N-methylformanilide

Repeated dose toxicity: oral

Administrative data

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

data from handbook or collection of data

Data source

Reference

Reference Type:

review article or handbook

Title:

Toxicology and carcinogenesis studies of trans-cinnamaldehyde in f344/n rats and b6c3f1 mice (feed studies)

Author:

NATIONAL TOXICOLOGY PROGRAM

Year:

2004

Bibliographic source:

NATIONAL TOXICOLOGY PROGRAM February 2004.

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: As mention below

Principles of method if other than guideline:

In a Repeated dose toxicity study, test chemical was evaluated for its toxic potential in F344/N male and female rats by oral feed for 2 years.

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

(2E)-3-phenylprop-2-enal

Cas Number:

14371-10-9

Molecular formula:

C9H8O

IUPAC Name:

(2E)-3-phenylprop-2-enal

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic Laboratory Animals and Services
(Germantown, NY)
- Age at study initiation: 6 weeks old
- Weight at study initiation: no data
- Fasting period before study: no data
- Housing: Male rats were housed two or three per cage,
female rats were housed five per cage.
Polycarbonate (Lab Products, Inc., Maywood, NJ) changed twice weekly (rats and female mice) or once weekly (male mice)
- Diet (e.g. ad libitum): (Zeigler Brothers,
Inc., Gardners, PA) Feed available ad libitum. feed was irradiated
- Water (e.g. ad libitum): Tap water (Columbus municipal supply) via automatic watering system (Edstrom Industries, Inc., Waterford, WI) available ad libitum.

- Acclimation period: Male rats was quarantined for 11 days and female rats were quarantined for 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72° ± 3° F
- Humidity (%):50% ± 15%
- Air changes (per hr): 10/hour
- Photoperiod (hrs dark / hrs light): 12 hours/day

Other: Five male and five female rats and mice were randomly selected for parasite evaluation and gross observation of disease.

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: Diet, (Zeigler Brothers, Inc., Gardners, PA) Feed available ad libitum. feed was irradiated

Details on oral exposure:

Details on oral exposure
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): 3 weeks by mixing microencapsulated trans-cinnamaldehyde
with nonirradiated NTP-2000 feed
- Mixing appropriate amounts with (Type of food): Placebo and/or loaded microcapsules were combined with feed to a concentration of 1.25% (2-year studies) in the diet
- Storage temperature of food: Formulations were stored in plastic buckets at 5° C (2-year studies) for up to 5 weeks.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

by the analytical chemistry
laboratory using HPLC by methods

Duration of treatment / exposure:

105 or 106 (females) weeks

Frequency of treatment:

Daily

Doses / concentrations

Remarks:

0, 1000, 2100, or 4100 ppm delivered average daily doses of approximately 50, 100, or 200 mg/kg body weight to males and females.

No. of animals per sex per dose:

Total number of animals 400 animals
0mg/kg= (50 males and 50 females)
50mg/kg= (50 males and 50 females)
100mg/kg= (50 males and 50 females)
200mg/kg= (50 males and 50 females)

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

Observations and examinations performed & frequency
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Observed twice daily; animals were weighed at the beginning, on day 8, every 4 weeks thereafter, and at the end of the studies.

- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
Clinical findings were recorded on day 36, thereafter every 4 weeks and at the end of the studies.

BODY WEIGHT: Yes / No / No data
- Time schedule for examinations:

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, Feed consumption was recorded by cage for a 1-week period approximately every 4 weeks.

- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / Not specified

URINALYSIS: Yes
- Time schedule for collection of urine: Urine was collected during a 24-hour period from 10 male and 10 female rats from each group at 2 weeks and 3, 12, and 18 months.

- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes,

Urine samples were placed on ice, urine volume and creatinine concentration were measured, and then the samples were frozen pending shipment to Battelle Toxicology Northwest (Richland, WA) for hippuric acid quantitation.

Parameters evaluated included creatinine and hippuric acid concentrations and volume.

Sacrifice and pathology:

Sacrifice and pathology
GROSS PATHOLOGY: Yes, Necropsies were performed on all animals using carbon dioxide asphyxiation technique.

All organs and tissues were grossly examined for lesions, and all major tissues were fixed and preserved in 10% formalin neutral buffered further processed and trimmed and embedded in paraffin and sectioned to a thickness of 4 to 6 μm and stained with hematoxylin and eosin for microscopic examination of the tissue.

HISTOPATHOLOGY: Yes
Complete histopathology was performed on all rats. In addition to gross lesions and tissue masses, the following tissues were examined: adrenal gland, bone with marrow, brain, clitoral gland, esophagus, gallbladder (mice), heart with aorta, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum),
kidney, liver, lung, lymph nodes (mandibular and/or mesenteric), mammary gland (except male mice), nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, skin, spleen, stomach (forestomach and glandular),
testis with epididymis and seminal vesicle, thymus, thyroid gland, trachea, urinary bladder, and uterus.

