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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2019-10-24 to 2019-11-07
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions

Data source

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

Materials and methods

Test guideline
Qualifier:
no guideline required
Principles of method if other than guideline:
Preliminary dose range-finding study designed to allow selection of appropriate dose levels for a subsequent OECD Guideline 421 study.
GLP compliance:
no
Remarks:
Preliminary dose range-finding 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: 99.49%
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) protected from humidity (store in a tightly closed container)
- Stability under storage conditions: stable for at least five days at room temperature
- Stability under test conditions: Stability of the test item in the vehicle was assessed under the conditions employed on the study during the analytical method validation. In that study, the formulation samples in the 2-250 mg/mL concentration range (using PEG 400 as vehicle) were proven as being stable for at least 5 days when stored at room temperature.

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

OTHER SPECIFICS
-Appearance: Clear yellow liquid
-Purity: 99.49%

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl:WI Wistar rats
Details on species / strain selection:
The rat is regarded as suitable species for toxicology and reproduction studies. Wistar rat was selected due to experience with this rat strain in toxicity and reproductive toxicity studies and its’ known fertility.
Sex:
male/female
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)
- Females (if applicable) nulliparous and non-pregnant:yes
- Age at study initiation: Young adult rats, 14 weeks old at the start
- Weight at study initiation: Males: 447 - 489 g; females: 269 - 303 g
- Fasting period before study: Not specified
- Housing: 2 animals of the same sex per cage
- Diet (e.g. ad libitum): ssniff® SM R/M "Autoclavable complete diet for rats and mice – breeding and maintenance" (ssniff Spezialdiäten GmbH (D-59494 Soest, Germany)) ad libitum
- Water (e.g. ad libitum): tap water from municipal supply, as for human consumption from 500 mL bottle, ad libitum
- Acclimation period: 35 days

DETAILS OF FOOD AND WATER QUALITY: The standard content of the diet as provided by the Supplier and a copy of the Certificate of Analysis (batch number: 746 55782, expiry date: 30 April 2020) was archived with the raw data at Charles River Laboratories Hungary Kft. Water quality control analysis was performed once every three months and microbiological assessment was performed monthly by Veszprém County Institute of
State Public Health and Medical Officer Service (ÁNTSZ, H-8200 Veszprém, József Attila u. 36., Hungary). The quality control results are included in the raw data and archived at Charles River Laboratories Hungary Kft. The food and water were considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.8 - 23.8°C (target: 22 ± 3 °C)
- Humidity (%): 30 - 62% (target: 30 - 70%)
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 2019-09-19 To: 2019-11-07

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The oral route was selected as it is one of the possible routes of human exposure and will be the route used in the upcoming repeated dose studies for which this dose range-finding study will provide data to inform dose selection
Vehicle:
polyethylene glycol
Remarks:
PEG (400)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test material was formulated at appropriate concentrations in the vehicle (as a visibly stable homogenous formulation) in the Pharmacy of Charles River Laboratories Hungary Kft. Formulations were prepared daily.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on the results of a short solubility test performed at the Test Facility, PEG 400
was selected as vehicle for this study in agreement with the Sponsor.
- Concentration in vehicle: 0, 50, 100, or 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/Kg bw
- Lot/batch no. (if required): Acros (Batch # A0401858)
- 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 and/or homogeneity were made on study samples, performed using a validated HPLC method. Duplicate samples were taken from test material formulations once during the study, one set to analyse and one set as a back-up, if required for any confirmatory analyses. Similarly, one sample was taken in duplicate from the middle of the vehicle control solution for concentration measurement. Acceptance criteria of the concentration analysis were 100 ± 15% of the nominal concentration.
Duration of treatment / exposure:
14 consecutive days
Frequency of treatment:
once daily via oral gavage
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1 - Control
Dose / conc.:
250 mg/kg bw/day (nominal)
Remarks:
Group 2 - Low Dose
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
Group 3 - Mid Dose
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Group 4 - High Dose
No. of animals per sex per dose:
4/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose selection was based on available data from dietary and repeated dose studies provided by the Sponsor. In these studies, no effect was observed at 220 mg/Kg bw/day (Trubeck Laboratories (1958a)) when animals were dosed orally (by gavage), and no adverse effects were observed in dietary studies with a product with similar chemistry at up to about 5000 mg/Kg bw/day (EFSA Journal, Dec 2016). Based on these results, a high dose of 1000 mg/kg bw/day was considered appropriate for this DRF study. Lower doses were set with a constant ratio of 2.

