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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:
2020-02-25 to 2021-06-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
Deviations had no impact on the results or integrity of the study
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
(2E)-2-methyl-3-phenylacrylaldehyde
EC Number:
701-219-0
Cas Number:
15174-47-7
Molecular formula:
C10H10O
IUPAC Name:
(2E)-2-methyl-3-phenylacrylaldehyde
Test material form:
liquid
Details on test material:
Name of substance: (2E)-2-methyl-3-phenylacrylaldehyde
CAS#: 15174-47-7
EC#: 701-219-0
Batch/Lot No.: A190809B
Appearance: Clear yellow liquid
Purity: 99.49%
Expiry date: 2021-08-12
Storage conditions: Controlled room temperature (15-25°C, ≤70% relative humidity), under inert gas
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Emerald Performance Materials (1499 SE Tech Center Place, Suite 300, Vancouver, WA 98683); Batch number: A190809B
- Purity, including information on contaminants, isomers, etc.: 99.49%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25°C, ≤70% relative humidity), under inert gas
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: stable up to 5 days at room temperature
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: stable up to 5 days at room temperature

FORM AS APPLIED IN THE TEST: Liquid

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Hannover Wistar rats (CRL:WI(Han))
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH (Sandhofer Weg 7, D-97633 Sulzfeld, Germany) from SPF colony
- Age at study initiation: Young adult females, 13-14 weeks old at mating
- Weight at study initiation: 205 - 261 g at onset of treatment
- Fasting period before study: Not specified
- Housing: Type II polycarbonate cages were used during mating and gestation period and Type III polycarbonate cages were used during the acclimatisation period. Successfully mated animals were housed individually.
- Diet (e.g. ad libitum): ssniff® SM R/M-Z+H “Autoclavable Complete Feed for Rats and Mice – Breeding and Maintenance” (Ssniff Spezialdiäten GmbH, D-59494 Soest, Germany) ad libitum
- Water (e.g. ad libitum): and tap water (in water bottles) as for human consumption ad libitum
- Acclimation period:at least 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.1-24.0°C (target: 22 ± 3°C)
- Humidity (%): 20-90% (target: 30-70%)
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 2020-02-26 To: 2020-04-01

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
Poly(ethylene glycol) 400
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test material was formulated in the vehicle (PEG400), as a visibly stable homogenous formulation at the appropriate concentrations according to the dose level and volume selected. The formulations were stirred for at least one hour after formulating. Formulations were prepared fresh prior to administration to animals or at the appropriate frequency to allow their use according to stability assessment results (up to 5 days). A constant volume of 5 mL/Kg body weight was administered to all dose groups, including the controls. The individual volume of the treatment was based on the most recent individual body weight of the animals.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on the available information provided by the Sponsor as well as results of the trial formulation, PEG400 was selected as vehicle for this study in agreement with the Sponsor
- Concentration in vehicle: 0, 30, 60, or 120 mg/mL for the 0, 150, 300, and 600 mg/Kg bw/day dose levels, respectively.
- Amount of vehicle (if gavage): 5 mL/Kg body weight
- Lot/batch no. (if required): Source: Sigma-Aldrich/ACROS Organics; Batch Number: BCBZ1233/A0402800
- Purity: Not specified
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of test material formulations for concentration were performed on the day of preparation in the Analytical Laboratory of Charles River Laboratories Hungary Kft. Duplicate samples were taken from three different place of the formulations two times during the study (at the first treatment day and at the last week of the treatment), one set to analyse (which was collected in replicates as practical) and one set as a back-up, if required for any confirmatory analyses. Similarly, duplicate samples were taken from the middle of the vehicle control formulation for concentration measurement.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused: After acclimation, the females were paired according to their oestrus cycle with males in the morning for approximately 2 hours (1 male : 1 female)
- M/F ratio per cage: 1:1
- Length of cohabitation: 2 hours
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.- Not specified
- Further matings after two unsuccessful attempts: Not specified
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: Not specified

