N-ethyl-N-(3-methylphenyl)propionamide

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:

4 July to 30 August 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N-ethyl-N-(3-methylphenyl)propionamide

EC Number:

423-640-6

EC Name:

N-ethyl-N-(3-methylphenyl)propionamide

Cas Number:

179911-08-1

Molecular formula:

C12H17NO

IUPAC Name:

N-ethyl-N-(3-methylphenyl)propanamide

Test material form:

liquid

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Alpk:APfSD

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Rodent Breeding Unit, Zeneca Pharmaceuticals, Alderley Park
- Age at study initiation: 5 weeks
- Weight at study initiation: 100 - 150g
- Fasting period before study: Not specified
- Housing: 5 per cage, sexes separately, in multiple rat racks suitable for animals of this strain and weight range expected during the course of the study.
The rats were transferred to clean cages and racks as necessary during the study
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY:
Diet supplied by Special Diet Services Limited, Witham, Essex, UK was available ad libitum.
Mains water was supplied by an automatic system ad libitum, except during week 3. During
week 3, water was administered in bottles for determination of water consumption.
Each batch of diet is routinely analysed for composition and for the presence of contaminants.
Water is also periodically analysed for the presence of contaminants. No contaminants were found to be present in the diet or water at levels considered to be capable of interfering with the purpose or outcome of the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21±2°C
- Humidity (%): 55±15%
- Air changes (per hr): 15 changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours dark

IN-LIFE DATES: From: 4 July 1996 To: 30 August 1996

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The dosing preparations were prepared weekly. For each dose level, a weighed amount of test item (corrected for test substance purity of 99.5% w/w) was added to the appropriate amount of com oil, mixed for at least 30 minutes using a magnetic stirrer and subdivided into
aliquots. Fresh aliquots were used for each day of the study.


VEHICLE
- Concentration in vehicle: 0.5% w/v, 1.5% w/v, 10% w/v,
- Amount of vehicle (if gavage): 1.0 ml/100g

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of the vehicle control and of the low dose (0.5% w/v) and high dose (10% w/v) formulations prepared during weeks 1 and 3 were returned to the Responsible Practitioner, Unilever ESL, for analysis for achieved concentration prior to the start of dosing with each preparation. Homogeneity and chemical stability of test item in com oil were determined separately at Unilever ESL (Unilever Study Number AH960174). Homogeneity and chemical stability were stated by the Sponsor to be satisfactory and covered the range of dosing concentrations and conditions of storage used on this study.

Samples of corn oil containing the test item were diluted to be within the standard range and analysed by GC with FID detection.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

150 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 (five males and five females)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: the dose levels were selected based on the results of a preliminary sub-acute oral dosing study in the Alpk:APfSD strain rat, carried out in this laboratory. In this study, the test substance is tested at 50, 150 and 1000 mg/kg bw/day.
- Rationale for animal assignment (if not random): random
- Post-exposure recovery period in satellite groups: 14 days after the end of administration (0 and 1000 mg/kg bw/day satellite groups).

Positive control:

Not applicable

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: daily

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/rat/day: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: daily during week 3

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at terminaison on days 29 (main study) and 57 (recovery groups)
- Anaesthetic used for blood collection: Yes (EDTA)
- Animals fasted: Not specified
- How many animals: all
- Parameters checked
red blood cell count; haemoglobin; haematocrit; mean cell volume; mean cell haemoglobin; red cell distribution width; total white cell count; differential count; platelet count; mean cell haemoglobin concentration;

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at terminaison on days 29 (main study) and 57 (recovery groups)
- Animals fasted: Not specified
- How many animals: all
- Parameters checked
urea; creatinine; glucose; albumin; total protein; cholesterol; triglycerides; sodium; potassium; magnesium; chloride; calcium; phosphorus (as phosphate); total bilirubin; albumin/globulin (A/G) ratio; alkaline phosphatase activity; aspartate aminotransferase activities; alanine aminotransferase activity; gamma-glutamyl transferase activity; creatine kinase activity

OTHER:
Organ weights
adrenal glands; brain; epididymides; heart; kidneys; liver; ovaries; spleen; testes; thymus.