- Rationale for animal assignment: All animals were weighed on the day before the start of the treatment period and randomly allocated to study groups. The results of the randomization were checked using a computer program to verify the homogeneity and deviations between the groups. Males and females were randomised separately.

- Fasting period before blood sampling for clinical biochemistry: On Day 14, after an overnight period of food deprivation, blood samples were collected
from all animals immediately prior to the scheduled necropsy by heart puncture under pentobarbital anaesthesia.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: inspected for signs of morbidity and mortality twice daily (at the beginning and end of each working day)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily, before and after treatment, at the beginning and towards the end of the working day as practical. Animals were monitored for any clinical signs, including pertinent behavioural changes, signs of toxicity including mortality, 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), observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep or coma.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight of each animal was recorded with precision of 1 g at randomisation, then on Days 0, 7, 13, and 14 (fasted, prior to necropsy), where possible.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Food consumption was recorded with precision of 1 g at the start (Day 0) and then on Days 7 and 13.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not specified

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 14, immediately prior to the scheduled necropsy
- Anaesthetic used for blood collection: Yes (pentobarbital)
- Animals fasted: Yes
- How many animals: all surviving animals
- Parameters checked in table [No.2] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 14, immediately prior to the scheduled necropsy
- Animals fasted: Yes
- How many animals: all surviving animals
- Parameters checked in table [No.3] were examined.

PLASMA/SERUM HORMONES/LIPIDS: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
All surviving animals were euthanized under pentobarbital anaesthesia by exsanguination. After exsanguination, the external appearance was examined, cranium, thoracic and abdominal cavities were opened, and the appearance of the tissues and organs were observed macroscopically

GROSS PATHOLOGY: Yes (see table 4). The organs listed in Table 4 were trimmed of fat and weighed in all surviving animals after completion of the 14-day treatment. Paired organs were weighed together. Absolute organ weights were measured, and relative organ weights to the body and brain weights were calculated.

HISTOPATHOLOGY: No histopathology evaluation was performed. On completion of the macroscopic examination, the tissues and organs listed in Table 5 were retained from surviving animals. Eyes with the optic nerve, testes and epididymides were retained in modified Davidson’s fixative. All other organs were retained in 10% buffered formalin solution.
Statistics:
Data were collected using the software PROVANTIS v.9. Group means and standard deviations were calculated from numerical data obtained in the study. The statistical evaluation of appropriate data was performed with the statistical program package of SAS 9.2 software package (within the validated Provantis system). The following decision tree was applied automatically for statistical evaluation of continuous numeric data:

The normality and heterogeneity of variance between groups were checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log-transformed when justified). Where both tests showed no significant heterogeneity, an Anova / Ancova (one-way analysis of variance) test was carried out. If the obtained result was positive, Dunnett (Multiple Range) test was used to assess the significance of inter-group differences; identifying differences of <0.05 or <0.01 as appropriate. This parametric analysis is the better option when the normality and heterogeneity assumptions implicit in the tests are adequate.

