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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 March 2017 - 20 April 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
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:
Esterification products of acrylic acid and 4,4'-isopropylidenediphenol ethoxylated
EC Number:
701-362-9
Molecular formula:
(C2 H4 O)x (C2 H4 O)y C21 H20 O4
IUPAC Name:
Esterification products of acrylic acid and 4,4'-isopropylidenediphenol ethoxylated
Details on test material:
SID Change
Previous CAS: 64401-02-1
Previous EC: 613-584-2

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: 10-11 weeks old
- Mean body weight at the beginning of the treatment period: a mean body weight of 315 g (range: 232 g to 360 g)
- Fasting period before study: no
- Housing: The animals were individually housed in polycarbonate cages (Tecniplast 2154, 940 cm2)
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: for a period of 4 or 5 days before the beginning of the treatment period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): about 8 to 15 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h

IN-LIFE DATES: 27 March 2017 to 20 April 2017.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING FORMULATIONS:
- Emulsion in the vehicle
- Concentration in vehicle: 10, 50 and 200 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 UV detection (HPLC/UV)
Test item concentrations:remained within an acceptable range of variation (-4.2% to +2.6%) when compared to the nominal values.
Homogeneity: the dose formulations containing the test item in corn oil at 2 and 200 mg/mL were found to be homogeneous. They are therefore considered to be suitable for routine administration in GLP Toxicological studies, based on a daily preparation.
Stability: no stability available, dose formulations prepared daily.
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 Days 6 to 20 p.c., inclusive.
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
250 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 females (groups 2, 3 and 4).
For group 1 (control group), at animal receipt, one female presented a malformation at the inferior lip. This female was excluded from the study.
Only 23 females instead of 24 were therefore assigned to the control group.
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for dose level selection:
The dose levels were selected in agreement with the Sponsor, on the basis of the results of a previous OECD 421 study (5 weeks of treatment for males, about 6 weeks for females in total) performed in the same species and strain at 50, 250 and 1000 mg/kg/day. In females of this study, there were no unscheduled deaths and no toxicologically significant effects on mean body weight, mean food consumption or mean reproductive and litter parameters. However, adverse findings were noted in females at 1000 mg/kg/day: transient round back and piloerection in Weeks 2/3 and higher mean cholesterol level at the end of the treatment period.

- 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 once a day as part of the routine examinations. From the start of the treatment period, each animal was observed once a day, at approximately the same time, for the recording of clinical signs.

BODY WEIGHT:
- Time schedule: the body weight of each female was recorded on Days 2, 4, 6, 9, 12, 15, 18 and 21 p.c.

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

POST-MORTEM MACROSCOPIC EXAMINATION:
- Sacrifice on Day 21 post-coitum.
- Examined: principal thoracic and abdominal organs.
Ovaries and uterine content:
The ovaries and 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,
- placental weight.
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: body weight, sex
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).
Indices:
% Pre-implantation loss = 100 * (Number of corpora lutea - Number of implantation sites) / Number of corpora lutea
% Post-implantation loss = 100 * (Number of implantation sites - Number of live fetuses) / Number of implantation sites
Historical control data:
Cf attached document

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
See table 1.
At 50 mg/kg/day, one female had a mass on mammary gland from Day 16 p.c. As this observation was limited to one animal from the low-dose group only, a relationship to the test item treatment was considered unlikely.

At 250 mg/kg/day, there were no clinical signs.

