3-methyl-5-phenylpent-2-enenitrile

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-07-24 to 2015-11-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

3-methyl-5-phenylpent-2-enenitrile

EC Number:

299-682-2

EC Name:

3-methyl-5-phenylpent-2-enenitrile

Cas Number:

93893-89-1

Molecular formula:

C12 H13 N

IUPAC Name:

(2E)-3-methyl-5-phenylpent-2-enenitrile

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 6 weeks
- Weight at study initiation: male: 201.1 - 215.4 g, female: 142.6 - 157.2 g
- Housing: single, Stainless wire mesh cage, 260W*350D*210H (mm)
- Diet: ad libitum, Pelleted rodent chow, Teklad Certified Irradiated Global 18% Protein Rodent Diet 2918C, Harlan Laboratories Inc. U.S.A.
- Water: ad libitum, tap water, filtered and irradiated by ultraviolet light
- Acclimation period: in quarantine room 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.2-22.6
- Humidity (%): 46.6-56.7
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The required amount of the test substance was weighed on electronic balances and placed in a bottle. A small amount of vehicle, corn oil, was added and mixed using a vortex mixer until dissolved. The vehicle was gradually added to yield the desired concentrations (2, 10 and 50 mg/mL).The dosing formulations were stored in a refrigerator (4.4 to 6.0°C). These dosing formulations were used within 7 days.

VEHICLE
- Concentration in vehicle: 2, 10 and 50 mg/mL
- Amount of vehicle: 5 mL/kg bw
- Lot/batch no: MKBS6944V

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses of the dosing formulations were conducted using a Gas Chromatography (GC-2010 series, Shimadzu Corp., Japan).
Analyses of the dosing formulations were conducted based on the method used in the study of “An Analytical Method Validation, Homogeneity and Stability Study of test substance Dosing Formulations (Biotoxtech Study No.: B15255)” and samples were taken three times from the middle of each dosing formulation and analyzed for verification of dose level concentration prior to dosing. As a result, the accuracies at 10, 50 and 250 mg/kg/day were 108.70, 106.10 and 113.04 %, respectively. These results were within the acceptable range (range: ± 15 % of nominal values).

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

once per day

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on the result of the dose range finding study (supporting study IUCLID section 7.5.1), related clinical signs, decreased body weights, decreased platelet count (PLT) and increased liver weights were observed in the 250 mg/kg bw/day group. Therefore, 250 mg/kg bw/day was selected as the high dose level. Mid and low dose levels were selected at 50 and 10 mg/kg bw/day, respectively.

Positive control:

No positive control was done.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 1/day for clinical signs, 2/day for mortality and moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before dosing and once weekly

BODY WEIGHT: Yes
- Time schedule for examinations: prior to dosing (day 1), 1/week during dosing, day of necorpsy

FOOD CONSUMPTION AND COMPOUND INTAKE :
Food consumption was recorded once from the day of group assignment to the first day of dosing. During the study period, mean daily food consumption was calculated using the total amount of food consumed for 7 days. Six-day food consumption was calculated for Week 4.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 29 (prior to necropsy)
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes, 18 h
- How many animals: all animals

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day 29 (prior to necropsy)
- Animals fasted: Yes, 18 h
- How many animals: all animals

URINALYSIS: Yes
- Time schedule for collection of urine: week 4 (fresh, 3 h and 24 h urine)
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at week 4
- Dose groups that were examined: all animals
- Battery of functions tested: sensory activity / grip strength / motor activity

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

Organ weights: individual: brain, liver, heart, spleen, thymus, uterus, prostate+seminal vesicle with coagulating gland paired: kidney, testis, ovary, adrenal, epididymis, thyroid an parathyriod

HISTOPATHOLOGY: Yes
Brain, Pituitary, Thyroid and parathyroida), Thymus, Lung including bronchi, Trachea, Heart, Liver, Spleen, Kidney, Adrenal, Salivary gland (submandibular, sublingual and parotid glands), Esophagus, Stomach, Duodenum, JejunumIleum, Cecum, Colon, Rectum, Pancreas, Testis, Epididymis, Prostate, Seminal vesicle with coagulating gland, Ovary, Uterus and cervix, Vagina, Urinary bladder, Submandibular lymph node, Mesenteric lymph node, Skeletal muscle (thigh), Sciatic nerve, Eye including optic nerve and Harderian gland, Mammary gland: inguinal, Skin: inguinal, Sternum including bone marrow, Femur including bone marrow, Spinal cord