If either of the Shapiro-Wilk or Levene tests showed significance on the data, then the ANOVA type approach was not valid, and a non-parametric analysis was required. A Kruskal-Wallis analysis of variance was used after Rank Transformation. If there was a positive result, the inter-group comparisons were performed using Dunn test; identifying differences of <0.05 or <0.01 as appropriate. As there was only one surviving female and two surviving males in the High dose group, statistics were not conducted.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Soft faeces was present in all animals in the study but not considered to be a treatment-related effect since this is a common finding associated with PEG 400 as a vehicle. No other symptoms were observed in the control, low-, or mid-dose groups. Animals in the 1000 mg/Kg bw/day dose group were symptom-free until Day 3. Apart from the animals found dead on Day 4 (symptoms observed were laboured respiration, increased salivation and lethargy), the following symptoms were present Day 4 onwards: hunched back, piloerection, decreased activity, noisy respiration, recumbence, ataxia.
Mortality:
mortality observed, treatment-related
Description (incidence):
2 out of 4 males and 3 out of 4 females in the 1000 mg/Kg bw/day dose group were found dead during the study. With the exception of one female (found dead on Day 10), the treatment-related animal deaths occurred in the first week of the treatment, on Day 4 or Day 6.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There was no effect of treatment on body weight observed in any of the animals in the low- and mid-dose groups when compared to the control animals. Markedly lower body weight gain was observed in the surviving high dose (1000 mg/Kg bw/day) male animals.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Measured food consumption values in the 250 (low-dose) and 500 mg/Kg bw/day (mid-dose) dose groups were comparable to the control group. However, lower food consumption (approximately -40%) was observed in the surviving high dose animals.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
A minor increase in the percentage of reticulocytes was observed in mid- and high-dose groups, reaching statistical significance in the mid-dose group in both sexes. However, this reticulocyte increase was beyond the historical control range in the case of only one mid-dose female animal and therefore considered as a non-adverse effect of the test material.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related effects were observed on the clinical parameters evaluated in the study. The observed significant differences were considered to be incidental, and due to the low group sizes.
Endocrine findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
No treatment-related changes were observed in the male organ weights in any of the dose groups. In the surviving high-dose female, the measured weight of the adrenal gland was the highest measured value among all females. This value being at the upper end of the historical control range may represent a stress-related adrenal weight change. In the mid-dose females, significantly higher liver weight (absolute and relative to body or brain weight) were observed when compared to the control mean. In the surviving high-dose female, the increase compared to the control mean was higher than the mid-dose, indicating a treatment-related effect. All other organ weight parameters were within the expected range.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Red, dark red, or pale discolouration (multifocal to diffuse) was observed in the glandular or the non-glandular mucosal region of the stomach of the animals found dead. All other findings were considered to be incidental, the consequence of cannibalism by cage mates, or post-mortem changes. In the surviving animals, thickness of the non-glandular stomach region was present in one of four low-dose males, all four mid-dose males, one of two high-dose males, and three of four mid-dose females, as well as in the surviving high dose female rat. No other macroscopic findings were present in any of the animals.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Effect levels

Key result
Dose descriptor:
other: MTD
Effect level:
< 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
gross pathology
mortality
organ weights and organ / body weight ratios

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Any other information on results incl. tables

All test material formulations were shown to be homogeneous and they were found to be in the range of 95 to 98% of nominal concentrations. No test material was detected in the negative (vehicle) control sample.

Table 6. Summary of Total Incidence of Clinical Signs

Observation Type:

All Types

Dose Groups

Males

Females

From

Day 0 to 14

Control

(0 mg/Kg/day)

250 mg/Kg/day

500 mg/Kg/day

1000 mg/Kg/day

Control

(0 mg/Kg/day)

250 mg/Kg/day

500 mg/Kg/day

1000 mg/Kg/day

Total Number of Animals

4

4

4

4

4

4

4

4

 

Normal

 

  Number of Animals Affected

4

4

4

4

4

4

4

4

  % of Affected Animals

100

100

100

100

100

100

100

100

  Number of Times Recorded

92

32

32

32

92

32

32

32

 

Activity decreased

 

  Number of Animals Affected

0

0

0

1

0

0

0

2

  % of Affected Animals

0

0

0

25

0

0

0

50

  Number of Times Recorded

0

0

0

3

0

0

0

9

 

Ataxia

 

  Number of Animals Affected

0

0

0

0

0

0

0

1

  % of Affected Animals

0

0

0

0

0

0

0

25

  Number of Times Recorded

0

0

0

0

0

0

0

2

 

Faeces soft

 

  Number of Animals Affected

4

4

4

4

4

4

4

2

  % of Affected Animals

100

100

100

100

100

100

100

50

  Number of Times Recorded

24

80

80

48

24

80

80

33

 

Found Dead

 

  Number of Animals Affected

0

0

0

2

0

0

0

3

  % of Affected Animals

0

0

0

50

0

0

0

75

  Number of Times Recorded

0

0

0

2

0

0

0

3

 

Hunched Back

 

  Number of Animals Affected

0

0

0

2

0

0

0

2

  % of Affected Animals

0

0

0

50

0

0

0

50

  Number of Times Recorded

0

0

0

16

0

0

0

11

 