At 1000 mg/kg/day, almost all the females had ptyalism for 1 to 13 days of treatment. This test item-related finding was considered as non-adverse (not critical, sign of discomfort).
One female started showing round back and piloerection on Day 19 p.c. and reddish vaginal discharge was found on Day 21 p.c. This animal also had a strongly reduced food consumption from Day 15 p.c. and lost 15% of body weight from Days 15 to 18 p.c. with a low body weight gain thereafter. At hysterectomy, only implantation scars were found and at necropsy the female had a segmental aplasia of the right uterine horn and no right kidney/ureter (congenital anomalies), as well as an enlarged spleen. In absence of similar findings on the clinical condition of the other females of the group and in presence of the congenital anomalies in this female, a test item effect was considered to be unlikely.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No unscheduled death occurred during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
See table 2.
At 50 and 250 mg/kg/day, there were no effects on mean body weight and mean body weight change.
At 1000 mg/kg/day, there was a lower mean body weight gain vs. controls at the beginning of the treatment (Days 9 to 12 p.c., p<0.001). This effect were considered to be test item treatment-related and non adverse, in absence of effect on mean body weight and in view of the slight and transient effect on mean body weight change. It correlated with the effects on mean net body weight change.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
See table 3.
There were no effects on mean food consumption, except a slightly higher mean food consumption at 1000 mg/kg/day between Days 18 and 21 p.c. (p<0.05). In view of the limited difference from controls, this was considered of no biologically significance and unlikely test item-related.
Food efficiency:
not examined
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, non-treatment-related
Description (incidence and severity):
See table 4.
None of the necropsy findings were ascribed to the test item treatment: they were noted in isolated cases, were not dose-related and/or were congenital anomalies.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Mean gravid uterus and carcass weights, and mean net body weight changes
See table 5.
At 50 and 250 mg/kg/day, there were no effects on mean carcass and gravid uterus weights and on mean net body weight change from Day 6 p.c.
At 1000 mg/kg/day, the mean net body weight change from Day 6 p.c. was lower than in controls (p<0.05), correlating with the effects on mean body weight change. This effect was considered to be test item-related and non-adverse (no toxicologically significant effects on mean carcass weight and slight difference) and test item treatment-related. There were no effects on mean gravid uterus weight.

Maternal developmental toxicity

Number of abortions:
not examined
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
See table 6.
There were no test item treatment-related findings.
Total litter losses by resorption:
effects observed, non-treatment-related
Description (incidence and severity):
See table 6.
There were no test item treatment-related findings.

At 250 mg/kg/day, the higher mean number of late resorptions when compared with controls was due to one female with 100% of implantation loss.
At 1000 mg/kg/day, the higher mean number of implantation scars when compared with controls was due to one female only (with implantation scars only).
Early or late resorptions:
effects observed, non-treatment-related
Description (incidence and severity):
See table 6.
There were no test item treatment-related findings.

At 250 mg/kg/day, the higher mean number of late resorptions when compared with controls was due to one female with 100% of implantation loss.
At 1000 mg/kg/day, the higher mean number of implantation scars when compared with controls was due to one female only (with implantation scars only).
Dead fetuses:
no effects observed
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:
effects observed, non-treatment-related
Description (incidence and severity):
See table 6.
On Day 21 p.c., there were 20/23, 23/24, 20/24 and 22/24 females pregnant with live fetuses at 0, 50, 250 and 1000 mg/kg/day, respectively.
The other females were non-pregnant with the exception of one female at 250 mg/kg/day and one at 1000 mg/kg/day which had implantation scars/total resorptions.
Other effects:
not specified

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
See table 7.
At 1000 mg/kg/day when compared with controls, female fetuses had a lower mean body weight (p<0.01), which was included in Historical Control Data. This slight effect was considered to be test item-related and non-adverse.
Similar trend was observed in males at the same dose or in females at the mid-dose but without statistical significance and within the range of the Historical Control range. This trend was therefore considered to be not toxicologically significant.

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:
not examined
Changes in sex ratio:
no effects observed
Description (incidence and severity):
See table 8.
There were no effects on mean sex ratio.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
See table 6.
There were no test item treatment-related findings.
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Variations
See table 10.
There were no external variations in fetuses at 50 and 250 mg/kg/day.
At 1000 mg/kg/day one litter had three fetuses with small mandible and protruding tongue. They were associated with cleft palate. Considering that these variations were observed in only one litter and were also observed in Historical Control Data at similar litter incidence, a relationship to the test-item was considered unlikely.

