N-benzylprop-2-enamide

Endpoint summary

Description of key information

In a 28-day oral toxicity study rats (5/sex/dose) received the substance at 0, 5, 15 or 50 mg/kg bw in propylene glycol. Analyses of the formulations showed that the doses were prepared accurately. A few slight changes observed in biochemistry (reduced creatinine levels in males, increased total bilirubin and potassium levels in females and reduced sodium and chloride levels in females) were within historical control ranges and not supported by histopathological findings. No treatment related effects were found for clinical signs, functional observations, body weight, food consumption, haematology, organ weights, macroscopy and histopathology. The NOAEL is therefore set at the highest dose tested, 50 mg/kg bw.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 November 2007 to 07 January 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3050

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

recognised by the international guidelines

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzbach, Germany
- Strain: Crl:WI (Han) (outbred, SPF quality)
- Females nulliparous and non-pregnant: yes
- Age at study initiation: ca 6 weeks
- Weight at study initiation: males 144-171 g; females 125-140 g
- Housing: 5/cage (Macrolon MIV) saw dust bedding, paper as enrichment
- Diet: Pelleted Rodent Diet ad libitum (SM R/M-Z SSNIFF Soest Germany)
- Water: tap water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.9-23.1 ˚C
- Humidity (%): 29-94%
- Air changes (per hr): ca 15/h
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on oral exposure:

VEHICLE
- Amount of vehicle (if gavage): <= 5 mL/kg
- Justification for choice of vehicle: laboratory trial

MAXIMUM DOSE VOLUME APPLIED: 5 mL/kg

DOSAGE PREPARATION: prepared within 4 hours before dosing (homogeneity assessed visually) adjusted for specific gravity (1.036)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Instrument Alliance Separation Module 2695 (Waters, Milford, MA, USA)
Detector Dual lambda Absorbance Detector (Waters)
Column 150 mm X 4.6 mm i.d., Symmetry Shield RP-18 dp, 5 µm (Waters)
Injection volume 10 µL
Mobile phase 45/55 (v/v) methanol/water
Flow 1.0 mL/min
UV detection 210 nm

Sample analysis:
Calibration (range 0.955-25.5 mg/L) based on 4 concentrations Linear with r > 0.99 (no details)
Recovery: at 1 mg/g 99-102% of nominal, at 100 mg/g 95-104% of nominal
Homogenicity: CV at 5 mg/kg bw 2.3%; CV at 50 mg/kg bw 0.88
Stability over 5 hours: rel difference at 5 mg/kg bw 1.9%; at 50 mg/kg bw -0.067%
Accuracy: in controls not detected; treatment groups 96-98% of nominal

Duration of treatment / exposure:

28 days

Frequency of treatment:

once daily

Dose / conc.:

5 mg/kg bw/day (nominal)

Remarks:

measured concentration 97% of nominal

Dose / conc.:

15 mg/kg bw/day (nominal)

Remarks:

measured concentration 96% of nominal

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

measured concentration 98% of nominal

No. of animals per sex per dose:

5/sex/dose group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on 10-day dose range finding study with 3 females at 0, 50, 150, 500 and 1000 mg/kg bw. At 500 and 1000 mg/kg bw all animals were killed in extremis on day 4 and 3 resp. showing a.o. lethargy, piloerection, hunched posture, abnormal gait and/or uncoordinated movements. Macroscopy revealed reddish discoloration of the fore-stomach.
At 150 mg/kg bw animals had breathing rales, piloerection, chromodacryorrhea and/or salivation and weight loss. Macroscopy showed no abnormalities (no effects seen on liver and kidney). At 50 mg/kg bw animals showed hunched posture and salivation. Bodyweight gain was decreased.There were no macroscopic abnormalities.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily

MORTALITY: yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: in standard arena weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: Yes
- Time schedule for examinations: weekly

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: immediately before scheduled necropsy
- Anaesthetic used for blood collection: No
- Animals fasted: Yes overnight (maximum 20 h)
- How many animals: all
- Parameters checked: White blood cells, Differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils), Red blood cells, Reticulocytes, Red blood cell distribution width, Haemoglobin, Haematocrit, Mean corpuscular volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Platelets, PT and APTT

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: immediately before scheduled necropsy
- Anaesthetic used for blood collection: No
- Animals fasted: Yes overnight (maximum 20 h)
- How many animals: all
- Parameters checked: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Total Protein, Albumin, Total Bilirubin, Bile acids, Urea, Creatinine, Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during week 4
- Dose groups that were examined: all
- Battery of functions tested: motor activity, grip strength, hearing ability, pupil reflex, static righting reflex

