Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2019-12-17 to 2020-01-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Dose range-finding toxicity study conducted to select appropriate dose levels for an OECD Guideline 414 developmental toxicity study in rats.
GLP compliance:
no
Remarks:
Dose range-finding toxicity study
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:
Substance Name: (2E)-2-methyl-3-phenylacrylaldehyde
EC No.: 701-219-0
Batch/Lot No.: A190809B
Appearance: Clear yellow liquid
Purity: 97%
Expiry date: 12 August 2021
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: Emerald Performance Materials (1499 SE Tech Center Place, Suite 300 Vancouver, WA 98683); Batch/Lot No.: A190809B
- Expiration date of the lot/batch: 2021-08-12
- Purity test date: 2019-08-13

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 under storage conditions: stable for at least five days at room temperature
- Stability under test conditions: Based on the results from the stability assessment from the analytical validation study (19/205-316AN [3]), the test item was shown to be stable in the vehicle for at least five days at room temperature

FORM AS APPLIED IN THE TEST (if different from that of starting material) : Liquid

OTHER SPECIFICS
-Appearance: Clear yellow liquid
-Purity: 97%

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Hannover Wistar, 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 female rats, nulliparous and non-pregnant, at least 15 weeks old at mating.
- Weight at study initiation: 202-243 g
- Fasting period before study: Not specified
- Housing: Standard laboratory conditions; individual housing (Type II polycarbonate cages used during mating and gestation period and Type III polycarbonate cages used during the acclimatisation period). Successfully mated animals were housed individually. Deep wood sawdust was use as bedding to allow digging and other normal rodent activities. Nest building material was also added into the cages.
- Diet (e.g. ad libitum): ssniff® SM Autoclavable Complete Diet for Rats/Mice – Breeding and Maintenance (Batch number: 746 55782, Expiry date: 30 April 2020, (Batch number: 232 58297, Expiry date: 30 July 2020 Ssniff Spezialdiäten GmbH, D-59494 Soest, Germany) ad libitum
- Water (e.g. ad libitum): tap water (in water bottles) ad libitum
- Acclimation period: at least 23 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.9-23.7°C (target: 22 ± 3°C)
- Humidity (%): 24-55% (target: 30-70%)
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light):

IN-LIFE DATES: From: 2019-11-21 To 2020-01-12:

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Remarks:
PEG 400
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test material was formulated in the vehicle (PEG 400) at appropriate concentrations according to the dose level and volume selected, in the Pharmacy of Charles River Laboratories Hungary Kft. Formulations were prepared at the appropriate frequency to allow their use according to stability assessment from the analytical validation study (19/205-316AN [3]). Please see Table 1 (experimental design) in the section 'any other information on materials and method incl. tables'.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on the Sponsor’s information and previous studies, PEG 400 was selected as the vehicle for this study in agreement with the Sponsor.
- Concentration in vehicle: 0, 25, 50, 100, and 150 mg/mL for the control, low-, mid 1-, mid 2-, and high-dose groups, respectively.
- Amount of vehicle (if gavage): 5 mL/Kg
- Lot/batch no. (if required): BCBZ1233
- Purity: Not specified
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of test item formulations for concentration and homogeneity was performed at in the Analytical Laboratory of Charles River Laboratories Hungary Ltd. (Study code: 19/205-316AN [3]). Duplicate samples were taken from the test item formulations once during the study. Similarly, duplicate samples were taken from the vehicle control formulation for concentration measurement. Acceptance criteria of the concentration analysis was 100 ± 15% of the nominal concentration.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused: females were paired according to their oestrus cycle with males in the morning for approximately 2-3 hours (1 male - 1 female) until at least 6 sperm positive females/group were attained
- M/F ratio per cage: 1:1
- Length of cohabitation: approximately 2-3 hours (1 male - 1 female) until at least 6 sperm positive females/group were attained
- 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
Duration of treatment / exposure:
Daily from gestation day 6 (GD 6) to gestation day 19 (GD 19)
Frequency of treatment:
Once daily
Duration of test:
From gestation day 6 (GD 6) to gestation day 19 (GD 19)
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Remarks:
Control
Dose / conc.:
125 mg/kg bw/day
Remarks:
Low Dose
Dose / conc.:
250 mg/kg bw/day
Remarks:
Mid Dose 1
Dose / conc.:
500 mg/kg bw/day
Remarks:
Mid Dose 2
Dose / conc.:
750 mg/kg bw/day
Remarks:
High Dose
No. of animals per sex per dose:
6 mated females/group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were set by the Sponsor in agreement with the Study Director based on the available data, including the results of a 14-day dose range finding study (DRF) in non-pregnant rats (19/205-209PE), where >50% mortality was observed at 1000 mg/Kg/day, with the aim of inducing toxic effects but no death or suffering at the highest dose (750 mg/Kg bw/day). The remaining dose levels (500, 250, 125 mg/Kg bw/day) were spaced with a factor of 2.