Malformations
See table 11.
At 50 mg/kg/day, one fetus displayed various severe external malformations. Taking into account that these malformations affected only one fetus, were observed in controls or Historical Control Data for most of them and did not occur in mid-dose or high-dose group, this finding was considered to be not test item treatment-related.
At 250 mg/kg/day, there were no fetal external malformations.
At 1000 mg/kg/day, three fetuses from the same litter had a cleft palate (and small mandible/protruding tongue, see above). Considering that this malformation was observed in only one litter and was also observed in Historical Control Data at similar litter incidence, it was considered to be not test item treatment-related.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
Cartilages
There were no test item treatment-related findings at cartilage examinations.
All cartilages were present and had a normal morphology with the exceptions of one fetus at 250 mg/kg/day (absent rib) and another one at 1000 mg/kg/day (misshapen carpal joint). These findings were considered to be not test item treatment-related.

Variations
See table 14.
At 1000 mg/kg/day and when compared with controls, there were higher fetal and litter incidences of incomplete ossification of cervical and thoracic vertebrae centra (including bipartite and dumbbell ossification of thoracic vertebrae centra) and of unossification of the 1st metatarsal bones. Ossification of metacarpal bones and caudal vertebrae centra also had the tendency to be delayed. The fetal and litter incidences of unossified 1st metatarsal bone and of unossified/incomplete ossified metacarpal(s) were outside the Historical Control Data (as well as the litter incidence of bipartite ossification of thoracic vertebrae and the fetal incidence of incompletely ossified caudal vertebrae centra).

Higher litter and fetal incidences of some of these bones tended also, and in a dose-related manner, to be higher at 50 and 250 mg/kg/day than in controls but they remained within the Historical Control Data.

These ossification delays were considered to be test item-related and non-adverse, associated with a slight effect on mean fetal body weight at 1000 mg/kg/day..

Other differences from controls were considered as not test item treatment-related as they were isolated, recorded at lower/similar incidences than/as in control groups and/or HCD, and/or were not dose-related.
The incomplete ossifications noted on head, hindlimb and forlimb bones described in the table above were only observed in 2 fetuses from the same litter already mentioned.

Malformations
See table 15.
At 50 and 250 mg/kg/day, there were no skeletal malformations.

At 1000 mg/kg/day, one fetus had head (absent nasal bone and split palate) and hindpaw malformations (unossified tibia and fibula), as well as another fetus from the same litter (split palate, short tibia and short fibula). One fetus from another litter displayed supernumerary digit(s) (right forepaw).
Almost all skeletal malformations noted at 1000 mg/kg/day were observed in 2 fetuses from the same litter. The litter incidence of split palate noted in both these fetuses was within Historical Control Data, and a third fetus from the same litter was also affected (see external malformations, skeleton not examined) while there was no other litter in the group with split/cleft palate. In addition, these 2 fetuses also had ossification delays on head, limb and/or metatarsal bones (variations), and/or misshapen carpal joint, which all were also not seen in any other fetuses from the study.
Therefore, malformations noted in these fetuses from the same litter were likely due to a litter bias.
The digit malformation of the fetus from another litter was not similar (different morphological pattern) to the limb malformations in litter mentioned above, and despite the fact that it can not be found in our Historical Control Data, it can occur spontaneously in laboratory rats (Lang 1993, Morita et al. 1987).
Therefore, a relationship between skeletal malformations in litters at 1000 mg/kg/day and the test item treatment was considered to be unlikely.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Variations
See table 12.
None of the soft tissue variations were considered to be related to the test item treatment: they were not dose-related and/or recorded at incidences lower than Historical Control Data and/or controls.