Tissue microscopic examination
abnormal tissue; adrenal gland; brain; caecum; cervix; colon; duodenum; epididymis; heart; ileum; jejunum; kidney; liver; lung; lymph nodes (cervical and mesenteric); ovary; pituitary; prostate gland; rectum; sciatic nerve; seminal vesicles; spinal cord; spleen; sternum ( bone marrow); stomach; testis; thymus; thyroid gland; trachea;urinary bladder; uterus.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Detailed clinical assessments (during which each rat was removed from its cage and physically examined for changes in general health status) and quantitative assessments of landing foot splay, muscle weakness (fore- and hindlimb grip strength) and sensory perception (tail-flick test) were made in weeks -1 and 4 for all animals, and during week 8 for the recovery groups. The observations were made by one observer who was 'blind' with respect to the animals treatment, and recorded on a computer system by personnel not directly involved in the clinical observations. The presence and/or absence of all listed observations was recorded and the degree of condition noted (slight, moderate or extreme) where appropriate.

HISTOPATHOLOGY: Yes
All submitted tissues from the control and high dose groups (main study and recovery groups), kidneys and liver from the low and mid dose groups and all macroscopic abnormalities were examined by light microscopy. In addition, the kidneys from main study males from the control and high dose groups were also examined by light microscopy for alpha-2-µ globulin

Statistics:

Bodyweights were considered by analysis of covariance on initial (day I) bodyweight, separately for males and females.

No statistical analysis was performed for food or water consumption as there was only one observation per group.

Haematology and blood clinical chemistry were considered by analysis of variance. Male and female data were analysed together and the results examined to determine whether any differences between control and treated groups were consistent between the sexes.

Motor activity measurements, time to tail-flick (in the sensory function test), landing foot splay and grip strength were considered by analysis of variance, separately for males and females.

Organ weights were considered by analysis of variance and analysis of covariance on final bodyweight, separately for males and females.

All analyses were carried out separately for main study and recovery animals.

Analyses of variance and covariance were carried out using the GLM procedure in SAS (1989). Least-squares means for each group were calculated using the LSMEAN option in SAS PROC GLM. Unbiased estimates of differences from control were provided by the difference between each treatment group least-squares mean and the control group least­squares mean. Differences from control were tested statistically by comparing each treatment group least squares mean with the control group least-squares mean using a two-sided Student's t-test, based on the error mean square in the analysis.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical signs considered to be related to treatment were seen for males receiving 150 or 1000 mg/kg/day and for females receiving 1000 mg/kg/day.

Salivation, signs of salivation adn /or staining around the mouth or nose were seen at an increased incidence for males receiving 150 or 1000 mg/kg/day during the dosing period. An increased incidence of salivation was also appreent for females which received 1000 mg/kg/day.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Throughout the study, there was no evidence of any adverse effects on body weight for main study animals receiving 50, 150, 1000 mg/kg/day or for recovery group animals at 1000 mg/kg/day.

Food consumption and compound intake (if feeding study):

no effects observed

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

effects observed, non-treatment-related

Description (incidence and severity):

During week 3, cage mean water consumption for males and females receiving 1000 mg/kg/day was slightly higher than that of concurrent controls.

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

There were no changes in any of the haematology parameters measured or effects on any of the quantitative components of the Functional observational batter, or on locomotor activity that could be attributed to administration of Agarbois.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related changes in several blood clinical chemistry paratmeters (e.g. plasma albumin/globin ratio, plasma total protein, cholesterol and or triglyceride levels) and increased liver weights, were seen for main study animals which received 1000 mg/kg/day.

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There was no evidence of any treatment-related effects on landing foot splay, on time to tail flick, on forelimbe or hindlime grip strength, on locomotor activitey.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

treatment-related changes were seen for main study males and females which received 150 mg, or 1000 mg/kg/day.

Group mean liver weight for main study males and females receiving 1000 mg/kg/day was statistically significantly higher than that of concurrent controls. When adjusted for terminal body weight, group mean liver weight for main study males and females receiving 1000 mg.kg.day was approximately 22% and 29 % highet than that of concurrent controls Group mean liver weight for males receiving 150 mg/kg/day was also higher than that of concurrent controls, although this difference only attained statistically sinificance when adjusted for terminal bodyweight. Group mean liver weight adjusted for terminal body weight for males receiving 150 mg/kg/day was approximatley 11% highter than that of concurrent controls.