- Rationale for animal assignment (if not random): Sperm-positive, assumed pregnant females were allocated to the experimental groups (on each mating day) in such a way that the group averages of the body weight were as similar as possible. Provantis software was used to verify homogeneity/variation among/within groups.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Cage-side (general) clinical observations were made twice daily (at the beginning and end of each working day) and once before necropsy (in the morning).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each working day). Animals were monitored for any changes including pertinent behavioural changes and signs of toxicity including 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), 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” (intrauterine 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.

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 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): Not specified

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
Before expected delivery, on GD 20, Caesarean section was performed on each treated dam. Sodium pentobarbital (Euthanimal, 400 mg/mL sodium pentobarbital solution; Supplier: Alfasan Nederland B.V., The Netherlands; Batch number: 1811347-03, Expiry date: 31 December 2021) administered by intramuscular injection and followed by exsanguination was used for euthanasia.
- Organs examined: The dams’ viscera were examined macroscopically for any structural abnormalities or pathological changes.
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

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. The number of corpora lutea in each ovary and implantation sites in each uterine horn, the number of live foetuses, early and late embryonic death and foetal death were counted, the number and percentage of pre- and post-implantation losses were calculated. The degree of resorption was described in order to estimate the relative time of death of the conceptus. The placentas were examined macroscopically.
Fetal examinations:
- External examinations: Yes: [all per litter]
Each foetus was weighed individually (accuracy ±0.01 g) and subjected to external examination. The gender of foetuses was determined according to the appearance of the anogenital distance. All external abnormalities found during the foetal examinations were recorded and photographic records made.
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No
Statistics:
Please see section 'any other information on materials and methods incl. tables' for information on statistical analysis.
Indices:
Maternal Data:
1) Preimplantation loss (%, group mean):
(Number of corpora lutea - Number of implantations / Number of corpora lutea) x 100

2) Post-implantation loss (%, group mean):
(Number of implantations - Number of live foetuses / Number of implantations) x 100

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

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Transient signs of noisy respiration and piloerection were seen in the 250, 500, and 750 mg/Kg bw/day dose groups, with no clear dose-response. These observed, non-specific clinical signs were condidered to most probably be due to the treatment, or incidental findings, but were not considered as a systemic adverse treatment-related effect.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
One High dose group animal (5504) was found dead on GD 9. On the previous day, slight noisy respiration and piloerection was present in the animal. This death was considered to be treatment-related.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Lower body weights, not reaching statistical significance, were observed in females in the 750 mg/Kg bw/day dose group. While the High dose gained weight normally during the study, a slight overall body weight difference (approximately 4% lower when compared to the Control) was observed. Consequently, a body weight effect in the 750 mg/Kg bw/day dose group was considered to be treatment-related.

The observed lower body weight parameters in the 125 mg/Kg bw/day dose group were considered to be incidental findings due to the individual variety and lownumber of evaluated animals.

No treatment-related effect on body weight was observed in the 250 and 500 mg/Kg bw/day dose groups when compared to control and no treatment-related effect on the body weight gain parameters were observed in the study.

