item, daily by oral administration (gavage) throughout gestation (Day 6 to Day 20 p.c.) at dose levels of 100, 300 or 1000 mg/kg/day. An additional group of eight pregnant females received the vehicle control, corn oil, under the same experimental conditions. The dosing volume was 5 mL/kg/day.
At 1000 mg/kg/day, there were:
- ptyalism in all females,
- lower mean body weight gain at treatment initiation together with a lower mean food consumption,
- no effects on pre- and post-implantation losses, number of viable fetuses and fetal body weight,
- no external variations and malformations,
- increases in the absolute and relative mean liver and kidney weights.
At 300 mg/kg/day, there were ptyalism in 7/8 females associated with half-closed eyes in one of them.
At 100 mg/kg/day, there were no test item-related findings.

Thus, based on these data, the dose levels selected for the present study are: 100, 300 and 1000 mg/kg/day.

- Rationale for animal assignment: computerized stratification procedure.

Examinations

Maternal examinations:

MORTALITY/MORBIDITY:
- Time schedule: each animal was checked for mortality and morbidity once a day before the treatment period and at least twice a day during the treatment period, including weekends and public holidays.

CLINICAL SIGNS:
- Time schedule: from arrival, each animal was observed at least 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:
- Time schedule: the body weight of each female was recorded on Days 2, 4, 6, 9, 12, 15, 18 and 21 p.c.

FOOD CONSUMPTION:
- Time schedule: the quantity of food consumed by each female was recorded for the following intervals: Days 2-4, 4-6, 6-9, 9-12, 12-15, 15-18 and 18-21 p.c.

POST-MORTEM EXAMINATION:
- Sacrifice on Day 21 post coitum
- Examined: principal thoracic and abdominal organs

PRESERVATION OF TISSUES
Macroscopic lesions observed in females were sampled and kept preserved in 10% buffered formalin (or in another appropriate fixative). Since remarkable macroscopic lesions were observed in test item-treated group animals, the corresponding tissues (ovaries, placenta and lungs) of five control animals were sampled and preserved.
For all study animals, the stomach, the liver and the kidneys were also preserved in 10% buffered formalin.

ORGAN WEIGHTS
The body weight of each animal was recorded before euthanasia at the end of the treatment period. The organs specified in the Tissue Procedure Table were weighed wet as soon as possible after dissection.
The ratio of organ weight to net body weight was calculated.

PREPARATION OF HISTOLOGICAL SLIDES
All tissues required for microscopic examination were trimmed according to the RITA guidelines, when applicable (Ruehl-Fehlert et al., 2003; Kittel et al., 2004; Morawietz et al., 2004), embedded in paraffin wax, sectioned at a thickness of approximately 4 microns and stained with hematoxylin-eosin.
The tissue processing was performed at Citoxlab France.

MICROSCOPIC EXAMINATION
A microscopic examination was performed on the kidneys, the liver and the stomach from control and low-, intermediate- and high-dose females (groups 1 to 4).

BODY WEIGHT OF FETUSES
The body weight of each fetus was recorded.

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

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,
- Gross evaluation of placentas.

Fetal examinations:

- External examinations: Yes: all fetuses per litter
- Soft tissue examinations: Yes: half fetuses per litter
- Skeletal examinations: Yes: half fetuses per litter
- Head examinations: Yes: half fetuses per litter
- Other: fetal weight, fetal sex

Statistics:

Data will be compared by one-way analysis of variances and Dunnett test (mean values being considered as normally distributed, variances being considered as homogenous) or by Fisher’s exact probability test (proportions).

Indices:

% Pre-implantation loss = 100 * (Number of corpora lutea - Number of implantation sites) / Number of corpora lutea
% Post-implantation loss = 100 * (Number of implantation sites - Number of live fetuses) / Number of implantation sites

Historical control data:

Cf attached document

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

See table 1.
Test item-related clinical signs consisted of ptyalism, observed for 1 to 14 days in few females given 100 mg/kg/day, most females given 300 mg/kg/day and in all females given 1000 mg/kg/day. This clinical sign was considered to be non-adverse as it is commonly observed when oily dose formulations are administered by gavage.

The other clinical signs (namely bent tail, cutaneous lesion and/or scab) were considered to be unrelated to the test item, as they were not dose-related and they were transient and/or reported in isolated animals.