Increased Salivation

 

  Number of Animals Affected

0

0

0

1

0

0

0

0

  % of Affected Animals

0

0

0

25

0

0

0

0

  Number of Times Recorded

0

0

0

1

0

0

0

0

 

Laboured respiration

 

  Number of Animals Affected

0

0

0

1

0

0

0

0

  % of Affected Animals

0

0

0

25

0

0

0

0

  Number of Times Recorded

0

0

0

1

0

0

0

0

 

Lethargy

 

  Number of Animals Affected

0

0

0

0

0

0

0

1

  % of Affected Animals

0

0

0

0

0

0

0

25

  Number of Times Recorded

0

0

0

0

0

0

0

1

 

Piloerection

 

  Number of Animals Affected

0

0

0

3

0

0

0

2

  % of Affected Animals

0

0

0

75

0

0

0

50

  Number of Times Recorded

0

0

0

9

0

0

0

10

 

Recumbency

 

  Number of Animals Affected

0

0

0

1

0

0

0

0

  % of Affected Animals

0

0

0

25

0

0

0

0

  Number of Times Recorded

0

0

0

2

0

0

0

0

 

Terminal Euthanasia

 

  Number of Animals Affected

4

4

4

2

4

4

4

1

  % of Affected Animals

100

100

100

50

100

100

100

25

  Number of Times Recorded

4

4

4

2

4

4

4

1

 

Table 7. Summary of Bodyweight Data

Day(s) Relative to Start Date

Dose Groups

Males

Females

Control

(0 mg/Kg/day)

250 mg/Kg/day

500 mg/Kg/day

1000 mg/Kg/day

Control

(0 mg/Kg/day)

250 mg/Kg/day

500 mg/Kg/day

1000 mg/Kg/day

0

Mean

471.3 I1

470.0

467.8

470.0

286.3 I1

293.0

293.3

289.8

SD

19.6

19.2

14.1

19.0

14.1

15.1

17.0

17.5

Max

493

489

485

493

305

311

313

311

Min

447

453

452

452

272

277

275

269

N

4

4

4

4

4

4

4

4

% Diff

 

-0.3

-0.7

-0.3

 

2.4

2.4

1.2

 

7

Mean

471.0 I1

470.8

462.5

448.5

289.3 I1

294.5

294.3

286.5

SD

27.9

19.7

20.9

27.6

7.2

16.4

19.0

30.4

Max

498

492

491

468

297

310

315

308

Min

435

449

445

429

280

272

271

265

N

4

4

4

2

4

4

4

2

% Diff

 

-0.1

-1.8

-4.8

 

1.8

1.7

-1.0

 

13

Mean

482.0 I1

476.5

471.5

451.5

298.8 R2

301.5

305.5

302.0

SD

34.0

24.1

26.1

29.0

6.6

7.8

20.2

 

Max

514

501

506

472

307

310

328

302

Min

439

452

448

431

293

292

286

302

N

4

4

4

2

4

4

4

1

% Diff

 

-1.1

-2.2

-6.3

 

0.9

2.3

1.1

Statistical Test: Citoxlab DT Transformation: Automatic

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

2 [R - Automatic Transformation: Rank]

 

Table 8. Summary of Food Consumption Data

Day(s) Relative to Start Date

Dose Groups

Males

Females

Control

(0 mg/Kg/day)

250 mg/Kg/day

500 mg/Kg/day

1000 mg/Kg/day

Control

(0 mg/Kg/day)

250 mg/Kg/day

500 mg/Kg/day

1000 mg/Kg/day

0 - 7

Mean

27.04 R1

25.75

23.25

15.31

17.54 R1

19.32

17.25

11.13

SD

 

 

 

 

 

 

 

 

Max

27.0

25.8

23.3

15.3

17.5

19.3

17.3

11.1

Min

27.0

25.8

23.3

15.3

17.5

19.3

17.3

11.1

N

1

1

1

1

1

1

1

1

% Diff

 

-4.8

-14.0

-43.4

 

10.2

-1.6

-36.6

 

7 - 13

Mean

28.46 R1

26.88

27.92

-

20.00 R1

21.25

20.42

-

SD

 