Malformations
See table 13.
None of the soft tissue malformations were considered to be related to the test item treatment: they were not dose-related, were noted in one same litter only and/or had similar incidences as Historical Control Data.
Other effects:
no effects observed
Description (incidence and severity):
Placental weight
See table 9.
There were no effects on mean placental weight.
Details on embryotoxic / teratogenic effects:
There were no toxicologically significant differences in the incidence of litters with malformed fetuses between groups, nor any dose-relationship in the incidence of each malformation.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
other: skeletal variations

Fetal abnormalities

Abnormalities:
effects observed, non-treatment-related

Overall developmental toxicity

Developmental effects observed:
no

Any other information on results incl. tables

Table 1: Clinical signs

 

Dose level (mg/kg/day)

0

50

250

1000

Ptyalism

 

 

 

20

Piloerection

 

 

 

1a

Round back

 

 

 

1a

Reddish vaginal discharge

1

 

 

1a

Mass on mammary gland

 

1

 

 

Scab on abdomen or neck

1

 

 

1

No remarkable observations

18/20

22/23

21/21

3/23

a: same female.

Table 2: Body weight

 

Dose level (mg/kg/day)

0

50

250

1000

Body weight

 

 

 

 

Day 6p.c.

317

317

316

314

 

 

(0)

(0)

(-1)

Day 9p.c.

328

330

327

322

 

 

(+1)

(0)

(-2)

Day 12p.c.

351

353

348

337

 

 

(+1)

(-1)

(-4)

Day 21p.c.

462

469

469

445

 

 

(+2)

(+2)

(-4)

Body weight change

 

 

 

 

Days 6 - 9p.c.

+11

+13

+11

+8

Days 9 - 12p.c.

+22

+22

+21

+15#

Days 6 - 21p.c.

+145

+152

+154

+131*

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

(): % from controls.

Table 3: Food consumption

 

Dose level (mg/kg/day)

0

50

250

1000

Days 6 - 9p.c.

22

23

23

22

 

 

(+5)

(+5)

(0)

Days 9 - 12p.c.

24

25

25

23

 

 

(+4)

(+4)

(-4)

Days 12 - 15p.c.

26

27

28

27

 

 

(+4)

(+8)

(+4)

Days 15 - 18p.c.

28

29

30

29

 

 

(+4)

(+7)

(+4)

Days 18 - 21p.c.

30

30

31

32*

 

 

(0)

(+3)

(+7)

Statistical significance:*:p<0.05.

(): % from controls.

Table 4: Maternal necropsy findings

 

Dose level (mg/kg/day)

0

50

250

1000

Spleen: enlarged

 

 

1a

1b

Liver: reduced in size + mass on median lobe

 

1

 

 

Mammary gland: palpable mass

 

1

 

 

Vagina: colored contents

 

 

1a

 

Placenta(s): enlarged

1

 

1

1

Colored material around placenta

 

 

 

1

Placentas fused

1

 

 

1

Kidney: enlarged (unilateral)

 

 

1c

 

Kidney: absence (unilateral)

 

1d

1c

1b

Ureter: absence (unilateral)

 

1d

1c

1b

Uterus: absence of uterine horn (unilateral)

 

1d

1c

 

Uterus: segmental aplasia of uterine horn (unilateral)

 

 

 

1b

No remarkable observations

18/20

20/23

18/21

19/23

a: same female, total resorption.

b: same female, implantation scars only.

c: same female.

d: same female.

Table 5: Gravid uterus weight, carcass weight and net body weight change

 

Dose level (mg/kg/day)

0

50

250

1000

Gravid uterus weight

93.3

96.9

100.2

96.8

 

 

(+4)

(+7)

(+4)

Carcass weight

368.4

372.1

369.0

348.1

 

 

(+1)

(0)

(-6)

Net body weight change from Day 6p.c.

+51.5

+54.9

+53.4

+33.9**

Statistical significance: **: p<0.01.

(): % from controls.