Description (incidence and severity):

n/a

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

See table 2.
At 1000 mg/kg/day, when compared with controls, statistically significant lower mean body weight gain was recorded throughout the treatment period (+132 g vs. +155 g, p<0.01). This resulted in lower mean body weight gain on Days 6-9 p.c. (+5 g vs. +15 g, p<0.001), on Days 15-18 p.c. (+39 g vs. +48 g, p<0.01) and on Days 18-21 p.c. (+41 g vs. +49 g, not statistically significant). These changes were considered to be test item-related and adverse in view of the marked magnitude recorded at the start of the treatment period and as they lead to a statistically significant lower mean body weight on completion of the study (425 g vs. 454 g in controls, p<0.05).

At 300 mg/kg/day and when compared with controls, statistically significant lower mean body weight gain was noted on Days 15-18 p.c. (+42 g vs. +48 g, p<0.05). As this effect was transient, of slight magnitude (as mainly due to higher body weight gain of one control female: +85 g) and did not impact the mean body weight, this was considered to be of minor toxicological importance.

There were no effects on mean body weight change in the 100 mg/kg/day group.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

See table 3.
At 1000 mg/kg/day and when compared with controls, mean food consumption was markedly lower (-22%; p<0.001) during the first 3 days of treatment. Although this difference was transient, this was associated with a low mean body weight gain and was therefore considered to be test item-related and adverse.

There were no effects on mean food consumption in the 100 and 300 mg/kg/day groups.

Description (incidence and severity):

n/a

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:

effects observed, treatment-related

Description (incidence and severity):

See table 4.
Increased absolute and relative-to-body kidney (+7% and +14% vs. controls, p<0.05 and 0.01, respectively) and/or relative-to-body liver weights (+13% vs. controls, p<0.01) were noted in females treated at 1000 mg/kg/day.

The increased liver weights correlated with the hepatocellular vacuolation seen at microscopic examination. This difference was thus considered to be related to the test item administration.

The increased kidney weights had no microscopic correlates. A relationship to test item could not be excluded in spite of the low magnitude of these differences and of the absence of microscopic correlates.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Thickened mucosa was noted in the stomach from 22/24 females treated at 1000 mg/kg/day (corresponding to 20/22 pregnant females). This change was considered to be related to the test item administration and correlated with the hyperplasia of squamous cells and hyperkeratosis seen in these animals.

The other changes were considered to be part of the spontaneous background in the rats of these strain and age.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See table 5.
Liver
Increased severity and incidence of hepatocellular vacuolation was noted in females treated at 1000 mg/kg/day, ranging from minimal to moderate grades.
These small coalescing vacuoles in enlarged hepatocytes were consistent with increased glycogen storage. In the absence of degeneration, this test item-related finding was considered to be non-adverse.
No differences were noted at 100 or 300 mg/kg/day.

. Forestomach
Minimal to moderate, focal or multifocal ulceration was noted in females treated at 1000 mg/kg/day. This was accompanied by slight to moderate subacute inflammation up to the muscle layer in these animals, with occasional granuloma and bacteria (opportunistic).
In view of the extension of these ulcers associated with secondary inflammation affecting the mucosa and the submucosa (up to the muscle layer), these findings were considered to be adverse.

In addition, minimal to moderate hyperplasia of squamous cells and hyperkeratosis were noted in females treated at 300 or 1000 mg/kg/day. These findings were considered to be non-adverse in view of their moderate magnitude and of their nature.

. Kidney
There were no test item-related findings.

The other isolated findings in kidneys, liver or stomach were considered not to be related to the test item administration as they were not dose-related, were of low magnitude and/or commonly seen in the untreated rat kept under laboratory condition.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

See table 6.
Uterus weight, carcass weight and net body weight change from Day 6 post coitum:
There were no effects on mean gravid uterus weight.

At 1000 mg/kg/day and when compared with controls, there was a slight to moderate decrease in mean carcass weight (-6%, p<0.05) and in mean net body weight change (-28%, p<0.01). As these findings were associated with lower mean body weight and mean body weight changes in view of its magnitude, the difference in net body weight change was considered to be adverse.

Maternal developmental toxicity

Description (incidence and severity):

n/a

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

See table 7.
Number of dams with pre-implantation loss = 13, 16, 15, 15, at 0, 100, 300 or 1000 mg/kg/day, respectively
Number of dams with post-implantation loss = 17, 14, 11, 16, at 0, 100, 300 or 1000 mg/kg/day, respectively

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

See table 7.
No dams with total resorption.