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Ovaries, Adrenal glands, Pancreas, Aorta, Peyer's patches [jejunum, ileum] if detectable, Brain [cerebellum, mid-brain, cortex], Pituitary gland, Caecum, Ileum, Jejunum, Duodenum, Colon, Rectum, Preputial gland, Cervix, Prostate gland, Clitoral gland, Salivary glands - mandibular, sublingual, Sciatic nerve, Skeletal muscle, Epididymides, Seminal vesicles including coagulating gland, Eyes with optic nerve and Harderian gland, Skin, Female mammary gland area, Spinal cord -cervical, midthoracic, lumbar, Femur including joint, Spleen, Heart, Sternum with bone marrow, Stomach, Testes, Kidneys, Thymus, Larynx, Thyroid including parathyroid, Lacrimal gland, exorbital, Tongue, Liver, Trachea, Lung, infused with formalin, Urinary bladder, Lymph nodes - mandibular, mesenteric, Uterus, Nasopharynx, Vagina, Oesophagus, All gross lesions

ORGAN WEIGHTS: Adrenal glands, Spleen, Brain, Testes, Epididymides, Thymus, Heart, Kidneys,

HISTOPATHOLOGY: Ovaries, Adrenal glands, Pancreas, Aorta, Peyer's patches [jejunum, ileum], Brain [cerebellum, mid-brain, cortex], Pituitary gland, Caecum, Ileum, Jejunum, Duodenum, Colon, Rectum, Cervix, Prostate gland, Salivary glands - mandibular, sublingual, Sciatic nerve, Epididymides, Seminal vesicles including coagulating gland, Eyes with optic nerve and Harderian gland, Skin, Female mammary gland area, Spinal cord -cervical, midthoracic, lumbar, Spleen, Heart, Sternum with bone marrow, Stomach, Testes, Kidneys, Thymus, Larynx, Thyroid including parathyroid [if detectable], Liver, Trachea, Lung, infused with formalin, Urinary bladder, Lymph nodes - mandibular, mesenteric, Uterus, Vagina, Oesophagus, All gross lesions

Statistics:

Dunnett-test, Steel-test, The Fisher Exact-test

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Salivation was observed along females at 15 mg/kg bw and males and females at 50 mg/kg bw.
This is considered related to gavage treatment.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Slight changes (within normal ranges) during part of the study in males and females at 5 and 15 mg/kg bw. No relationship with dose

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

females 50 mg/kg bw: sign lower reticulocyte counts
males 15 mg/kg bw: sign lower rel neutrophil counts, sign higher rel. lymphocyte counts, lower MCHC

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

males 50 mg/kg bw: sign reduced creatine levels
females at 50 mg/kg bw: sign. reduced sodium and chloride level, sign increased total bilirubin and potassium levels
females at 15 mg/kg bw: sign.lower inorganic phosphate levels (no relation with dose)

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

motor activity, grip strength, hearing ability, pupil reflex and static righting reflex were within normal limits at all doses

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

sign increased liver and kidney weights at 50 mg/kg bw in males

Gross pathological findings:

no effects observed

Description (incidence and severity):

No abnormalities observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No microscopic findings

Histopathological findings: neoplastic:

not examined

Details on results:

see table

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: absence of adverse effects at the highest dose tested

Critical effects observed:

no

 

 

Dose (mg/kg bw)	0		5		15		50		Treatment related
Endpoint	M	F	M	F	M	F	M	F
Mortality	0/5	0/5	0/5	0/5	0/5	0/5	0/5	0/5	no
Clinical signs$ Salivation						2/5	5/5	5/5	yes (see remark)
Body weight	NTRE
Body weight gain d 1-28					↓27 %				no
Food consumption	NTRE
Behavioral effects grip strength, hearing ability, pupil reflex, static righting reflex	NTRE
Motoractivity	NTRE
Haematology					neutr↓(18%) Lymp↑ (4%) MCHC ↓ (3%)			Ret↓ (16%)	no
Clinical biochemistry						Phos ↓( 18%)	Creat ↓( 8%)	TB ↑ (28%) Na, Cl ↓(2-3%) K ↑ (11%)	no
Organ weights r= relative to BW							Kidney r↑ (19%) Liverr↑(8%)		no
Macroscopy	No findings
Histopathology	NTRE

NTRE= no treatment related effects

↑/↓= significantly increased/decreased

Conclusions:

The NOAEL of the substance in a subacute oral study is 50 mg/kg bw (the highest dose tested)

Executive summary:

In a 28-day oral toxicity study rats (5/sex/dose) received the substance at 0, 5, 15 or 50 mg/kg bw in propylene glycol. Analyses of the formulations showed that the doses were prepared accurately. A few slight changes observed in biochemistry (reduced creatinine levels in males, increased total bilirubin and potassium levels in females and reduced sodium and chloride levels in females) were within historical control ranges and not supported by histopathological findings. No treatment related effects were found for clinical signs, functional observations, body weight, food consumption, haematology, organ weights, macroscopy and histopathology. The NOAEL is therefore set at the highest dose tested, 50 mg/kg bw.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

subacute

Species:

rat

System:

other: no adverse effecst observed at the highest dose tested

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available information the substance does not need to be classified for repeated dose toxicity according to EU Regulation 1272/2008 (CLP).