 

 

-

 

 

 

-

Max

28.5

26.9

27.9

-

20.0

21.3

20.4

-

Min

28.5

26.9

27.9

-

20.0

21.3

20.4

-

N

1

1

1

-

1

1

1

-

% Diff

 

-5.6

-1.9

-

 

6.3

2.1

-

 

0 - 13

Mean

27.69 R1

26.27

25.40

15.31

18.67 R1

20.21

18.71

11.13

SD

 

 

 

 

 

 

 

 

Max

27.7

26.3

25.4

15.3

18.7

20.2

18.7

11.1

Min

27.7

26.3

25.4

15.3

18.7

20.2

18.7

11.1

N

1

1

1

1

1

1

1

1

% Diff

 

-5.1

-8.3

-44.7

 

8.2

0.2

-40.4

Statistical Test: Citoxlab DT Transformation: Automatic

1 [R - Automatic Transformation: Rank]

 

Table 9. Summary of Select Haematological Data

Day 14 Relative to

Start Date

Dose Groups

Males

Females

Control

(0 mg/Kg/day)

250 mg/Kg/day

500 mg/Kg/day

1000 mg/Kg/day

Control

(0 mg/Kg/day)

250 mg/Kg/day

500 mg/Kg/day

1000 mg/Kg/day

Reticulocyt.

Rel. (%)

Mean

2.38 R k1

2.58

3.05 u2

3.20 u2

2.45 I, a1

2.78

3.95 d2

4.50 n3

SD

0.15

0.17

0.35

0.57

0.52

0.49

0.97

-

Max

2.5

2.8

3.4

3.6

2.9

3.2

5.3

4.5

Min

2.2

2.4

2.7

2.8

2.0

2.3

3.0

4.5

N

4

4

4

2

4

4

4

1

% Diff

 

8.4

28.4

34.7

 

13.3

61.2

83.7

1 [R,k - Automatic Transformation: Rank, (All Groups) Test: Kruskal-Wallis p < 0.05]

1 [I,a - Automatic Transformation: Identity (No Transformation), (All Groups) Test: Analysis of Variance p < 0.05]

2 [u - Test: Dunn 2 Sided p < 0.05]

2 [d - Test: Dunnett 2 Sided p < 0.05]

3 [n - Inappropriate for statistics]

 

Table 10. Summary of Select Organ Weight Data

Day 14 Relative to

Start Date

Dose Groups

Males

Females

Control

(0 mg/Kg/day)

250 mg/Kg/day

500 mg/Kg/day

1000 mg/Kg/day

Control

(0 mg/Kg/day)

250 mg/Kg/day

500 mg/Kg/day

1000 mg/Kg/day

Adrenal Glands

(g)

Mean

0.0703 I1

0.0620

0.0755

0.0690

0.0918 I, a2

0.0930

0.0803

0.1080 n3

SD

0.0057

0.0052

0.0097

0.0000

0.0091

0.0054

0.0087

-

Max

0.077

0.068

0.085

0.069

0.103

0.099

0.092

0.108

Min

0.065

0.056

0.062

0.069

0.081

0.086

0.071

0.108

N

4

4

4

2

4

4

4

1

% Diff

-

-11.7

7.5

-1.8

-

1.4

-12.5

17.7

 

Adrenals

/BW.

(%)

Mean

0.0156 I1

0.0138

0.0170

0.0162

0.0332 L4

0.0326

0.0286

0.0374

SD

0.0025

0.0015

0.0022

0.0009

0.0041

0.0006

0.0043

-

Max

0.019

0.016

0.019

0.017

0.039

0.033

0.035

0.037

Min

0.013

0.012

0.014

0.016

0.029

0.032

0.025

0.037

N

4

4

4

2

4

4

4

1

% Diff

-

-11.5

9.0

3.7

-

-1.7

-13.9

12.7

 

Adrenals

/Brain

(%)

Mean

3.177 I1

2.805

3.431

3.159

4.498 I1

4.437

3.978

5.118

SD

0.283

0.270

0.450

0.072

0.421

0.182

0.479

-

Max

3.48

3.04

3.76

3.21

4.88

4.65

4.51

5.12

Min

2.86

2.41

2.77

3.11

3.93

4.22

3.43

5.12

N

4

2

4

2

4

4

4

1

% Diff

-

-11.7

8.0

-0.6

-

-1.4

-11.6

13.8

 