The sperm-positive, assumed pregnant females were allocated to each experimental groups (on each mating day) in such a way that the group averages of the body weight were as similar as possible. Females paired with the same male were allocated to different groups on the same mating day. A computer software PROVANTIS v9 was used to verify homogeneity/variation among/within groups.
Duration of treatment / exposure:
Daily from gestation day 6 (GD 6) to gestation day 19 (GD 19)
Frequency of treatment:
Daliy
Duration of test:
From GD 0 to GD 20 (Caesarean section and necropsy)
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Remarks:
Group 1 (Control)
Dose / conc.:
150 mg/kg bw/day
Remarks:
Group 2
Dose / conc.:
300 mg/kg bw/day
Remarks:
Group 3
Dose / conc.:
600 mg/kg bw/day
Remarks:
Group 4
No. of animals per sex per dose:
24 mated females/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected by the Sponsor in consultation with the Study Director based on the results of two dose range finding (DRF) studies (CRL study codes 19/205-209PE and 19/205-105PE), with the aim of inducing toxic effects but ideally no death or suffering at the highest dose and a NOAEL at one of the lower doses. In the DRF study with non-pregnant rats (study code: 19/205-209PE), 2 out of 4 males and 3 out of 4 females from the High dose group (1000 mg/Kg bw/day) were found dead. In the DRF study with pregnant Hannover Wistar rats (Study code: 19/205-105PE), 1 out of 6 animals in the High dose group (750 mg/Kg bw/day) was found dead. For all of the animals found dead, no clear signs of test material related toxicity preceded the death of the animals. Therefore, it was considered that, apart from mortality in 1 out of 6 pregnant animals, there were minimal signs of toxicity at 750 mg/Kg bw/day, and no significant toxicity was seen at 500 mg/Kg/day. Therefore, an intermediate dose of 600 mg/Kg bw/day was selected as the High dose for this OECD 414 study as the highest dose level compatible with the study objectives. Lower doses were set with a factor of 2. Based on this information, the doses of 600, 300, and 150 mg/Kg bw/day are deemed suitable for the purpose of the study.

- Rationale for animal assignment: The sperm-positive, assumed pregnant females were allocated to each experimental groups (on each mating day) in such a way that the group averages of the body weight were as similar as possible. Females paired with the same male were allocated to different groups on the same mating day. A computer software PROVANTIS v9 was used to verify homogeneity/variation among/within groups.

- Fasting period before blood sampling for (rat) dam thyroid hormones: Not specified

- Time of day for (rat) dam blood sampling: time not specified but blood samples were taken from all dams at termination and centrifuged within 30 minutes of collection (1600 g / approx. 3000 rpm, 10 minutes, 4 ºC)

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each working day, on GD0 only one inspection was conducted). General clinical observations were made twice daily (at the beginning and end of each working day). Only one general clinical observation was made on the first day (in the afternoon), on the afternoon on those days when detailed clinical observation was made in the morning. Furthermore, clinical observation (detailed) was made only once on necropsy days (in the morning).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were made on all animals at the onset of treatment (GD 6) then at least weekly.
Pertinent behavioural changes and all signs of toxicity including mortality were recorded including onset, degree and duration of signs as applicable. Signs evaluated were include, but not be limited to, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), bizarre behaviour (e.g. self-mutilation, walking backwards) was also recorded. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma. On GD 13 and/or 14, the sperm positive females were examined for the presence of vaginal bleeding or “placental sign” (intra-uterine extravasation of blood as an early sign of pregnancy in rat, which is considered to confirm implantation).

BODY WEIGHT: Yes
- Time schedule for examinations: body weight of each animal was recorded on GD 0, 3, 6, 8, 10, 12, 14, 16, 18 and 20. Body weight gain was calculated for each interval, including GD0-6, GD6-20 and GD0-20.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes; Food was measured with precision of ± 1 g on GD 0, 3, 6, 8, 10, 12, 14, 16, 18 and 20.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes; Food consumption was also calculated for each interval, including GD 0-6, GD 6-20 and GD 0-20.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: The dams’ viscera were examined macroscopically for any structural abnormalities or pathological changes; all the gross findings were retained in 10% buffered formalin solution (or modified Davidson fixative, in case of the eyes, if any abnormalities noted) for possible future evaluation. The weight of the thyroid gland with parathyroid glands for all dams were measured. As a paired organ, it was weighted together. Absolute organ weights were measured, and relative organ weights to the body weights were calculated and reported. The ovaries and uterus were removed and the pregnancy status ascertained. The uterus including the cervix was weighed and examined for early and late embryonic or foetal deaths and for the number of live foetuses. Gravid-uterine weights were not obtained from animal/s found dead during the study. The corrected body weight was calculated (body weight on GD20 minus weight of the gravid uterus).