Table 6: Hysterectomy data

 

Dose level (mg/kg/day)

0

50

250

1000

Number of females with live fetuses

20

23

20

22

Mean number ofcorpora lutea

14.9

15.7

15.8

15.2

Mean number of implantation sites

12.4

12.8

13.0

12.8

Mean pre-implantation loss (%)

17.5

18.0

16.8

15.5

Mean number of fetuses

11.1

11.9

11.8

11.5

Mean dead fetuses (%)

0.0

0.0

0.0

0.0

Mean number of implantation scars

0.0

0.0

0.0

0.4

Mean number of early resorptions

1.0

0.8

0.6

0.8

Mean number of late resorptions

0.2

0.1

0.7

0.1

Mean post-implantation loss (%)

10.1

7.7

8.3

11.1

 

Table 7: Fetal body weight

 

Dose level (mg/kg/day)

0

50

250

1000

HCD

Mean fetal body weight
(males and females)

5.91

5.80

5.77

5.64

[5.5-5.9]

 

 

(-2)

(-2)

(-5)

 

males

6.06

5.97

5.99

5.83

[5.7-6.1]

 

 

(-1)

(-1)

(-4)

 

females

5.77

5.64

5.55

5.44**

[5.4-5.7]

 

 

(-2)

(-4)

(-6)

 

(): % from controls.

**: p<0.01

HCD: Historical Control Data (Sprague-Dawley rats from the Supplier Janvier; signed in December 2016): [min study mean; max study mean].

Table 8: Fetal sex ratio

 

Dose level (mg/kg/day)

0

50

250

1000

Mean percentage of male fetuses

47.9

50.3

48.7

51.4

Table 9: Placental weight

 

Dose level (mg/kg/day)

0

50

250

1000

Mean placental weight

0.69

0.68

0.69

0.69

 

Table 10: Fetal external variations

 

Dose level (mg/kg/day)

0

50

250

1000

HCD

Number of litters

20

23

20

22

388

Number of fetuses

223

273

247

265

5000

Small mandible, F (L)

1.1 (4.5)a

0.4 (5.0)b

Protruding tongue, F (L)

1.1 (4.5)a

0.4 (4.8)

Litters with external variation, n (%)

1 (4.5)

6 (1.5)

Fetuses with external variation, n (%)

 

3 (1.1)

7 (0.1)

F: fetal incidence (%).

L: litter incidence (%).

a: same fetuses;b: referred as short mandible in HCD.

HCD: Historical Control Data (Sprague-Dawley rats from the Supplier Janvier; signed in December 2016): max fetal incidence (max litter incidence) for the findings.

 

Table 11: Fetal skeletal malformations

 

Dose level (mg/kg/day)

0

50

250

1000

HCD

Number of litters

20

23

20

22

388

Number of fetuses

117

143

131

138

2596

Nasal: absent

 

 

 

0.7 (4.5)a

-

Palate: split

 

 

 

1.4 (4.5)a, b

0.7 (5.0)

Fused sternebra(e)

0.9 (5.0)

 

 

 

1.3 (8.7)

Absent sternebra(e)

0.9 (5.0)

 

 

 

-

Forepaw: supernumerary digit(s)

 

 

 

0.7 (4.5)

-

Tibia: unossified (unilateral)

 

 

 

0.7 (4.5)a

-

Tibia: short

 

 

 

0.7 (4.5)b

-

Fibula: unossified (unilateral)

 

 

 

0.7 (4.5)a

-

Fibula: short

 

 

 

0.7 (4.5)b

-

Litters with skeletal malformation, n (%)

1 (5.0)

0 (0.0)

0 (0.0)

2 (9.1)

15 (3.9)

Fetuses with skeletal malformation, n (%)

1 (0.9)

0 (0.0)

0 (0.0)

3 (2.2)

18 (0.7)

F: fetal incidence; L: litter incidence.

a and b: two fetuses from the same litter.

HCD: Historical Control Data (Sprague-Dawley rats from the Supplier Janvier; signed in December 2016): max fetal incidence (max litter incidence) for the findings; -: none in HCD.