Statistics:

Statistical analysis was performed using SAS Program (version 9.3, SAS Institute Inc., U.S.A.).
Body weight, food consumption, functional observations (hindlimb landing foot splay, grip strength and motor activity), urine volume, hematology, clinical chemistry and organ weight data were analyzed using Bartlett’s test for homogeneity of variance (significance level: 0.05). One-way analysis of variance (ANOVA) was employed on homogeneous data; then, if significant, Dunnett’s test was applied for multiple comparisons (significance levels: 0.05 and 0.01, two-tailed). Kruskal-Wallis test was employed on heterogeneous data; then, if significant, Steel test was applied for multiple comparisons (significance levels: 0.05 and 0.01, two-tailed).

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In the 50 mg/kg bw/day group, salivation was sporadically observed before and/or after dosing in five males and four females from Day 8 to Day 28. In the 250 mg/kg bw/day groups, salivation was sporadically observed before and/or after dosing in all males and females from Day 2 to Day 28. Irregular respiration was sporadically observed in all males (from Day 2 to Day 9 and on Day 25) and females (from Day 1 to Day 6 and on Days 26 and 27) and it was observed occasionally until afternoon on those days. Reddish urine was sporadically observed in all males on Days 16, 18, 19, 23, 24, 26 and 28 and in one female on Days 23, 24 and 27. A decrease of fecal volume was observed in two females on Day 3. Clinical signs (irregular respiration, reddish urine and decrease of fecal volume) observed in the 250 mg/kg bw/day group were considered to be test substance-related effects. Salivation observed temporarily before or after dosing was considered to be a secondary change caused by stimulation of the test substance, since it was not accompanied by morphologic changes in the salivary glands.

Mortality:

mortality observed, treatment-related

Description (incidence):

All animals survived the duration of the study. The mortality was not affected by the test substance.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant decreases in the body weight were observed in both sexes of the 250 mg/kg bw/day groups from Week 1 to Week 4 and statistically significant decreases in body weight gain were observed in males (from Week 1 to Week 3) and females (at Week 1) in the 250 mg/kg bw/day groups when compared to the control groups. The mean body weights at the end of the dosing period (Week 4) for males and females in the 250 mg/kg bw/day groups were 20.6 % and 16.7 % below than the control group, respectively. These changes were considered to be test substance-related effects. There were no statistically significant differences in the body weight and body weight gain in males of the 10 and 50 mg/kg bw/day groups when compared to the control group. Statistically significant decreases in the body weight and body weight gain were observed in females of the 10 and 50 mg/kg bw/day groups at Week 1 when compared to the control group. However, it was not observed from Week 2.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In the 250 mg/kg bw/day group, statistically significant decreases in food consumption were observed in males (Weeks 1 and 2) and females (Weeks 1 and 3) and statistically significant decreases in relative food consumption were observed in both sexes at Week 1 when compared to the control group. The decreases in food consumption and relative food consumption were correlated with the decreases in the mean body weight. There were no statistically significant differences in the food consumption and relative food consumption in both sexes in the 10 mg/kg bw/day groups and in males of the 50 mg/kg bw/day group when compared to the control groups. Statistically significant decreases in food consumption and relative food consumption were observed in females of the 50 mg/kg bw/day group at Week 1 when compared to the control group. However, it was not observed from Week 2.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