During the macroscopic examination of dams, the thyroid gland with parathyroid glands were retained from all animals and the organ weights recorded. The organs were preserved in 10% buffered formalin solution.

The retained thyroids were embedded in paraffin wax, sections were cut at 4-6 µm by microtome and transferred to slides. Tissue sections were stained with haematoxylin-eosin/phloxine and examined by light microscope. Parathyroid glands were examined histologically only if present in routine sections.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Blood sampling:
- Plasma: Yes
For thyroid hormone analysis, blood samples were taken from all dams at termination by sublingual vein into tubes containing K3-EDTA as anticoagulant and centrifuged within 30 minutes of collection (1600 g / approx. 3000 rpm, 10 minutes, 4 ºC). The resulting plasma was divided in three aliquots (volume target of 125 µL for the first aliquot, 75 µL for the second aliquot and remaining plasma for the third aliquot) and stored in an ultrafreezer (-80 ± 10 °C). The levels of the thyroid hormone (T3 and T4) and thyroid stimulating hormone (TSH) were determined respectively by LC-MS/MS or Luminex MAP® technology.
- Serum: Not specified
- Volume collected : plasma was divided in three aliquots (volume target of 125 µL for the first aliquot, 75 µL for the second aliquot and remaining plasma for the third aliquot)
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
- Anogenital distance of all live rodent pups: Yes, the anogenital distance of each foetus was measured

After ensuring humane death, each fully developed foetus was weighed individually (accuracy ±0.01 g) and subjected to external examination, plus an additional examination of the great arteries. The anogenital distance of each foetus were measured, and the sex of each assigned based on the distance. The sex of the foetuses was reconfirmed by examining the internal sex organs. Thereafter, the foetuses were individually identified; approximately half of each litter were subjected to detailed visceral examination, and the other half were processed for skeletal examination.

For the foetuses subjected to visceral examination, the abdominal and thoracic region was opened and the thymus and great arteries were freshly examined by means of a dissecting microscope. The rest of the body was fixed in Sannomiya mixture (a mixture of 920 mL concentrated isopropanol, 30 g sulfosalicylic acid, and 50 mL acetic acid), then, after fixation, the body was micro-dissected by means of a dissecting microscope. The heads were examined by Wilson's free-hand razor blade method.

For the foetuses subjected to skeletal examination, the abdominal region was opened, and the viscera and skin of foetuses were removed and the cadaver was fixed in Alcian-blue - acetic acid – ethanol/isopropanol mixture. After fixation in isopropanol the skeletons were stained by KOH-Alizarin red-S method and examined by means of a dissecting microscope.

All abnormalities (external, soft tissue and skeletal malformations, and variations) found during the foetal examinations were recorded.


Statistics:
For information on statistics, please see 'Any other information on materials and methods incl. tables'.
Indices:
Caesarean Section and Necropsy Data:
 
1) Pre-implantation loss (%, group mean): (Number of corpora lutea - Number of implantations / Number of corpora lutea) x100
 
2) Post-implantation loss (%, group mean): (Number of implantations - Number of live foetuses / Number of implantations) x100
 
Foetal Data:
 
1) Sex distribution (%, group mean): (Number of male (female) foetuses / Number of foetuses) x 100
 
2) External abnormalities/litter (%, group mean): (Number of foetuses with abnormality / Number of foetuses) x 100
           
3) Visceral abnormalities/litter (%, group mean): (Number of foetuses with abnormality / Number of examined foetuses) x 100
           
4) Skeletal abnormalities/litter (%, group mean): (Number of foetuses with abnormality / Number of examined foetuses) x 100
Historical control data:
Historical control data is provided in Appendix 9 of the final study report.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Piloerection was observed in 1 out of 23 animals in the Mid dose group, and 11 out of 24 animals in the High dose group. Laboured or noisy respiration was present in 1 out of 23 animals in the Mid dose and 3 out of 24 animals in the High dose group. These effects were considered to be treatment-related.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One mortality was observed on Day 10 (Low dose female). Although no specific reason for death was identified at necropsy, the absence of evidence of any dose related systemic toxicity in the study or in this animal strongly suggests that the death was not treatment related.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There was no statistically significant lower body weight or lower overall weight gains observed in any dose group. A slightly reduced body weight gain was observed in the high dose group. A slight but statistically significant lower corrected body weight gain, and net body weight gain was observed in animals in the high dose group (Day10-12) only. No treatment-related effect on the body weight parameters were observed in animals in the low- or mid- dose group when compared to controls.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Statistically significant treatment-related reduction of the overall food consumption (Day 6-20) was observed in the high dose group animals.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Endocrine findings:
no effects observed
Description (incidence and severity):
There were no statistically significant changes in the concentration of the T3, T4 and TSH level between the control and the treated groups.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no statistical differences in the thyroid and parathyroid organ weights between the control and the dose groups.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Although the cause of death of the low dose female (animal# 2511) was not identified, there was no evidence for any adverse toxic effect observed in this animal, and no indications of any systemic toxicity in other animals that could suggest any relationship between the death and treatment with the test material.