Table 12: Fetal soft tissue variations

Dose level (mg/kg/day)

0

50

250

1000

HCD

Number of litters

20

23

20

22

387

Number of fetuses

106

130

116

127

2404

Dilated cerebral ventricle, F (L)

0.9 (5.0)

 

 

 

0.8 (4.8)

Enlarged atrial chamber, F (L)

 

0.8 (4.3)

 

 

-

Malpositioned subclavian artery, F (L)

 

 

0.9 (5.0)

 

-

Short innominate artery, F (L)

3.8 (15.0)

1.5 (8.7)

4.3 (10.0)

0.8 (4.5)

3.7 (22.7)

Absent innominate artery, F (L)

 

 

 

2.4 (9.1)

5.1 (25.0)

Lung: colored area, F (L)

 

0.8 (4.3)

 

 

-

Dilated renal pelvis, F (L)

 

1.5 (8.7)

 

2.4 (13.6)

9.5 (28.6)

Dilated uterine horn, F (L)

 

 

 

0.8 (4.5)

-

Dilated ureter, F (L)

0.9 (5.0)

1.5 (8.7)

 

1.6 (9.1)

7.1 (28.0)

Litters with soft tissue variation, n (%)

4 (20.0)

6 (26.1)

3 (15.0)

7 (31.8)

86 (22.2)

Fetuses with soft tissue variation, n (%)

6 (5.7)

6 (4.6)

6 (5.2)

9 (7.1)

119 (5.0)

F: fetal incidence; L: litter incidence.

HCD: Historical Control Data (Sprague-Dawley rats from the Supplier Janvier; signed in December 2016): max fetal incidence (max litter incidence) for the findings; -: none in HCD.

Table 13: Fetal soft tissue malformations

 

Dose level (mg/kg/day)

0

50

250

1000

HCD

Number of litters

20

23

20

22

387

Number of fetuses

106

130

116

127

2404

Cleft palate, F (L)

 

 

 

0.8 (4.5)

b

Absent kidney, F (L)

 

 

1.7 (5.0)a

 

0.7 (4.5)

Absent ureter, F (L)

 

 

1.7 (5.0)a

 

-

Litters with soft tissue malformation, n (%)

0 (0.0)

0 (0.0)

1 (5.0)

1 (4.5)

 

7 (1.8)

Fetuses with soft tissue malformation, n (%)

0 (0.0)

0 (0.0)

2 (1.7)

1 (0.8)

 

13 (0.5)

F: fetal incidence; L: litter incidence, a: 2 fetuses from the same litter

b: not found in soft tissue malformations of HCD, but finding seen in one HCD fetus as external and skeletal malformations.

HCD: Historical Control Data (Sprague-Dawley rats from the Supplier Janvier; signed in December 2016): max fetal incidence (max litter incidence) for the findings; -: none in HCD.

Table 14: Fetal skeletal variations

 

Dose level (mg/kg/day)

0

50

250

1000

HCD

Number of litters

20

23

20

22

388

Number of fetuses

117

143

131

138

2596

Vertebrae

 

 

 

 

 

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

0.9 (5.0)

0.7 (4.3)

2.3 (15.0)

15.9a(50.0)b

18.3 (54.2)

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

1.7 (5.0)

3.5 (21.7)

9.2c(30.0)

14.5a(45.5)b

29.6 (87.5)

Thoracic vertebra(e): bipartite ossification of centrum

0.9 (5.0)

 

3.1 (10.0)

3.6 (18.2)

5.0 (16.7)

Thoracic vertebrae: dumbbell

ossification of centrum

1.7 (10.0)

 

3.1 (10.0)

4.3 (18.2)

6.5 (30.0)

Caudal vertebrae: incomplete ossification of centrum

0.9 (5.0)

 

0.8 (5.0)

2.9 (9.1)

1.4 (10.0)

Metatarsal bone

 

 

 

 

 

Unossified 1stmetatarsal, F (L)

12.0 (45.0)

19.6 (69.6)

20.6 (55.0)

42.0a(86.4)b

36.8 (72.7)

Metatarsal: unossified

 