Statistics:

The probability of survival was estimated by the prod
uct- limit procedure of Kaplan and Meier (1958) and is
presented in the form of graphs. Animals found dead of
other than natural causes or missing were censored from
the survival analyses; animals dying from natural causes
were not censored. Statistical analyses for possible
dose-related effects on survival used Cox’s (1972)
method for testing two groups for equality and Tarone’s
(1975) life table test to identify dose-related trends. All
reported P values for the survival analyses are two sided.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical findings related to toxicity related to test chemical exposure in any group 50, 100, or 200 mg/kg body weight of treated animals compare to control.

Mortality:

mortality observed, treatment-related

Description (incidence):

Mortality of 200 mg/kg males was greater than that of the vehicle control group; mortality in other exposed groups of males and of exposed females was similar to that of the vehicle control groups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights in 200mg/kg males were significantly less than those of the vehicle controls throughout the study however in 200mg/kg females body weight were significantly less after week 18 as compared to control treated group.

Mean body weights of 100mg/kg males were less after
week 94 compared to control treated group.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Feed consumption by 100mg/kg and 200mg/kg males and 200mg/kg females was less than that by the vehicle controls at the beginning and end of the study.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

no effects observed

Description (incidence and severity):

Hippuric acid excretion in urine expressed as the hippuric acid to creatinine ratio was proportional to dose indicating that neither absorption, metabolism, nor excretion was saturated in either male or female rats exposed to dosed feed containing 50 ,100and 200mg/kg of test chemical.

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Description (incidence and severity):

No Gross lesions were observed.

Neuropathological findings:

not specified

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

The incidences of adenoma of the preputial gland (vehicle control, 5/50; 50mg/kg, 1/49; 100mg/kg, 2/50; 200 mg/kg, 0/50) and prostate gland (4/50, 0/49, 0/49, 0/50) in 200 mg/kg males were significantly decreased compared to those in the vehicle controls

The incidence of mononuclear cell leukemia in 200mg/kg males was significantly decreased (18/50, 15/50, 21/50, 9/50), was considered unrelated test chemical exposure.

Effect levels

Dose descriptor:

NOAEL

Effect level:

100 other: mg/kg/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Mortality, body weight, food consumption and urinalysis and gross pathology.

Remarks on result:

other: No toxic effects were observed

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The NOAEL for chronic study (2 years) of test chemical in F344/N male and female rats was considered to be 100mg/kg.

Executive summary:

The present study aimed to performed chronic study (2 years) of the test chemical inF344/N male and female rats. In this chronic study the vehicle control treated and dietary doses equivalent to 50, 100 and 200mg/kg/bw/day was exposed to male and female F344/N rats. The exposure of test chemical to the animals was performed for105 (Male) and 106 (females) weeks. The animals were observed throughout the study duration for mortality, change in body weight, food consumption, clinical signs and urinalysis, gross and histopathology at the end of the study.Mortality of 200 mg/kg males was greater than that of the vehicle control group but not significantly however mortality in other exposed groups of males and of exposed females was similar to that of the vehicle control groups. No clinical signs of toxicity observed in any animals exposed to test chemical. In the body weight study mean body weights of 200mg/kg males were less than those of the vehicle controls throughout the study however in 200mg/kg females body weight were significantly less after week 18 as compared to control treated group.Mean body weights of 100mg/kg males were less afterweek 94 compared to control treated group. At the beginning and end of the study feed consumption by 100mg/kg and 200mg/kg males and 200mg/kg females was less than that by the vehicle controls.In urinalysis Hippuric acid excretion in urine expressed as the hippuric acid to creatinine ratio was proportional to dose indicating that neither absorption, metabolism, nor excretion was saturated in either male or female rats exposed to dosed feed containing 50mg/kg to 200mg/kg of test chemical. No visible lesions were observed in gross pathology However in histopathology The incidences of adenoma of the preputial gland (vehicle control, 5/50; 50mg/kg, 1/49; 100mg/kg, 2/50; 200 mg/kg, 0/50) and prostate gland (4/50, 0/49, 0/49, 0/50) in 200 mg/kg males were significantly decreased compared to those in the vehicle controls. The incidence of mononuclear cell leukemia in 200mg/kg males was significantly decreased (18/50, 15/50, 21/50, 9/50), was considered not related to test chemical exposure. Thus the NOAELfor chronic study (2 years)in F344/N male and female ratsof test chemicalwas considered to be 100mg/kg.