Thickness of the non-glandular region of the stomach was observed in 2 out of 23 animals in the mid dose group and 23 out of 24 animals in the high dose group at terminal necropsy. This indicates a local irritant effect of the test material.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Histopathological examination did not reveal any statistical differences between the control and the dose groups.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
Mean value of the implantation number in the high dose group was statistically significantly higher (p<0.05) compared to the control group but within the historical control range.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
effects observed, treatment-related
Description (incidence and severity):
Mean number of early embryonic loss and of dead foetuses were outside of the historical control range for the high dose group. Mean number of viable foetuses was out of the historical control range in case of the control and low dose group.
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
The number of confirmed pregnant, evaluated dams was 24 in the control and the high dose groups, 23 in the low- and mid- dose groups.
Other effects:
no effects observed
Description (incidence and severity):
No abnormalities were observed on the placentas of any animals in the control, low-, mid-, or high-dose groups.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
ca. 300 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean foetal weight per litter in the high dose group was statistically significantly decreased (p<0.01) compared to the control but within the historical control range.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
effects observed, treatment-related
Description (incidence and severity):
Number of the low dose male foetuses was statistically and significantly lower than the control group and therefore the number of the low dose female foetuses was statistically significantly higher compared to the control group.
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
A statistically significant lower mean foetal body weights/litter value was recorded in the high dose group (p<0.01). This was out of the historical control range (mean ± SD 3.36 ± 0. 23) and therefore, considered as an adverse effect of the test material.
Anogenital distance of all rodent fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
A statistically significant decrease (p<0.05) between the mean anogenital distances of the litters in case of low dose males was observed but the data were within the historical control range (15) and there was no dose response. It was concluded that there was no treatment-related effect on anogenital distance.
Changes in postnatal survival:
no effects observed
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Malformation/s observed in the control group (Jaw, lower, absent; Eye protruding), in the low dose group (Subcutaneous Oedema, Generalized) and variation in the mid dose group (Subcutaneous Oedema, Localized) were considered to be incidental findings, not related to the treatment. There were no such findings in the high dose group.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Most of the skeletal findings correspond with the current historical control (HC) or the concurrent study control data, or were considered to be incidental findings without dose response. Based on the skeletal findings, the number of malformed / intact foetuses were comparable with the control in the low-, mid-, and high-dose groups. The number of variations were higher in all treated groups.

The only finding where a possible increase was observed was Unossified Sternebra. The difference was not statistically significant is of equivocal association with treatment. This finding is commonly associated with maternal body weight or food intake which was seen in the high dose dams. In this context, since slight maternal toxicity is normally accompanied by slightly retarded ossification, the observation may be related to treatment, but was not considered to represent a direct effect of test material on foetal development.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
All the visceral findings were consistent in general nature and incidence with the concurrent study control data / existing historical control data or showed incidental occurrence, therefore considered as incidental findings. Visceral findings, the number of malformed / variant / intact foetuses were comparable with the control in the low-, mid-, and high-dose groups and comparable to the historical control data.
Other effects:
not examined

Effect levels (fetuses)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Foetotoxicity
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 600 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Teratogenicity
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 600 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Embryotoxicity

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
600 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
not specified

Any other information on results incl. tables

The mean concentration of all formulations was found to be in the range of 94-100% of their nominal concentrations (30, 60,and 120 mg/mL). No test material was detected in the vehicle control formulations.

Table 2. Summary of the dose formulation analysis

Formulation

03 March 2020

27 March 2020

Mean measured concentration (mg/mL)

Relative
to the nominal
concentration (%)

Measured concentration (mg/mL)

Relative
to the nominal
concentration (%)

Control

not detected

-

not detected

-

30 mg/mL

28.2 ± 1.85

94

29.7 ± 0.435

99

60 mg/mL

59.7 ± 0.44

100

60.3 ± 0.493

100

120 mg/mL

115.3 ± 1.36

96

118.6 ± 5.375

99

Note: Samples collected freshly on the days indicated in the header of the table.