 

 

1.4 (4.5)d,e

5.4 (21.7)

Metacarpal bone

 

 

 

 

 

Incomplete ossification of metacarpal(s)

1.7 (10.0)

0.7 (4.3)

0.8 (5.0)

4.3 (22.7)

2.2 (10.0)

Unossified metacarpal(s)

0.9 (5.0)

 

1.5 (10.0)

3.6 (18.2)f

2.9 (15.0)

Head

 

 

 

 

 

Nasal: incomplete ossification

 

 

 

0.7 (4.5)d

0.8 (5.6)

Incisive bone: incomplete ossification

 

 

 

1.4 (4.5)d,e

0.7 (5.0)

Maxilla: incomplete ossification

 

 

 

1.4 (4.5)d,e

0.7 (5.0)

Forelimb

 

 

 

 

 

Radius: incomplete ossification

 

 

 

0.7 (4.5)e

-

Hindlimb

 

 

 

 

 

Tibia: incomplete ossification

 

 

 

0.7 (4.5)e

-

Fibula: incomplete ossification

 

 

 

0.7 (4.5)e

-

Litters with skeletal variation in total, n (%)

17 (85.0)

23 (100.0)

17 (85.0)

21 (95.5)

345 (88.9)

Fetuses with skeletal variation in total, n (%)

72 (61.5)

96 (67.1)

65 (49.6)

96 (69.6)

1267 (48.8)

F: fetal incidence; L: litter incidence.

a: p<0.001 for the number of affected fetuses or litters;b: p<0.01 for the number of affected fetuses or litters;c: p<0.05 for the number of affected fetuses or litters.

d and e: tow fetuses from the same litter; f: including these two fetuses

HCD: Historical Control Data (Sprague-Dawley rats from the Supplier Janvier; signed in December 2016): max fetal incidence (max litter incidence) for the findings.

Table 15: Fetal skeletal malformations

 

Dose level (mg/kg/day)

0

50

250

1000

HCD

Number of litters

20

23

20

22

388

Number of fetuses

117

143

131

138

2596

Nasal: absent

 

 

 

0.7 (4.5)a

-

Palate: split

 

 

 

1.4 (4.5)a, b

0.7 (5.0)

Fused sternebra(e)

0.9 (5.0)

 

 

 

1.3 (8.7)

Absent sternebra(e)

0.9 (5.0)

 

 

 

-

Forepaw: supernumerary digit(s)

 

 

 

0.7 (4.5)

-

Tibia: unossified (unilateral)

 

 

 

0.7 (4.5)a

-

Tibia: short

 

 

 

0.7 (4.5)b

-

Fibula: unossified (unilateral)

 

 

 

0.7 (4.5)a

-

Fibula: short

 

 

 

0.7 (4.5)b

-

Litters with skeletal malformation, n (%)

1 (5.0)

0 (0.0)

0 (0.0)

2 (9.1)

15 (3.9)

Fetuses with skeletal malformation, n (%)

1 (0.9)

0 (0.0)

0 (0.0)

3 (2.2)

18 (0.7)

F: fetal incidence; L: litter incidence.

a and b: fetuses from the same litter.

HCD: Historical Control Data (Sprague-Dawley rats from the Supplier Janvier; signed in December 2016): max fetal incidence (max litter incidence) for the findings; -: none in HCD

Applicant's summary and conclusion

Conclusions:
The test item, Ethoxylated bisphenol A diacrylate, was administered by gavage, once daily, from Days 6 to 20 p.c. inclusive, to pregnant Sprague-Dawley rats at dosages of 50, 250 and 1000 mg/kg/day.

At 1000 mg/kg/day, lower fetal body weight (female fetuses) and increased fetal ossification delays were observed in presence of some signs of limited maternal toxicity (i.e. lower body weight gain and net body weight gain from Day 6 p.c.).
At 50 and 250 mg/kg/day, there were signs of slight fetal ossification delays.