Early or late resorptions:

no effects observed

Description (incidence and severity):

See table 7.
Number of dams with resorptions = 17, 14, 11, 16, at 0, 100, 300 or 1000 mg/kg/day, respectively

Dead fetuses:

no effects observed

Description (incidence and severity):

See table 7.
No dead fetuses.

Changes in pregnancy duration:

not examined

Description (incidence and severity):

No early deliveries.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

See table 14.
As the test item was administered after implantation, the absence of gestation for 2 females given the high dose level was considered to fortuitous and not to be test item-related.

Other effects:

not specified

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

gross pathology

histopathology: non-neoplastic

other: net body weight

Maternal abnormalities

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

other: forestomach, liver

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

See table 8.
There were no effects on mean fetal body weight.

Description (incidence and severity):

n/a

Changes in sex ratio:

no effects observed

Description (incidence and severity):

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

Description (incidence and severity):

n/a

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

There were no external malformations.

Skeletal malformations:

no effects observed

Description (incidence and severity):

There were no skeletal malformations.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

See table 11.
There were no test item-treatment related soft tissue malformations.

In the control and in the 100, 300 and 1000 mg/kg/day groups, two litters had three fetuses, one litter had one fetus, two litters had two fetuses and one litter had one fetus with marked dilated ureter, respectively.

In the control group, one litter had a malformed fetus with absent aortic arch.

These tissue malformations were observed in control and/or treated groups with an incidence similar to the Historical Control Data and/or without any dose relationship. Therefore they were considered to be unrelated to the test item.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

External variations
See table 9.
There were no test item-treatment related external variations.
In the 1000 mg/kg/day group, one litter had a fetus with local edema. As this finding was isolated and not associated with additional findings at fetal examination, a test-item treatment relationship was considered to be unlikely.

In the 100 mg/kg/day group, one litter had a fetus with limb hyperflexion. As this finding was isolated, not associated with additional findings at fetal examination and not observed at higher doses, it was considered to be not related to the test item.

Soft tissue variations
See table 10.
There were no test item treatment-related soft tissue variations.

In the 1000 mg/kg/day group, one litter had a fetus with small kidney. As this finding was isolated and not associated with additional findings at fetal examination, a test-item treatment relationship was considered to be unlikely.

When compared with controls, dilated ureter and/or short or absent innominate artery were observed at a higher incidence in the 1000 mg/kg/day group. As these variations were noted, with a poor dose-relationship, with an isolated incidence and/or with an incidence similar or close to the Historical Control Data, they were therefore considered to be unrelated to the test item treatment.

As the other tissue variations were not observed at the high dose level or were noted at a lower incidence than that recorded in controls, they were also considered to be unrelated to the test item treatment.

Skeletal examinations
Cartilage
See table 12.
In the 1000 mg/kg/day group and when compared with controls, there were statistically significant increases in the incidence of the non ossified cartilage (cartilage present) [i.e. cervical and/or thoracic vertebra(e)]. These incidences were within the range of the Historical Control Data, with the exception of cervical vertebra(e) cartilage for which litter and fetal incidences were above the upper limit of the Historical Control Data.
Increases in the incidences of non ossified cartilage of sternebra(e) and metacarpal bone were also noted but to a lesser extent (not statistically significant) and within the range of the Historical Control Data.
These findings were considered to be related to the incomplete ossification and test item-related but not adverse (as the bones concerned were present).
When compared with controls, the presence of cartilage was observed at a slightly higher fetal and/or litter incidence at 1000 mg/kg/day [i.e. cartilage of hyoid, bipartite cartilage of thoracic vertebra(e), cartilage of caudal vertebra(e), cartilage of metatarsal bone(s) and cartilage of distal phalanx hindpaw]. As these findings were noted, with a poor dose-relationship, with an isolated incidence and/or with an incidence similar or lower than the Historical Control Data, they were therefore considered to be unrelated to the test item treatment.

Skeletal variations
See table 13.
In the 1000 mg/kg/day group and when compared with controls, there were statistically significant increases in the litter and fetal incidences of bones with incomplete ossification [i.e. incomplete ossification of centrum for cervical vertebra(e) and bipartite ossification of centrum for thoracic vertebra(e)].
These incidences were within the range of the Historical Control Data, with the exception of bipartite ossification of centrum for thoracic vertebra(e) for which litter and fetal incidences were above the upper limit of the Historical Control Data. Increases in the incidences of incomplete frontal ossification, incomplete 6th sternebra ossification and unossification of metacarpal(s) were also noted with an incidence higher than Historical Control Data but to a lesser extent (not statistically significant).
Although these findings did not impact mean fetal body weights, they were considered to be test item-related but not adverse (there are signs of on-going development as the cartilages were present).