Table 3. Selected body weight parameters

Parameters

Historical control

Dose (mg/kg bw/day)

 

0

150

300

600

Number of evaluated dams

507

24

23

23

24

 

Body weight gain GD 6-20 (g)

83.1±14.1

83.7

84.3

82.7

75.1

NS

Corrected body weight gain GD0-20 (g)

46.1±11.8

53.6

54.5

56.6

45.0#

DN

Net body weight gain (g)

24.9±9.8

30.1

31.0

29.1

22.0#

DN

Notes: Historical control data is mean ± 1SD. Body weight data were rounded to one decimal place.

Corrected and net weight / weight gains refer to body weight values minus the weight of the gravid uterus.

DN: #= p<0.05; ##= p<0.01; Dunnett two sided test,

NS: Not significant

Table 4. Food Consumption Diagram - Female Animals

 

 

Day(s) Relative to Start Date

0 to 3

3 to 6

6 to 8

8 to 10

10 to 12

12 to 14

14 to 16

16 to 18

18 to 20

0

mg/Kg bw/day

Mean

16.75

20.03

18.56

19.94

22.92

22.02

22.90

23.75

21.90

N

24

24

24

24

24

24

24

24

24

 

150

mg/Kg bw/day

Mean

17.30

20.80

19.85

20.59

22.41

22.04

23.11

25.11

22.26

N

23

23

23

23

23

23

23

23

23

 

300

mg/Kg bw/day

Mean

17.04

21.10

19.61

21.26

22.20

21.48

23.17

25.41

22.76

N

23

23

23

23

23

23

23

23

23

 

600

mg/Kg bw/day

Mean

17.21

20.04

17.44

17.19

18.69

18.44

21.13

24.00

22.44

N

24

24

24

24

24

24

24

24

24

Table 5. Summary of Thyroid Hormone Concentrations

Sex: Female

 

Group 1

0 mg/Kg bw/day

Group 2

150 mg/Kg bw/day

Group 3

300 mg/Kg bw/day

Group 4

600 mg/Kg bw/day

Adult

T3 Conc.

(ng/mL)

Mean

0.2746 L+

0.2784

0.2771

0.2720

SD

0.0503

0.0496

0.0515

0.0495

Max

0.370

0.362

0.415

0.370

Min

0.200

0.200

0.200

0.200

N

24

23

23

24

% diff

-

1.4

0.9

-1.0

 

Adult

T4 Conc.

(ng/mL)

Mean

17.30 I+

18.19

16.98

17.98

SD

3.49

4.25

3.68

3.66

Max

26.4

28.2

23.4

26.2

Min

11.2

11.9

9.8

12.5

N

24

23

23

24

% diff

-

5.1

-1.9

3.9

 

Adult

TSH.

(pg/mL)

Mean

330.9 R+

365.0

411.3

511.8

SD

313.1

310.6

334.5

469.5

Max

1116

1161

1173

1736

Min

120

120

120

120

N

24

23

23

24

% diff

-

10.3

24.3

54.7

L: Automatic Transformation: Log

I: Automatic Transformation: Identity (No Transformation)

R: Automatic Transformation: Rank

Table 6. Summary of Necropsy Data (Females)

 

Group 1

0 mg/Kg bw/day

Group 2

150 mg/Kg bw/day

Group 3

300 mg/Kg bw/day

Group 4

600 mg/Kg bw/day

Evaluated Animals

Number of Animals:

24

23

23

24

Number of Completed Animals:

24

23

23

24

Stomach

 

  Submitted

0

0

2

23

  Thickness; diffuse, non-glandular region; mucosa

-

-

2

23

 

Thyroid Gland + Parathyroid Gland

 

  Submitted

24

23

23

23

  Normal

24

23

22

24

  Small; bilateral

0

0

1

0

Non-pregnant Animals

Number of Animals:

-

-

1

-

Number of Completed Animals:

-

-

1

-

Thyroid Gland + Parathyroid Gland

 

  Submitted

-

-

1

-

  Normal

-

-

1

-

Found Dead Animals

Number of Animals:

1

-

-

-

Number of Completed Animals:

1

-

-

-

Lungs

 

  Submitted

1

-

-

-

  Discoloration; dark red, diffuse, all lobes

1

-

-

-

  Non collapsed

1

-

-

-

 