Test substance-related changes were not observed in both sexes in the 10, 50 and 250 mg/kg bw/day groups.
All differences in hematology parameters were judged to be produced due to biological variation or were considered unrelated to the dosing of test substance because of a lack of dose-response, inconsistency in related parameters in both sexes and related parameters, and small magnitude. Therefore, they were considered not to be of toxicologically significance.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Decreased triglyceride (TG) was evident in both sexes in the 250 mg/kg bw/day groups. It was correlated with a histopathological finding in the liver. Other differences in clinical chemistry parameters were judged to be produced due to biological variation or were considered unrelated to the dosing of test substance because of inconsistency in both sexes and related parameters, and small magnitude. Therefore, they were considered not to be of toxicologically significance.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no test substance-related effects in both sexes in the 10, 50 and 250 mg/kg bw/day groups.
All differences in urinalysis parameters were not considered to be toxicologically significant based on their small magnitude and a lack of dose-relationship.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Statistically significant decreases in the ambulatory counts and vertical counts were observed in males of the 250 mg/kg bw/day group at 10 minute intervals up to 50 minutes and in the total count when compared to the control group. These changes were considered to be test substance-related effects. There were no differences in visual response, proprioceptive stimuli, auditory, pain response, aerial righting reflex, hindlimb landing foot splay and grip strength in males and females of the 10, 50 and 250 mg/kg bw/day groups when compared to the control groups.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Test substance-related changes were not observed in both sexes in the 10, 50 and 250 mg/kg bw/day groups.
Other organ weight changes, some of which were statistically significant, were not considered to have toxicological importance because they were small changes and were considered to be related to the decrease of the body weight.

Gross pathological findings:

no effects observed

Description (incidence and severity):

The macroscopic findings were not observed in both sexes in the 10, 50 and 250 mg/kg bw/day groups.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related findings were observed in the liver and kidney of males, and in the ovary of females.
Microvesicular vacuolation of hepatocytes occurred in centrilobular zone in the liver of males and cystic follicles were observed in the ovary of females in the 250 mg/kg bw/day group. Hyaline droplets in epithelial cells of proximal tubules were observed in the kidney of males in the 50 and 250 mg/kg bw/day groups. The vacuoles in centrilobular hepatocytes were microvesicular, clear and spherical, and serum triglycerides (TG) level was significantly decreased in both sexes in the 250 mg/kg bw/day group, compared to the controls. Thus, there was a likelihood of association between decreased TG level and hepatocellular vacuolation in males of which the degree of decrease was larger than that of females. The cystic follicles in the ovary of females in the 250 mg/kg bw/day group were evident, however, the newly formed corpora lutea were observed within normal limits, and level of proficiency in reproductive tract staging was also considered to be within normal limits until the end of treatment period in this study. The hyaline droplets were characterized by polygonal or angular form and preferring proximal tubule, but they were not observed in those of females. These characters indicate that the droplet was formed by alpha2u-globulin that is sex- and speciesspecific. Other microscopic findings seen in various organs and tissues were considered to be incidental and of no toxicological significance.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

histopathology: non-neoplastic

Target system / organ toxicity

1

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

female reproductive system

Organ:

uterus

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

2

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

A 4 week repeated oral dose toxicity study was conducted in rats. A NOAEL of 50 mg/kg bw/day for male and female rats was established.

Executive summary:

A repeated oral dose study toxicity study for 28 days of application via gavage was conducted in rats. The study was conducted according to OECD Guideline 407. The test substance was administered to rats at 10, 50 and 250 mg/kg/day. No mortality was observed during the complete study in both sexes. Animals treated with 10 mg/kg bw/day or corn oil only (control group) showed no changes. Irregular respiration, reddish urine and/or decrease of fecal volume were observed in both sexes in the 250 mg/kg bw/day groups. Decreases in the body weight and/or food consumption were evident in males and females in the 250 mg/kg bw/day groups from Week 1 to Week 4 and body weight increments were -20.6 % in males and -16.7 % in females in the 250 mg/kg bw/day groups by Week 4. Decreased ambulatory counts and vertical counts were observed in males of the 250 mg/kg bw/day group at 10 minute intervals up to 50 minutes and in the total count. Decreased triglyceride (TG) was observed in both sexes in the 250 mg/kg bw/day groups. Histopathologically, microvesicular vacuolation of hepatocytes occurred in centrilobular zone in the liver of males and cystic follicles were observed in the ovary of females in the 250 mg/kg bw/day group. Hyaline droplets in epithelial cells of proximal tubules were observed in the kidney of males in the 50 and 250 mg/kg bw/day groups, but as this is a species- and sex-specific effect not occurring in humans the NOAEL was determined to be 50 mg/kg bw/day for both male and female rats in this oral repeated dose toxicity study.