Group mean heart weight for main study males receiving 1000 mg/kg/day was statistically sinificantly lower than that of concurrent controls. When adjusted for terminal body weight, group mean heart weight for there animals was approximately 13% lower than that of concurrent control.
following completion of the recovery period, group mean liver and heart weights for males and/or females which received 1000 mg/kg/day were similar to those to concurrent controls.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no macroscopic findigns that could be attributed to treatment with Agarbois.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The only microscopic findings considered possible to be related to administrtion of Agarbois were seen in the kidneys and liver of males receiving 1000 mg/kg/day.
An increased incidence and or severity of bilateral renal tubule basophilia, renal tubular hyaline droplet formation and minimal hepatic centrilobular hypertrophy was seen in main study males which received 1000 mg/kg/day .  Following completion of 1 28 day recovery period, an increased incidence and/or severity of bilateral renal tubule basophilia was still apparent in males which had received 1000 mg/kg/day/

Effect levels

1

Key result

Dose descriptor:

NOEL

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effect observed

2

Key result

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effect observed

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

Oral administration of 1000 mg Agarbois kg/day for 28 consecutive days produced treatment related clinical signs, changes in several blood clinical chemistry parameters, increases in liver weight and/or reductions in heart weight. Evidence of rapid reversibility of these changes was demonstrted following a 28 day recovery period. Water consumption was also slightly increased for animals receiving 1000 mg/kg.day during the dosing period.
An increased incidence and or severity of bilateral renal tubule basophilia, renal tubular hyaline droplet formation and minimal hepatic centrilobular hypertrophy was seen in main study males which received 1000 mg/kg/day . Following completion of 28 day recovery period, an increased incidence and/or severity of bilateral renal tubule basophilia was still apparent in males which had received 1000 mg/kg/day

Oral administration of 150 mg/kg/day for 28 days produced treatement related clinical signs, changes in some blood clinical chemistry parameters and small increase in liver weight which, in the absence of any treatement-related histopathological changes, were considered to be of no toxicological significance.

The no observed effect level (NOEL), following daily oral administration of Agarbois for 28 days is 50 mg/kg/day.

The no observed adverse effect level ( NOAEL), following daily oral adminsitration of Agarbois for 28 days, is 150 mg/kg/day.

Executive summary:

Administration of Agarbois at dose levels of up to 1000 mg/kg/day for 28 days producted no adverse clinical signs or effects on body weight on body weight or food comsuption, and all animals survivied to scheduled termination.

Treatment-related clinical signs (including an inccreased incidence of salivation, signs of slivation and /or staining around the mouth or nose) were seen for main study males receving 150 or 1000 mg/kgday and for amin study females receiving 1000 mg/kg/day, but were not seen for recovery group animals following completion of the dosing period.

There were no changes in any of the haematology parameters measured or effects on any of the quantitative components of the Functional observational batter, or on locomotor activity that could be attributed to administration of Agarbois.

Treatment-related changes in several blood clinical chemistry paratmeters (e.g. plasma albumin/globin ratio, plasma total protein, cholesterol and or triglyceride levels) and increased liver weights, were seen for main study animals which received 1000 mg/kg/day.

Oral administration of 1000 mg Agarbois kg/day for 28 consecutive days produced tretment related clinical signs, changes in several blood clinical chemistry parameters, increases in liver weight and/or reductions in heart weight. Evidence of rapid reversibility of these changes was demonstrted following a 28 day recovery period. Water consumption was also slightly increased for animals receiving 1000 mg/kg.day during the dosing period.

An increased incidence and or severity of bilateral renal tubule basophilia, renal tubular hyaline droplet formation and minimal hepatic centrilobular hypertrophy was seen in main study males which received 1000 mg/kg/day .  Following completion of 1 28 day recovery period, an increased incidence and/or severity of bilateral renal tubule basophilia was still apparent in males which had received 1000 mg/kg/day/

Oral administration of 150 mg/kg/day for 28 days produced treatement related clinical signs, changes in some blood clinical chemistry parameters and small increase in liver weight which, in the absence of any treatement-related histopathological changes, were considered to be of no toxicological significance.  

The no observed effect level (NOEL), following daily oral administration of Agarbois for 28 days is 50 mg/kg/day.

The no observed adverse effect level ( NOAEL), following daily oral adminsitration of Agarbois for 28 days, is 150 mg/kg/day.