Thyroid Gland + Parathyroid Gland

 

  Submitted

1

-

-

-

  Normal

1

-

-

-

Table 7. Summary of Organ Weight Data

Sex: Female

 

Group 1

0 mg/Kg bw/day

Group 2

150 mg/Kg bw/day

Group 3

300 mg/Kg bw/day

Group 4

600 mg/Kg bw/day

Terminal Body Weight

(g)

Mean

314.7 I1

317.5

316.5

303.5

SD

19.2

22.9

17.3

22.1

Max

358

372

346

337

Min

281

287

275

253

N

24

23

23

24

% diff

-

0.9

0.6

-3.6

 

Thyroid / Parathyroid

(g)

Mean

0.0199 I1

0.0202

0.0202

0.0198

SD

0.0035

0.0028

0.0029

0.0031

Max

0.027

0.025

0.025

0.025

Min

0.011

0.016

0.014

0.015

N

24

23

23

24

% diff

-

1.7

1.5

-0.2

 

Thyroid / Parathyroid / BW

(%)

 

Mean

0.00633 I1

0.00638

0.00638

0.00654

SD

0.00113

0.00089

0.00089

0.00094

Max

0.0085

0.0080

0.0076

0.0088

Min

0.0039

0.0048

0.0046

0.0050

N

24

23

23

24

% diff

-

0.9

0.9

3.3

1 [I - Automatic Transformation: Identity (No Transformation)]

Table 9. Summary of Intra-uterine Evaluation

Parameters

Dose (mg/kg bw/day)

HCD data

(min-max)

 

0

150

300

600

-

Number of evaluated dams

24

23

23

24

671

 

Mean number of corpora lutea

11.54

11.26

11.08

12.38

10.4-12.8

NS

Pre-implantation loss, mean

1.83

1.78

1.61

1.54

0.2-2.3

NS

Pre-implantation loss (%), mean

14.69

14.74

13.68

11.24

1.8-17.1

NS

Mean number of implantations

9.71

9.48

10.04

10.83+

9.8-11.7

D

Early embryonic loss, mean

0.58

0.39

0.43

0.88

0.0-0.8

NS

Early embryonic loss (%), mean

5.69

3.85

4.68

7.59

0.5-7.1

NS

Late embryonic loss, mean

0.00

0.00

0.00

0.08

0.0-0.3

NS

Late embryonic loss (%), mean

0.00

0.00

0.00

0.80

0.0-3.2

NS

Dead foetuses, mean

0.00

0.00

0.04

0.13

0.0-0.1

NS

Dead foetuses (%), mean

0.00

0.00

0.54

1.17

0.0-1.0

NS

Post-implantation loss, mean

0.58

0.39

0.48

1.08

0.1-1.1

NS

Post-implantation loss (%), mean

5.69

3.85

5.23

9.55

1.2-11.0

NS

Total intra-uterine mortality, mean

2.42

2.17

2.09

2.63

0.3-3.1

NS

Total intra-uterine mortality (%), mean

19.43

18.26

18.03

19.99

3.0-23.1

NS

Viable foetuses, mean

9.13

9.09

9.57

9.75

9.2-11.5

NS

Notes: Most important parameters are shown in bold.

NS: Statistically not significant when compared to the vehicle control.

D: += p<0.05; ++= p<0.01; Duncan multiple range test.  

Table 10. Examination of Viable Foetuses

Parameters

Dose (mg/Kg bw/day)

HCD data

(min-max)

 

0

150

300

600

-

Number of examined litters

24

23

23

24

671

 

Viable foetuses, mean

9.13

9.09

9.57

9.75

9.2-11.5

NS

Male foetuses, mean

5.08

3.65+

4.57

4.50

4.4-5.9

D

Female foetuses, mean

4.04

5.43+

5.00

5.25+

4.5-6.1

D

Total number of foetuses

219

209

220

234

-

NS

Total number of male foetuses

122

84**

105

108*

-

CH2

Total number of female foetuses

97

125**

115

126*

-

CH2

Sex distribution (% of males / females)

56/44

40/60**

48/52

46/54

-

D

Mean foetal weight / litter (g)

3.324

3.366

3.228

3.098**

2.73-3.57

D

Number of affected litters (with runts)

6

3

6

11

0-5

NS

Notes: Most important parameters are shown in bold.

NS: Statistically not significant when compared to the vehicle control.