Due to a technical error, incorrect body weight data was measured on GD 0 for animal number 5506 (750 mg/Kg bw/day dose group). The data were not used for dose volume administration, so it had no consequences on the conduct of the study. This data was outside the historical control range and showing significant difference from the study average for GD 0.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Lower food consumption, not reaching statistical significance, was observed in the 750 mg/Kg bw/day dose group, and was considered to be treatment-related.

No treatment-related effects were observed on food consumption in the rest of the dose groups through the study period.
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
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Thickening of the non-glandular mucosal region (diffuse or multifocal) was observed in 2 animals out of 5 in the 250 mg/Kg bw/day dose group and in all animals in the 500 and 750 mg/Kg bw/day dose groups. These local findings were considered to be treatment-related.

No treatment-related change was found in the High dose group animal (5504) found dead on GD 9. The observed changes were considered to be post-mortem changes.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
No significant differences were observed between the treated groups and the control animals.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
No significant differences were observed between the treated groups and the control animals.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
No significant differences were observed between the treated groups and the control animals.
Early or late resorptions:
no effects observed
Description (incidence and severity):
No significant differences were observed between the treated groups and the control animals.
Dead fetuses:
no effects observed
Description (incidence and severity):
No significant differences were observed between the treated groups and the control animals.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Thirty females (6 for each group) were mated in the study. The number of confirmed pregnant, evaluated dams was 6 in the Control, 3 in the 125, 5 in the 250, 6 in the 500, and 5 in the 750 mg/Kg bw/day dose groups, respectively.
Other effects:
not examined

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
mortality
other: Systemic Toxicity

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean foetal weight per litter was significantly lower in the 750 mg/Kg bw/day dose group, and comparable to the control mean value in other treated groups. The reduced foetal body weight was considered to be a secondary effect due to the observed maternal toxicity (lower food consumption and lower body weight parameters) in the high-dose group dams.
Reduction in number of live offspring:
effects observed, non-treatment-related
Description (incidence and severity):
Due to the difference in the number of evaluated animals, statistically significant differences were observed in the total number of foetuses (Low and High dose group), and the total number of male and female foetuses in the High dose group. These differences were considered to be incidental, and not treatment-related. The mean number of viable foetuses was comparable with the control mean in all treated groups.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There was no toxicologically significant difference in the sex distribution of foetuses between the control and treatment groups.
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
Mean foetal weight per litter was significantly lower in the 750 mg/Kg bw/day dose group, and comparable to the control mean value in other treated groups. The reduced foetal body weight was considered to be a secondary effect due to the observed maternal toxicity (lower food consumption and lower body weight parameters) in the high-dose group dams.
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
The mean number of viable foetuses was comparable with the control mean in all treated groups.
External malformations:
no effects observed
Description (incidence and severity):
No external abnormalities were observed in any of the treated groups when compared with the controls.
Skeletal malformations:
not examined
Visceral malformations:
not examined
Other effects:
no effects observed
Description (incidence and severity):
The number of retarded foetuses were comparable between the Control and treated groups and no abnormalities were observed in the placenta in any of the study groups.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

Table 2. Selected body weight parameters

Parameters

Dose (mg/Kg bw/day)

0

125

250

500

750

Number of evaluated dams

6

3

5

6

5

Body weight on GD20 (g)

316.2

310.0

319.6

317.0

292.2

Body weight gain GD6-20 (g)

72.8

71.0

78.6

67.5

62.2

Body weight gain GD0-20 (g)

89.5

91.3

97.2

88.8

83.0

Corrected body weight on GD20 (g)

266.3

252

258.8

259.7

237.2

Corrected body weight gain GD0-20 (g)

39.7

33.3

36.4

31.5

27.3

Net body weight gain GD6-20 (g)

23

13

17.8

10.2

7.2

Notes: 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.