When compared with controls, other variations were observed at a slightly higher fetal and/or litter incidence in the 1000 mg/kg/day group [i.e. incomplete ossification of parietal, supraoccipital and/or hyoid, unossified centrum of cervical vertebra(e), incomplete ossification of thoracic vertebra(e) centrum, dumbbell ossification of thoracic vertebra(e) centrum, unossified caudal vertebra(e) centrum, extra sternebral ossification site, ossification point on 14th thoracic vertebra(e), unossified 1st metatarsal and unossified hindpaw distal phalanx]. As these variations were noted, with a poor dose-relationship, with an isolated incidence and/or with an incidence similar or lower than the Historical Control Data, they were therefore considered to be unrelated to the test item treatment.

As the other skeletal variations were not observed at the high dose level or were noted at a lower incidence than that recorded in controls, they were also considered to be unrelated to the test item treatment.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

no

Any other information on results incl. tables

Table 1: Clinical signs

 

Dose level (mg/kg/day)	0	100	300	1000
Ptyalism		2	16	24 (22 pregnant females)
Number of affected animals	0/24	2/24	16/24	24/24 (22/22 pregnant females)

 

Table 2: Body weight and body weight change

Dose level (mg/kg/day)	0	100	300	1000
Body weight (g)
Day 6p.c.	299	296	296	293
	-	(-1)	(-1)	(-2)
Day 9p.c.	313	310	309	298
	-	(-1)	(-1)	(-5)
Day 12p.c.	334	332	332	321
	-	(-1)	(-1)	(-4)
Day 15p.c.	356	352	354	344
	-	(-1)	(-1)	(-3)
Day 18p.c.	404	396	396	384
	-	(-2)	(-2)	(-5)
Day 21p.c.	454	446	447	425*
	-	(-2)	(-2)	(-6)
Body weight change (g)
Days 6 - 9p.c.	+15	+14	+13	+5^#
Days 9 - 12p.c.	+21	+21	+23	+24
Days 12 - 15p.c.	+22	+21	+22	+23
Days 15 - 18p.c.	+48	+44	+42*	+39**
Days 18 - 21p.c.	+49	+50	+51	+41
Days 6 - 21p.c.	+155	+149	+151	+132**
	-	(-4)	(-3)	(-15)

p.c.   : post-coitum.

-       : not applicable.

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

Statistically significant: *: p<0.05, **: p<0.01 and ^#: p<0.001.

 

Table 3: Food consumption

Dose level (mg/kg/day)	0	100	300	1000
. Days 6 - 9p.c.	23	22	22	18^#
. Days 9 - 12p.c.	24	24	24	23
. Days 12 - 15p.c.	26	26	25	26
. Days 15 - 18p.c.	31	29	29	30
. Days 18 - 21p.c.	30	29	30	29

p.c.   : post-coitum.

Statistically significant: ^#: p<0.001.

 

Table 4: Organ weights

Sex	Female
Group	2	3	4
Dose-level (mg/kg/day)	100	300	1000
Exam. animals / Num. of animals	24/24	24/24	24/24
- Final body weight	0	-1	-6*
- Kidneys
.absolute	0	+1	+7*
.relative-to-body	0	+2	+14**
- Liver
.absolute	-1	+2	+7
.relative-to-body	-1	+3	+13**

Statistically significant from controls: *: p<0.05, **: p<0.01.

The significance concerned the organ weights values and not the percentages

Table 5: Microscopic examination

Group	1	2	3	4
Dose-level (mg/kg/day)	0	100	300	1000
Number of females per group	24	24	24	24
Liver; vacuolation; hepatocyte
. grade 1	1	1	2	5
. grade 2	-	-	-	5
. grade 3	-	-	-	2
Forestomach; ulcer
. grade 1	-	-	-	1
. grade 2	-	-	-	6
. grade 3	-	-	-	8
Forestomach; inflammation
. grade 2	-	-	1	8
. grade 3	-	-	-	8
Forestomach, hyperplasia; squamous cells
. grade 1	-	-	2	-
. grade 2	-	-	1	11
. grade 3	-	-	1	13
Forestomach, hyperkeratosis
. grade 1	-	-	6	-
. grade 2	-	-	1	17
. grade 3	-	-	-	7

 

Table 6: Net body weight change

 

Dose level (mg/kg/day)	0	100	300	1000
Gravid uterus weight	102	95 (-7)	97 (-5)	93 (-9)
Carcass weight	352	351 (0)	349 (-1)	332* (-6)
Net body weight change from Day 6 p.c.	+54	+55	+54	+39**

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

p.c.   : post-coitum.