D: += p<0.05; ++= p<0.01; Duncan multiple range test

CH2: #= p<0.05; ##= p<0.01 Chi square test

Runt: a foetus is considered to be a runt (growth-retarded) when their body weight is less than the control average weight – 2 standard deviations)

Table 11. Summary of Abnormalities

Parameter

Dose (mg/Kg bw/day)

0

150

300

600

External abnormalities

Total number of examined litters

24

23

23

24

Total number of examined foetuses

219

209

220

234

Total number of intact (normal) foetuses

218

208

219

234

Total number of foetuses / litters with malformation

1 / 1

1 / 1

0 / 0

0/ 0

Total number of foetuses / litters with variation

0 / 0

0 / 0

1 / 1

0 / 0

Visceral abnormalities

Total number of examined litters

24

23

23

24

Total number of examined foetuses

110

103

110

118

Total number of intact (normal) foetuses

105

101

107

118*CH

Total number of foetuses / litters with malformation

0 / 0

0 / 0

1 / 1

0 / 0

Total number of foetuses / litters with variation

5 /5

2 / 2

2 / 2

0 / 0*CH

Skeletal abnormalities

Total number of examined litters

24

23

23

24

Total number of examined foetuses

109

106

110

116

Total number of intact (normal) foetuses

103

98

102

105

Total number of foetuses / litters with malformation

1 / 1

0 / 0

1 / 1

1 / 1

Total number of foetuses / litters with variation

5 / 4

8 / 4

7 / 5

10 / 7

Notes:

CH2: *= p<0.05; **= p<0.01; Chi Square test

Numbers represent the number of abnormalities / number of affected litters.

No statistically significant differences were noted when compared to the control group (for External and Skeletal abnormalities)

Table 12. Details of Visceral Abnormalities

Parameter

Dose (mg/Kg bw/day)

HC data

0

150

300

600

Total number of examined litters

24

23

23

24

507

Total number of examined foetuses

110

103

110

118

2608

Visceral malformations

Situs Inversus, Total;

Lung abnormal lobation

Litter
incidence

n

0

0

1

0

4

%

0.0

0.0

4.3

0.0

0.60

Foetal
incidence

n

0

0

1

0

4

%

0.000

0.000

0.909

0.000

0.058

Visceral variations

Brachiocephalic trunk, short

Litter
incidence

n

1

0

0

0

33

%

4.2

0.0

0.0

0.0

4.93

Foetal
incidence

n

1

0

0

0

35

%

0.909

0.000

0.000

0.000

1.021

Ureter convoluted

Litter
incidence

n

3

0

0

0

10

%

12.5

0.0

0.0

0.0

1.49

Foetal
incidence

n

3

0

0

0

10

%

2.727

0.000

0.000

0.000

0.292

Thymic cord

Litter
incidence

n

1

2

2

0

66

%

4.2

8.7

8.7

0.0

9.85

Foetal
incidence

n

1

2

2

0

77

%

0.909

1.942

1.818

0.000

2.246

Notes: Numbers represent the number (n) or ratio (%) of abnormalities.

HC: historical control (data provided where considered useful)

No statistically significant differences were noted when compared to the control group

 

Table 13. Details of Skeletal Abnormalities

Parameter

Dose (mg/Kg bw/day)

HC data

0

150

300

600

Total number of examined litters

24

23

23

24

671

Total number of examined foetuses

109

106

110

116

3420

Skeletal Malformations

Mandible, premaxilla absent, nasal fused

Litter
incidence

n

1

0

0

0

--

%

4.2

0.0

0.0

0.0

--

Foetal
incidence

n

1

0

0

0

--

%

0.917

0.000

0.000

0.000

--

Limb Cartilages, fused (Scapula Acromion Process with Humerus Greater Tuberosity)

Litter
incidence

n

0

0

1

0

--

%

0.0

0.0

4.3

0.0

--

Foetal
incidence

n

0

0

1

0

--

%

0.000

0.000

0.909

0.000

--

Rib, branched

Litter
incidence

n

0

0

0

1

2

%

0.0

0.0

0.0

4.2

0.30

Foetal
incidence

n

0

0

0

1

2

%

0.000

0.000

0.000

0.862

0.058

Notes: Numbers represent the number (n) or ratio (%) of abnormalities.

HC: historical control (data provided where considered useful)

No statistically significant differences were noted compared to the control group

--: No data.