 

Table 3. Summary of pregnancy data

Parameters

Dose (mg/Kg bw/day)

0

125

250

500

750

Number of mated females

6

6

6

6

6

Pre-terminal death or euthanasia

0

0

0

0

1

Number of non-pregnant females

0

3

1

0

0

Number of females with ≤ 5 implantation sites

0

0

0

0

0

Number of evaluated females on GD20 (Caesarean section)

6

3

5

6

5

 

Table 4. Summary of the intrauterine evaluation

Parameters

Dose (mg/Kg bw/day)

0

125

250

500

750

Number of evaluated dams

6

3

5

6

5

Mean number of corpora lutea

12.00

5.17

10.67

12.33

11.20

Preimplantation loss, mean

1.83

0.33

0.80

1.67

0.40

Preimplantation loss (%), mean

14.32

3.70

5.95

12.85

3.08

Mean number of implantations

10.17

10.00

12.00

10.67

10.80

Early embryonic loss, mean

1.33

0.00

1.20

0.50

0.20

Early embryonic loss (%), mean

10.99

0.00

1.20

0.50

0.20

Late embryonic loss, mean

0.17

0.00

0.00

0.00

0.00

Late embryonic loss (%), mean

1.85

0.00

0.00

0.00

0.00

Dead foetuses, mean

0.00

0.00

0.00

0.00

0.20

Dead foetuses (%), mean

0.00

0.00

0.00

0.00

1.67

Postimplantation loss, mean

1.50

0.00

1.20

0.50

0.40

Postimplantation loss (%), mean

12.84

0.00

10.17

4.99

3.48

Total intrauterine mortality, mean

3.33

0.33

2.00

2.17

0.80

Total intrauterine mortality (%), mean

24.89

3.70

15.24

16.91

6.28

Viable foetuses, mean

8.67

10.00

10.80

10.17

10.40

Notes: Most important parameters are shown in bold.

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

NA: Not applicable

DN = Duncan's multiple range test; ** = p< 0.01

n=6 for Corpora lutea, but the Number of evaluated dams applied to the other parameters

 

Table 5. Examination of viable foetuses

Parameters

Dose (mg/Kg bw/day)

0

125

250

500

750

Number of examined litters

6

3

5

6

5

Viable foetuses, mean

8.67

10.00

10.80

10.17

10.40

Number of male foetuses, mean

3.17

3.33

4.40

5.00

5.80

Number of female foetuses, mean

5.50

6.67

6.40

5.17

4.60

Total number of foetuses

52

30*

54

61

52*

Total number of male foetuses

19

10

22

30

29*

Total number of female foetuses

33

20

32

31

23*

Sex distribution (% of males / females)

38/62

32/68

41/59

47/53

56/44

Mean foetal weight / litter (g)

3.266

3.497

3.365

3.275

3.017*

Number of foetuses with retarded body weight

1

0

0

1

1

Number of affected litters (with runts)

1

0

0

1

1

Notes: Most important parameters are shown in bold, ‘per litter’ parameters mainly used in evaluation.

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

NA: Not applicable

CH2: Chi Square Test; DN: Duncan's multiple range test; * =p<0.05

Applicant's summary and conclusion

Conclusions:
(2E)-2-methyl-3-phenylacrylaldehyde, when administered daily by oral gavage to pregnant Hannover Wistar rats from gestation days GD6 to GD19 up to 750 mg/Kg bw/day, was associated with maternal toxicity (mortality and slight, local effects in the stomach at 750 mg/Kg bw/day; and slight, local effect in the stomach at 250 and 500 mg/Kg bw/day). Although a slight secondary toxic effect was observed on the body weight of the pups at the 750 mg/Kg bw/day dose group, no foetotoxicity effect was observed in the rest of the animals (up to 500 mg/Kg bw/day). No embryotoxicity or teratogenicity effect was observed in this study. Therefore, a dose between the 500 and 750 mg/Kg bw/day (i.e. 600 mg/Kg body weight/day) was determined to be suitable as the high dose for the OECD Guideline 414 developmental toxicity study in rats.
Executive summary:

In a supporting dose range-finding toxicity study conducted to select appropriate dose levels for an OECD Guideline 414 developmental toxicity study in rats, the test material ((2E)-2-methyl-3-phenylacrylaldehyde; CAS# 15174-47-7) was administered once daily by oral gavage to rats (6 female Hannover Wistar, Crl:WI(Han / dose group) from Gestation Day 6 (GD6) up to and including GD19 (sperm positive day = day 0 of pregnancy, GD0) at doses of 0, 125, 250, 500, or 750 mg/Kg bw/day.