Liver

(g)

Mean

12.058 I1

12.458

13.433

12.355

8.0103

8.650

9.408 d4

11.270 n5

SD

0.829

1.560

0.324

0.276

0.420

0.604

0.712

-

Max

13.08

14.25

13.90

12.55

8.57

9.05

10.08

11.27

Min

11.05

10.47

13.16

12.16

7.59

7.76

8.72

11.27

N

4

4

4

2

4

4

4

1

% Diff

-

3.3

11.4

2.5

-

8.0

17.4

40.7

 

Liver

/BW.

(%)

Mean

2.657 I1

2.763

3.026

2.899

2.8933

3.033

3.328 dd+

3.900 n5

SD

0.138

0.260

0.075

0.218

0.227

0.134

0.093

-

Max

2.77

2.99

3.11

3.05

3.22

3.18

3.46

3.90

Min

2.46

2.43

2.94

2.74

2.70

2.86

3.25

3.90

N

4

4

4

2

4

4

4

1

% Diff

-

4.0

13.9

9.1

-

4.8

15.0

34.8

 

Liver

/Brain

(%)

Mean

545.28 R2

563.85

610.53

565.45

392.76 R, k+

412.64

465.33 u+

534.12 n5

SD

42.24

79.64

31.74

0.19

19.74

22.78

25.48

-

Max

600.0

639.0

655.7

565.6

411.2

433.0

481.2

534.1

Min

500.0

467.4

582.3

565.3

368.4

380.4

427.5

534.1

N

4

4

4

2

4

4

4

1

% Diff

-

3.4

12.0

3.7

-

5.1

18.5

36.0

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

2 [R - Automatic Transformation: Rank]

2 [I,a - Automatic Transformation: Identity (No Transformation), (All Groups) Test: Analysis of Variance p < 0.05]

3 [n - Inappropriate for statistics]

3 [I,aa - Automatic Transformation: Identity (No Transformation), (All Groups) Test: Analysis of Variance p < 0.01]

4 [L - Automatic Transformation: Log]

4 [d - Test: Dunnett 2 Sided p < 0.05]

5 [n - Inappropriate for statistics]

 

Table 11. Summary of Select Necropsy Data - Terminal Euthanised Animals

 

Dose Groups

Males

Females

Control

(0 mg/Kg/day)

250 mg/Kg/day

500 mg/Kg/day

1000 mg/Kg/day

Control

(0 mg/Kg/day)

250 mg/Kg/day

500 mg/Kg/day

1000 mg/Kg/day

Number of Animals

4

4

4

2

4

4

4

1

Number of Completed Animals

4

4

4

2

4

4

4

1

 

STOMACH

 

  Submitted

4

4

4

2

4

4

4

1

  Normal

4

3

0

1

4

4

1

0

Thickness; diffuse, non-                                         glandular region; mucosa

0

0

3

0

0 c1

0

3

0

Thickness; focal, non-glandular region; mucosa

0

0

1

0

0

0

0

0

Thickness; diffuse, non-glandular region; wall

0

1

0

1

0

0

0

1

 

Animals 4001,4003,4501,4503 and 4504 were found dead

1 [c - Group Factor Chi-Squared & Fisher's Exact: Test: Chi-Squared p < 0.05]

 

Table 12. Summary of Select Necropsy Data – Found Dead Animals

 

Dose Groups

Males (1000 mg/Kg bw/day)

Females (1000 mg/Kg bw/day)

Number of Animals

2

3

Number of Completed Animals

2

3

 

STOMACH

 