^(a)         : weights are rounded values.

Statistically significant: *: p<0.05 and **:p<0.01.

 

Table 7: Hysterectomy data

 

Dose level (mg/kg/day)	0	100	300	1000
Number of pregnant females at hysterectomy	24	24	24	22
Number of females with live fetuses at termination	24	24	24	22
Number of females with total resorption	0	0	0	0
Mean number ofcorpora lutea	14.3	14.0	13.7	13.6
Mean number of implantation sites	13.4	12.4	12.7	12.6
Mean pre-implantation loss (%)	5.7	10.7	7.6	7.1
Number of females with pre-implantation loss	13	16	15	15
Mean number of live fetuses	12.4	11.5	12.0	11.4
Dead fetuses (%)	0.0	0.0	0.0	0.0
Mean number of implantation scars	0.0	0.0	0.0	0.0
Mean number of early resorptions	0.8	0.8	0.7	1.0
Mean number of late resorptions	0.1	0.1	0.0	0.3
Mean number of resorptions + scars	1.0	0.9	0.7	1.2
Number of females with resorptions	17	14	11	16
Mean post-implantation loss (%)	7.2	6.7	5.3	9.9
Number of females with post-implantation loss	17	14	11	16

Table 8: Fetal body weight

Dose level (mg/kg/day)	0	100	300	1000
Mean fetal body weight (g)	5.85 -	5.68 (-3)	5.84 (0)	5.65(-3)
Mean fetal body weight of males (g)	6.02 -	5.81 (-3)	6.03 (0)	5.83 (-3)
Mean fetal body weight of females (g)	5.66 -	5.55 (-2)	5.69 (+1)	5.45 (-4)
Mean percentage of male fetuses (%)	54.6	49.7	46.9	52.5

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

-       : not applicable.

 

Table 9: Fetal external variations

Dose level (mg/kg/day)	0	100	300	1000	HCD
Dams with live fetuses, n	24	24	24	22	235
Live fetuses, n	298	277	288	251	2896
. local edema, L(F)	0 (0)	0 (0)	0 (0)	4.5 (0.4)	2.6 (0.2)^(a)
. limb hyperflexion, L(F)	0 (0)	4.2 (0.4)	0 (0)	0 (0)	0 (0)^(a)
Litters affected, n (%)^(c)	0 (0)	1 (4.2)	0 (0)	1 (4.5)	6 (2.6)^(b)
Fetuses affected, n (%)^(c)	0 (0)	1 (0.4)	0 (0)	1 (0.4)	7 (0.2)^(b)

n      : number.

HCD      : Historical Control Data (control data collected from 11 studies covering a period ranging from August 2016 to December 2017).
(a)   : upper litter (fetal) incidence per study; (b): mean number (percentage) of litters or fetuses affected.
(c)    :   all variations combined.

 

 

Table 10: Fetal soft tissue variations

 

Dose level (mg/kg/day)	0	100	300	1000	HCD
Dams with live fetuses, n	24	24	24	22	235
Live fetuses, n	145	133	137	119	1385
. kidney: small, L(F)	0 (0)	0 (0)	0 (0)	4.5 (0.8)	0 (0)
. ureter: dilated, L(F)	33.3 (9.0)	45.8 (16.5)	41.7 (16.1)	45.5 (11.8)	48.7 (15.1)^(a)
. vessels: short innominate artery, L(F)	4.2 (1.4)	12.5 (2.3)	8.3 (1.5)	13.6 (2.5)	20.8 (4.3)^(a)
. vessels: absent innominate artery, L(F)	20.8 (3.4)	12.5 (4.5)	12.5 (2.9)	22.7 (5.0)	20.8 (3.6)^(a)
Litters affected, n (%)^(c)	14 (58.3)	14 (58.3)	17 (70.8)	16 (72.7)	70 (29.8)^(b)
Fetuses affected, n (%)^(c)	26 (17.9)	32 (24.1)	35 (25.5)	24 (20.2)	132 (9.5)^(b)

n      : number.

HCD      : Historical Control Data (control data collected from 11 studies covering a period ranging from August 2016 to December 2017).