Under the experimental conditions and results of this study:
¿ the No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 1000 mg/kg/day,
¿ the NOAEL for embryo-fetal development was considered to be 1000 mg/kg/day.
Executive summary:

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

 

Methods

Three groups of 24 time-mated female Sprague-Dawley rats received the test item at 50, 250 or 1000 mg/kg/day by oral route (gavage) once daily from Days 6 to 20 p.c. A constant dosage-volume of 5 mL/kg/day was used. Another group of 23 rats received the vehicle alone (corn oil) under the same experimental conditions, and acted as a control group.

 

Formulation concentrations were checked in the first day and last week of treatment in the study.

The animals were checked daily for mortality and clinical signs. Body weight and food consumption were recorded every 2 to 3 days. On Day 21 p.c., females were sacrificed and submitted to a macroscopic post-mortem examination of principal thoracic and abdominal organs. Hysterectomy was performed and the numbers of corpora lutea, implantations, uterine scars, early and late resorptions, live and dead fetuses were recorded. Placenta weight was recorded. The fetuses were sexed, weighed and examined for external, soft tissues and skeletal (cartilages + bones) abnormalities.

 

Results

The test item concentrations in the dose formulations analyzed were within an acceptable range of variations when compared with the nominal values (± 15% required). No test item was observed in the control dose formulations.

 

On Day 21 p.c., 20/23, 23/24, 20/24 and 22/24 females pregnant with live fetuses at 0, 50, 250 and 1000 mg/kg/day, respectively.

 

There were no deaths in the study and the only test item-related clinical sign was ptyalism noted in almost all females treated at 1000 mg/kg/day.

 

There were no test item-related effects on mean food consumption and mean body weight at any tested dose. The mean body weight gain from Days 9 to 12 p.c. was reduced in females treated at 1000 mg/kg/day (+15 g vs. +22 g in controls, p<0.001 vs. controls).

 

There were no test item-related necropsy findings in females.

At 1000 mg/kg/day, correlating with the effects on mean body weight change, there was a lower mean net body weight change from Day 6 p.c. (+33.9 g vs. +51.5 g p<0.01 vs. controls) but no toxicologically significant effects on mean carcass weight. There were no effects on mean carcass weight and on mean net body weight change from Day 6 p.c. at 50 and 250 mg/kg/day.

 

All above mentioned findings in dams were considered to be test item-related and non-adverse.

 

There were no test item treatment-related effects on mean hysterectomy data, mean sex ratio, mean placental weight and mean gravid uterus weight.

At 1000 mg/kg/day when compared with controls, female fetuses had a lower mean body weight (-6%, p<0.01), considered to be test item-related and non-adverse.

At fetal examination, there were no test item treatment-related external or soft tissue variations and malformations, and no test item treatment-related effects on cartilages.

At 1000 mg/kg/day, there were fetal skeletal ossification delays (test item-related and non-adverse) in cervical and thoracic vertebra centra, 1stmetatarsal bones and to a lesser extent in metacarpal bones and caudal vertebra centra. Similar trend but to a lesser extent was observed in some of these bones at 50 and/or 250 mg/kg/day. At 1000 mg/kg/day, a relationship between litter skeletal malformations and the test item treatment was considered unlikely.

 

Conclusion

The test item, Ethoxylated bisphenol A diacrylate, was administered by gavage, once daily, from Days 6 to 20 p.c. inclusive, to pregnant Sprague-Dawley rats at dosages of 50, 250 and 1000 mg/kg/day.

 

At 1000 mg/kg/day, lower fetal body weight (female fetuses) and increased fetal ossification delays were observed in presence of some signs of limited maternal toxicity (i.e.lower body weight gain and net body weight gain from Day 6p.c.).

At 50 and 250 mg/kg/day, there were signs of slight fetal ossification delays.

 

Under the experimental conditions and results of this study:

.            the No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 1000 mg/kg/day,

.            the NOAEL for embryo-fetal development was considered to be 1000 mg/kg/day.