 

Parameters monitored during the study included mortality and clinical observations, body weight, body weight gain and individual food consumption. Gross macroscopic examination was performed at necropsy of dams (Caesarean sections) and examination of uterine contents were performed on gestation day 20 (GD20). Maternal reproductive parameters associated with uterine examination were evaluated, and the foetuses were weighed and examined for external abnormalities. Placentae were also examined macroscopically. Thirty sperm positive females were included in the study, at least six in each group. The number of confirmed pregnant dams was 6, 3, 5, 6, and 5 in the control (0), 125, 250, 500, and 750 mg/Kg bw/day dose groups, respectively.

 

One animal in the 750 mg/Kg bw/day dose group was found dead on GD 9, following slight noisy respiration and piloerection on the previous day. This death, consistent with mortalities in the DRF

study with non-pregnant rats (19/205-209PE), was considered to be treatment-related but no treatment-related changes were observed in this animal.

 

Transient signs of noisy respiration and/or piloerection were seen in the 250, 500, and 750 mg/Kg bw/day dose groups, albeit without a clear dose-response. These observed, non-specific clinical signs were determined to most probably be due to the treatment procedure, or incidental findings, and were not considered as an adverse treatment-related effect.

 

Lower body weights, not reaching statistical significance, were observed in females in the 750 mg/Kg bw/day dose group. Similarly, lower food consumption, not reaching statistical significance, was observed in the same dose group. These changes were considered to be treatment-related. Similar effects on body weight or food consumption parameters were not observed in the 125, 250, or 500 mg/Kg bw/day dose groups.

 

Thickening of the non-glandular mucosal region (diffuse or multifocal) was observed in 2 animals out of 5 in the 250 mg/Kg bw/day dose group and in all animals in the 500 and 750 mg/Kg bw/day dose

groups. These local findings were considered to be treatment-related.

 

Intrauterine parameters remained unaffected by treatment and no statistically significant difference in foetal death was observed in any treated group when compared to the control animals. The mean number of corpora lutea was comparable with the control in all treated groups and no significant difference was observed in the number of implantations of the treated groups when compared to the controls. The pre-implantation loss and early and the late embryonic loss values of the treated groups, and the number of dead foetuses were comparable with the control group. There was no statistically significant difference observed in the post-implantation loss between the treated and control groups.

 

Mean foetal weight per litter was significantly lower in the 750 mg/Kg bw/day dose group, but comparable to the control mean value in other treated groups. No toxicologically relevant adverse treatment-related effects were observed on the rest of the foetal parameters, number of retarded foetuses (runts), and in the sex distribution between the treated and control groups. Placentas were normal for all observed animals and no treatment-related effect was observed on the external development of foetuses through the study period.

 

(2E)-2-methyl-3-phenylacrylaldehyde, when administered daily by oral gavage to pregnant Hannover Wistar rats from gestation days GD6 to GD19 up to 750 mg/Kg bw/day, was associated with maternal toxicity (mortality and slight, local effects in the stomach at 750 mg/Kg bw/day; and slight, local effect in the stomach at 250 and 500 mg/Kg bw/day). Although a slight secondary toxic effect was observed on the body weight of the pups at the 750 mg/Kg bw/day dose group, no foetotoxicity effect was observed in the rest of the animals (up to 500 mg/Kg bw/day). No embryotoxicity or teratogenicity effect was observed in this study. Therefore, a dose between the 500 and 750 mg/Kg bw/day (i.e. 600 mg/Kg body weight/day) was determined to be suitable as the high dose for the OECD Guideline 414 developmental toxicity study in rats.