(a)   : upper litter (fetal) incidence per study; (b): mean number (percentage) of litters or fetuses affected.
(c)    : all variations combined.

 

Table 11: Fetal soft tissue malformations

 

Dose level (mg/kg/day)	0	100	300	1000	HCD
Dams with live fetuses, n	24	24	24	22	235
Live fetuses, n	145	133	137	119	1385
. vessels: absent aortic arch, L(F)	4.2 (0.7)	0 (0)	0 (0)	0 (0)	0 (0)
. ureter: marked dilated, L(F)	8.3 (2.1)	4.2 (0.8)	8.3 (1.5)	4.5 (0.8)	10.3 (2.2)^(a)
Litters affected, n (%)^(c)	3 (12.5)	1 (4.2)	2 (8.3)	1 (4.5)	7 (3.0)^(b)
Fetuses affected, n (%)^(c)	4 (2.8)	1 (0.8)	2 (1.5)	1 (0.8)	10 (0.7)^(b)

n      : number.

HCD      : Historical Control Data (control data collected from 11 studies covering a period ranging from August 2016 to December 2017).

(a)   : upper litter (fetal) incidence per study; (b): mean number (percentage) of litters or fetuses affected.
(c)    : all malformations combined.

 

Table 12: Fetal skeletal examination

Cartilage

 

Dose level (mg/kg/day)	0	100	300	1000	HCD
Dams with live fetuses, n	24	24	24	22	235
Live fetuses, n	153	144	151	132	1511
. cartilage of hyoid present, L(F)	8.3 (2.0)	12.5 (2.8)	0 (0)	9.1 (1.5)	12.5 (3.2)^(a)
. cartilage of cervical  vertebra(e) present, L(F)	0 (0)	4.2 (0.7)	0 (0)	22.7* (3.8*)	12.5 (3.4)^(a)
. cartilage of thoracic  vertebra(e) present, L(F)	20.8 (4.6)	4.2 (0.7)	41.7 (9.3)	50.0 (15.2**)	66.7 (19.3)^(a)
. cartilage of thoracic  vertebra(e) bipartite, L(F)	0 (0)	0 (0)	0 (0)	4.5 (0.8)	4.3 (0.7)^(a)
. cartilage of caudal  vertebra(e) present, L(F)	4.2 (0.7)	4.2 (0.7)	4.2 (0.7)	9.1 (1.5)	10.0 (1.7)^(a)
. cartilage of sternebra(e) present, L(F)	8.3 (1.3)	8.3 (1.4)	0 (0)	18.2 (3.0)	20.0 (4.3)^(a)
. cartilage of metacarpal  bone(s) present, L(F)	0 (0)	4.2 (0.7)	4.2 (0.7)	9.1 (1.5)	15.0 (2.6)^(a)
. cartilage of metatarsal  bone(s) present, L(F)	54.2 (16.3)	58.3 (19.4)	45.8 (11.9)	72.7 (25.8)	87.5 (47.3)^(a)
. hindpaw: cartilage of distal phalanx present, L(F)	66.7 (42.5)	54.2 (29.2)	54.2 (25.8)	77.3 (32.6)	95.7 (56.0)^(a)
Litters affected, n (%)^(c)	24 (100.0)	23 (95.8)	24 (100.0)	22 (100.0)	128 (54.5)^(b)
Fetuses affected, n (%)^(c)	122 (79.7)	96 (66.7)	109 (72.2)	96 (72.7)	584 (38.6)^(b)

n      : number.

HCD      : Historical Control Data (control data collected from 11 studies covering a period ranging from August 2016 to December 2017).

(a)   : upper litter (fetal) incidence by study; (b): mean number (percentage) of litters or fetuses affected.
(c)    : all cartilage findings combined.

Statistically significant:*: p<0.05 and **: p<0.01 for the number of fetuses or litters affected.