  Submitted

2

3

 Discoloration; red, diffuse, glandular; mucosa

1

0

 Discoloration; dark red, multifocal, glandular; mucosa

1

0

  Discoloration; pale, multifocal, non-glandular region; mucosa

1

1

 Discoloration; red, diffuse, non-glandular region; mucosa

0

1

 Dilatation

1

1

Applicant's summary and conclusion

Conclusions:
((2E)-2-methyl-3-phenylacrylaldehyde in PEG 400 (5 mL/Kg) when administered by oral gavage to Wistar rats for 14 consecutive days at dose levels of 250, 500 or 1000 mg/Kg/day resulted in mortality and clinical signs of distress, as well as local stomach effects, in the high-dose animals (1000 mg/Kg bw/day). No mortality or clinical signs were present in the low-dose (250 mg/Kg bw/day) and mid-dose (500 mg/Kg bw/day) animals. Male and female rats in the mid-dose group showed slight local effects in the stomach mucosa, while females also displayed increased liver weight. Based on these findings, 1000 mg/Kg bw/day is clearly above the MTD with >50% mortality.
Executive summary:

In a supporting preliminary dose range-finding toxicity study, Wistar rats (4/sex/dose) were exposed to the test material ((2E)-2-methyl-3-phenylacrylaldehyde; CAS# 15174-47-7) in Propylene Glycol (PEF 400) via oral gavage once daily for a period of 14 days at 0, 250, 500, or 1000 mg/Kg bw/day.

 

The animals were inspected for signs of morbidity and mortality twice daily and monitored for any clinical signs, including pertinent behavioural changes, signs of toxicity including mortality, 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), observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep or coma twice daily. The body weight of each animal was recorded at randomisation, then on Days 0, 7, 13, and 14 (fasted, prior to necropsy), where possible and food consumption was recorded at the start (Day 0) and then on Days 7 and 13. On Day 14, after an overnight period of food deprivation, blood samples (for haematology and clinical chemistry evaluation) were collected from all animals immediately prior to the scheduled necropsy by heart puncture under pentobarbital anaesthesia. A gross macroscopic examination was performed at necropsy for each animal with selected organs weighed, and select tissues preserved in fixative.

 

Treatment-related mortality was observed in 2 out of 4 male rats (on Days 4 and 6) and 3 out of 4 female rats (two on Day 4 and one on Day 10) in the high-dose group (1000 mg/Kg bw/day). Clinical symptoms such as hunched back, piloerection, decreased activity, noisy respiration, recumbence, and ataxia related to test material exposure were observed in the high-dose animals only, while soft faeces was present in all animals in the study but considered to be a consequence of the vehicle and not treatment-related. No effect of treatment on body weight was observed in any of the animals in the low- and mid-dose groups when compared to the control animals but marked lower body weight gain was observed in the surviving high-dose male rats. Lower food consumption was observed in the surviving high-dose animals.

 

A minor increase in the percentage of reticulocytes was observed in mid- (500 mg/Kg bw/day) and high-dose groups, reaching statistical significance in the mid-dose group in both sexes. However, this reticulocyte increase was beyond the historical control range in the case of only one mid-dose female animal and therefore considered as a non-adverse effect of the test material. Clinical chemistry parameters were unaffected by treatment.

 

Gross necropsy revealed red, dark red, or pale discolouration (multifocal to diffuse) in the glandular or the non-glandular mucosal region of the stomach of the animals found dead. All other findings were considered to be incidental, the consequence of cannibalism by cage mates, or post-mortem changes. In the surviving animals, thickness of the non-glandular stomach region was observed in one of four low-dose males, all four mid-dose males, one of two high-dose males, and three of four mid-dose females, as well as in the surviving high dose female rat. No treatment-related changes were observed in the organ weights of male rats in any of the dose groups. In the mid-dose group females, significantly higher liver weights (absolute and relative to body weight or brain weight) were observed when compared to control animals. Elevated liver weight and adrenal gland weight were also observed in the surviving female rat in the high-dose group.

 

((2E)-2-methyl-3-phenylacrylaldehyde in PEG 400 (5 mL/Kg) when administered by oral gavage to Wistar rats for 14 consecutive days at dose levels of 250, 500 or 1000 mg/Kg/day resulted in mortality and clinical signs of distress, as well as local stomach effects, in the high-dose animals (1000 mg/Kg

bw/day). No mortality or clinical signs were present in the low-dose (250 mg/Kg bw/day) and mid-dose (500 mg/Kg bw/day) animals. Male and female rats in the mid-dose group showed slight local effects in the stomach mucosa, while females also displayed increased liver weight. Based on these findings, 1000 mg/Kg bw/day is clearly above the MTD with >50% mortality.