Applicant's summary and conclusion

Conclusions:
Based on the results observed, the NOAEL for maternal toxicity was determined to be 300 mg/Kg bw/day, based on a significant decrease in corrected body weight and reduced food consumption observed at 600 mg/Kg bw/day. The NOAEL for embryotoxicity was determined to be 600 mg/Kg bw/day, based on the lack of any treatment-related intrauterine effect observed at the highest dose tested. The NOAEL for foetoxicity was determined to be 300 mg/Kg bw/day, based on significant treatment-related lower foetal body weight/litter at 600 mg/Kg bw/day. The NOAEL for teratogenicity was determined to be 600 mg/Kg bw/day, based on the lack of treatment-related malformations observed at the highest dose tested.
Executive summary:

A key OECD Guideline 414 pre-natal developmental toxicity study was conducted to assess the effects of the test material ((2E)-2-methyl-3-phenylacrylaldehyde) on the embryonic and foetal development (including the organogenesis period) of Hannover Wistar rats. 24 female rats (per dose) were administered the test material once daily by oral gavage at doses of 0, 150, 300, or 600 mg/Kg bw/day, from gestation day 6 (GD 6) up to and including gestation day 19 (GD 19). Sperm positive day was counted as day 0 of pregnancy (GD 0) and control dams were treated with the vehicle (PEG 400) only.

 

Parameters monitored during the study included mortality and clinical observations, body weight, body weight gain, and individual food consumption. Maternal reproductive parameters associated with uterine examination were evaluated, and placentas were examined macroscopically. Caesarean sections, necropsy of dams and examination of uterine contents were performed on GD 20. The foetuses were weighed and examined for external, visceral and skeletal abnormalities.

 

One mortality (low-dose female) was observed during the study on Day 10 but not considered to be treatment-related. Piloerection was observed in 1 out of 23 animals in the mid-dose group, and 11 out of 24 animals in the high-dose group. Laboured or noisy respiration was present in 1 out of 23 animals in the mid-dose and 3 out of 24 animals in the high-dose group. These effects were considered to be treatment-related.Aslight but statistically significantly lower corrected body weight gain was observed in animals in the high-dose group. However, the overall body weight gain was not

significantly different from control when not corrected for uterus weight. Statistically significant treatment-related reduction of food consumption was also observed in the high-dose group during the treatment period. There were no statistically significant changes observed in the concentration of the T3, T4 and TSH level between the control and treated animals.

 

No statistical differences in the thyroid and parathyroid organ weights were observed between the control and the dose groups. Gross necropsy revealed thickness of the non-glandular region of the stomach in 2 of 23 animals in the mid-dose and in 23 of 24 animals in the high-dose group, indicating local irritation following oral exposure to the test material. Histopathological examination did not reveal any statistical differences between the control and treated groups. Treatment-related statistically significant differences in intra-uterine parameters were not observed in treated animals when compared to the controls.

 

A significant difference in the sex distribution of foetuses was observed between the control and low- and high-dose group animals and there was a significant difference on anogenital distance in the low- dose group compared to the control. However, all data were within the historical control range of the laboratory. Foetal weight/litter in the high-dose group was statistically and significantly decreased when compared to the control, although not outside the historical control range. This weight reduction was considered to be an adverse treatment-related effect (possibly secondary to maternal toxicity). There was no statistically significant increase in number of litters with runts (limit for growth-retarded

foetuses) in any dose group. There were no treatment-related effects on external or skeletal development of foetuses observed through the study period. The number of visceral variations was higher in the treated groups when compared to the controls, reaching statistical significance in the high-dose group. These were ascribed to maternal toxicity. Foetal malformations observed in the study were all considered to be incidental as they showed no dose dependency and were therefore, not regarded as treatment-related.

 

Based on the results observed, the NOAEL for maternal toxicity was determined to be 300 mg/Kg bw/day, based on a significant decrease in corrected body weight and reduced food consumption observed at 600 mg/Kg bw/day. The NOAEL for embryotoxicity was determined to be 600 mg/Kg bw/day, based on the lack of any treatment-related intrauterine effect observed at the highest dose tested. The NOAEL for foetoxicity was determined to be 300 mg/Kg bw/day, based on significant treatment-related lower foetal body weight/litter at 600 mg/Kg bw/day. The NOAEL for teratogenicity was determined to be 600 mg/Kg bw/day, based on the lack of treatment-related malformations observed at the highest dose tested.