In bold: test item-related findings

 

Table 13: Fetal skeletal variations

 

 

Dose level (mg/kg/day)	0	100	300	1000	HCD
Dams with live fetuses, n	24	24	24	22	235
Live fetuses, n	153	144	151	132	1511
. parietal: incomplete  ossification, L(F)	0 (0)	0 (0)	0 (0)	4.5 (0.8)	10.5 (1.6)^(a)
. frontal: incomplete ossification, L(F)	0 (0)	0 (0)	4.2 (0.7)	9.1 (1.5)	5.0 (0.9)^(a)
. supraoccipital: incomplete ossification, L(F)	0 (0)	8.3 (2.1)	0 (0)	9.1 (1.5)	8.3 (3.2)^(a)
. hyoid: incomplete ossification, L(F)	8.3 (2.0)	12.5 (2.8)	0 (0)	9.1 (1.5)	30.0 (6.5)^(a)
. cervical vertebra(e): unossified centrum, L(F)	0 (0)	4.2 (0.7)	0 (0)	4.5 (0.8)	15.0 (2.9)^(a)
. cervical vertebra(e): incomplete  ossification of centrum, L(F)	0 (0)	0 (0)	0 (0)	18.2*(3.0*)	45.0 (14.5)^(a)
. thoracic vertebra(e): incomplete ossification of centrum, L(F)	20.8 (4.6)	4.2 (0.7)	37.5 (8.6)	31.8 (10.6)	50.0 (14.7)^(a)
. thoracic vertebra(e): dumbbell ossification of centrum, L(F)	0 (0)	0 (0)	0 (0)	4.5 (0.8)	25.0 (4.7)^(a)
. thoracic vertebra(e): bipartite ossification of centrum, L(F)	0 (0)	0 (0)	4.2 (0.7)	18.2*(3.8*)	8.7 (1.9)^(a)
. caudal vertebra(e): unossified centrum, L(F)	0 (0)	4.2 (0.7)	0 (0)	4.5 (0.8)	5.6 (0.9)^(a)
. extra sternebral ossification site, L(F)	16.7 (2.6)	12.5 (2.1)	4.2 (0.7)	22.7 (4.5)	25 (6.0)^(a)
. incomplete ossification of 6^thsternebra, L(F)	8.3 (1.3)	8.3 (1.4)	0 (0)	18.2 (3.0)	8.3 (2.2)^(a)
. rib: ossification point on 14^ththoracic vertebra(e), L(F)	4.2 (0.7)	0 (0)	4.2 (0.7)	9.1 (1.5)	21.1 (6.0)^(a)
. unossified metacarpal(s), L(F)	0 (0)	0 (0)	4.2 (0.7)	9.1 (1.5)	5.3 (0.9)^(a)
. unossified 1^stmetatarsal(s), L(F)	54.2 (16.3)	58.3 (19.4)	45.8 (11.9)	72.7 (25.8)	87.5 (47.3)^(a)
. hindpaw: unossified distal phalanx, L(F)	66.7 (42.5)	54.2 (29.2)	54.2 (25.8)	77.3 (32.6)	95.7 (56.0)^(a)
Litters affected, n (%)^(c)	24 (100.0)	23 (95.8)	24 (100.0)	22 (100.0)	194 (82.6)^(b)
Fetuses affected, n (%)^(c)	123 (80.4)	97 (67.4)	110 (72.8)	96 (72.7)	742 (49.1)^(b)

n      : number.

HCD      : Historical Control Data (control data collected from 11 studies covering a period ranging from August 2016 to December 2017).

(a)   : upper litter (fetal) incidence by study; (b): mean number (percentage) of litters or fetuses affected.
(c)    : all variations combined.

Statistically significant: *: p<0.05 for the number of fetuses or litters affected.

In bold: test item-related findings

 

Table 14: Pregnancy status

Dose level(mg/kg/day)	0	100	300	1000
Number of females	24	24	24	24
Non-pregnant females	0	0	0	2
Females with live fetuses at term	24	24	24	22

 

Applicant's summary and conclusion

Conclusions:

The test item was administered to time-mated Sprague-Dawley rats by gavage, once daily, from Day 6 to Day 20 p.c., inclusive, at the dose levels of 100, 300 and 1000 mg/kg/day. The control group received the vehicle, corn oil, 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 300 mg/kg/day based on adverse findings recorded at 1000 mg/kg/day (decreased body weight gain, reduced food intake, decreased net body weight change and ulcers on the forestomach mucosa),
- the No Observed Adverse Effect Level (NOAEL) for embryo-fetal development was considered to be 1000 mg/kg/day based on the absence of adverse effects at this dose level.

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 a dose level of 100, 300 or 1000 mg/kg/day, once daily from Day 6 to Day 20 p.c. inclusive. Another group of 24 time-mated female rats received the vehicle only, corn oil, 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 in the dose formulations were determined. The animals were checked daily for mortality and clinical signs. Body weight and food consumption were recorded at designated intervals. On Day 21 p.c., the females were euthanized 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). The liver and the kidneys of each dam were weighed and macroscopic lesions and samples of liver, kidney and stomach were collected and preserved in 10% buffered formalin.

 

Results

 

Chemical analyses

The concentrations of the test item in the dose formulations, performed in the first and the last weeks of the treatment period were -0.6% to +8.2% of the nominal concentrations andremained within an acceptable range of variations (± 10% of the nominal concentrations).

No test item was detected in the control dose formulation.

 

Pregnancy status

All females were pregnant with live fetuses, with the exception of two in the 1000 mg/kg/day group for which a test item relationship was excluded.

 

Mortality

There were no unscheduled deaths.

 

Clinical signs

Ptyalism was noted in 2/24 females given 100 mg/kg/day, 16/24 females given 300 mg/kg/day and 24/24 females (22/22 pregnant females) given 1000 mg/kg/day. While test-item treatment related, this finding was considered to be non-adverse as it is commonly observed when oily dose formulations are administered by gavage.

 

Body weight, body weight change and food consumption

At 1000 mg/kg/day and when compared with controls, a lower mean body weight gain was observed at initiation of the treatment (+5 g vs.+15 g in controls on Days 6-9 p.c.; p<0.001), and then on Days 15-18 p.c.(+39 g vs.+48 g, p<0.01) and on Days 18-21 p.c.(+41 g vs.+49 g, not statistically significant), impacting the mean body weight change for the whole treatment period (+132 g vs.+155 g, p<0.01).This was associated with a marked lower mean food consumption on Days 6-9 p.c.(18 g/animal/day vs. 23 g/animal/day in controls; p<0.001). In view of the marked magnitude recorded on body weight gain and food consumption at the beginning of the study and as the terminal mean body weight was affected (425 g vs. 454 g in controls, p<0.05), these findings were considered to be adverse.

At 300 mg/kg/day and when compared with controls, a slightly lower mean body weight gain was noted on Days 15-18 p.c. (+42 g vs. +48 g, p<0.05). This finding was considered to be non-adverse as it was transient, of slight magnitude and did not affect the mean body weight.

 

Net body weight change

At 1000 mg/kg/day and when compared with controls, lower carcass weight (-6%, p<0.05) and lower net body weight change (-28%, p<0.01) were noted. These findings were associated with lower mean body weight and mean body weight changes. Therefore and in view of its magnitude, the decreased net body weight change was considered to be adverse.

There were no test item-related effects on mean gravid uterus weight.

 

Macroscopic post-mortem examination, organ weight and microscopic examination

At 1000 mg/kg/day, non-adverse test item-related increased liver weights correlated with microscopic non-adverse increased hepatocellular vacuolation. In addition, thickened forestomach mucosa was noted and correlated with adverse ulcers, accompanied by inflammation, squamous cell hyperplasia and hyperkeratosis. Increased kidney weights were observed without microscopic correlates, and thus were considered to be equivocal and non-adverse changes. 

At 300 mg/kg/day, non-adverse microscopic hyperplasia of squamous cells and hyperkeratosis were noted in the forestomach.

At 100 mg/kg/day, there were no test item-related findings.

 

Hysterectomy data

There were no test item treatment-related effects.

 

Fetal body weight and sex-ratio

There were no effects on fetal body weight and sex-ratio at any tested dose.

 

Fetal examinations

External examination:

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

 

Soft tissue examination:

. there were no test item-related variations or malformations observed at soft tissue examination.

 

Cartilage and skeletal examinations:

. when compared with controls and/or Historical Control Data, non-adverse test item-related variations (delayed ossification) were noted with higher litter and/or fetal incidences at 1000 mg/kg/day [i.e. incomplete ossification of centrum for cervical vertebra(e), bipartite ossification of centrum for thoracic vertebra(e), incomplete frontal ossification, incomplete 6^thsternebra ossification and/or unossification of metacarpal(s)],

. there were no malformations observed at skeletal examination.

 

Conclusion

The test item was administered to time-mated Sprague-Dawley rats by gavage, once daily, from Day 6 to Day 20 p.c., inclusive, at the dose levels of 100, 300 and 1000 mg/kg/day.The control group received the vehicle, corn oil, 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 300 mg/kg/day based on adverse findings recorded at 1000 mg/kg/day (decreased body weight gain, reduced food intake, decreased net body weight change and ulcers on the forestomach mucosa),

. the No Observed Adverse Effect Level (NOAEL) for embryo-fetal development was considered to be 1000 mg/kg/day based on the absence of adverse effects at this dose level.