Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study reported in Japanese with a summary in English, but no data available concerning the year of performance or reporting.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Appearance: colorless to slightly yellow liquid
Purity: 99.4% (provided by Sansei and Chemical Research)

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
After accepting the animals were acclimated for 8 days at the conditions of the test environment;
Exposure started at 6 weeks of age;
Animals were divided into groups by random sampling;
Animals were individually labeled with an identification number;
Body weights at start of treatment: 129 - 144 g for males and 111 ~ 122 g for females.
Temperature of 23 ± ℃ 2, relative humidity 55± 10 %, ventilation rate 20 times / hour;
Day-night regime: Akira Akira 150 ~ 300 lux, 12 hours (7:00 am lighting, off 7 pm);
Housing: a cage made of metal mesh front floor;
Food: radiation sterilized (Oriental Yeast ),
Tap water was provided ad libitum;
Breeding cages were changed once every two weeks

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
A predetermined amount of test substance dissolved in corn oil (Nacalai Tesque Ltd.).
Administration: test substance solution dosed daily using an injection pump via a probe in the stomach at 0.5 ml per 100 g bw.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis showed that the actual concentrations in the dosing solutions ranged from 98.4 to 102 %, confirming that they had been properly prepared.
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 5, 25 and 125 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
After the exposure period of 28 days, extra five rats per sex of groups 0 and 125 mg / kg were held for a two-week recovery period.

Examinations

Observations and examinations performed and frequency:
1) Observation of general condition: all the animals were observed twice, i.e. every morning and in the afternoon, for the presence of toxic symptoms, abnormal behavior, and dead or moribund animals.
2) Body weight: weekly from the beginning until the end of treatment and after the recovery period.
3) Food consumption: Once a week, feed intake (g/week) was calculated.
4) Clinical pathology: conducted twice, i.e. at the end of treatment and at the end of the recovery period.
a. Hematological tests:
First, EDTA-3K was added to the samples as anticoagulant. Analysis included white blood cell count (WBC: dark-field plate method), number of red blood cells (RBC: dark-field plate method), hemoglobin (HGB: cyanmethemoglobin method), hematocrit (HCT), mean corpuscular volume ( MCV: RBC, HCT calculated) hemoglobin mean corpuscular (MCH: HGB, RBC calculated) concentrations of hemoglobin mean cell (MCHC: HGB, HCT calculated), platelet count (PLT: plate method darkfield) differential count using the automatic blood analyzer THMS H6000 (Technicon Corporation, USA). Reticulocyte (RC) for the rate calculation, Kyapirotto whole blood (Terumo Corporation) after staining, and microscopic examination of blood smears. After addition of sodium citrate on blood plasma, measurement of prothrombin time (Quick one stage method), activated partial thromboplastin time method (clot) and fibrinogen (thrombin time method), blood coagulation and automatic measuring equipment KC-40 (Amelung Germany Inc.) was measured using.

b. Blood chemistry:
Measurement of serum total protein (burette method), albumin (BCG method), A / G ratio (calculated), glucose (glucose oxidase method), triglycerides (enzymatic method), total cholesterol (enzymatic method), urea nitrogen (BUN: improved method urease), total bilirubin (dye method diazo), calcium (Arsenazo III dye method), inorganic phosphorus method (ammonium molybdate), Sodium (electrode method), potassium (electrode method) and chloride (electrode law) to EKTACHEM 700N (Kodak, USA), and creatinine (alkaline picric acid colorimetric method), glutamate oxaloacetate transaminase (GOT: Karmen Improvement Act), glutamate pyruvate transaminase (GPT: Karmen Improvement Act), γ-glutamyl transpeptidase (γ-GTP: Szasz modified method) and alkaline phosphatase (ALP: Bessey-Lowry-Brock Improvement Act) to CentrifiChem ENCOREII (Baker Inc., USA) .

c. Urinalysis:
Prior to the examination of blood, urine collection device for 24 hours (until 10 am to 10 am the next day) and urine collection, urine volume, color and turbidity after the test, the urine specific gravityurinometer was measured using UR-S ((Co) Atago). Also, urine sediment after centrifugation of applying Sternheimer staining variations, and microscopic examination.

Further, measurement of pH, occult blood, ketones, sugars, proteins, bilirubin and urobilinogen on, N-Marutisutikkusu SG test strips (Miles Sankyo (Co)) and CLINITEK 200 (Miles Inc., USA).
Sacrifice and pathology:
At the end of the period of treatment and of recovery, autopsy was performed on animals anesthetized with ether.
The brain, liver, kidney, spleen, adrenals, testes and ovaries were separated and weighed, relative organ weights (organ/body weight ratio) were calculated. Other organs dissected: pituitary gland eye, thyroid (including parathyroid gland), heart, lungs, stomach, bladder, bone marrow (femur) were observed for macroscopical changes in organ tissue and all organs were fixed in 10% neutral buffered formalin solution. Histopathological examination of organ tissue was performed on heart, liver, spleen, kidney, adrenal gland and femurof the high-dose group and control group were performed. Slice preparation was prepared according to conventional methods and stained with hematoxylin and eosin for microscopic examination.
Statistics:
Body weights, food consumption, hematology laboratory values, blood chemistry values, urinalysis values (only urine volume and urine specific gravity), organ weight and organ weight body weight ratio, were tested for variance (ANOVA) and subsequently a Bartlett's test was performed. After analysis of the distribution and variance the Dunnett's multiple comparison test was performed in case of equal numbers of samples in each group or the Duncan multiple range test when unequal sample sizes. In the case of unequal variance the Kruskal-Wallis rank test was used instead of the Bartlett test. Regarding the correlation between response and dose, the Jonckheere test was used to test the significance of the trend.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Mortality: During the 28-d treatment and the recovery period, no deaths occurred in any of the groups. Clincial signs: cyanosis in both sexes receiving the 125 mg/kg dose, which continued well into the recovery period.
Mortality:
mortality observed, treatment-related
Description (incidence):
Mortality: During the 28-d treatment and the recovery period, no deaths occurred in any of the groups. Clincial signs: cyanosis in both sexes receiving the 125 mg/kg dose, which continued well into the recovery period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
During the treatment period no significant effects on body weight were observed for any of the groups tested. At the end of the recovery period, mean male body weight of the 125 mg/kg was significantly higher than that observed in the control group.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
During the treatment period no significant effects on food consumption were observed for any of the groups tested. In the recovery period, male food consumption at 125 mg/kg was significantly higher than that of the control group.
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Anemia in both sexes given 25 and 125 mg/kg (lower red blood cell count and hemoglobin concentration ). In the 5-mg/kg group, hemoglobine concentration was significantly lower compared to the control and apparently dose related.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At 125 mg/kg, blood chemistry showed elevated total bilirubin in both sexes, high GOT in males, creatinine in females. At the end of the recovery period, all had returned to control values except for total bilirubin in males at 125 mg/kg, which was lower.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Increase in urine volume and yellowish-brown urine in both sexes given 125 mg/kg.
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Absolute and relative spleen weights had increased in both sexes receiving 125 mg/kg and females of the 25 mg/kg group (see remarks).
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Darkening of the spleen was recorded for all rats exposed to 25 and 125 mg/kg. Enlargement of the liver and blackening of the liver and kidney in both sexes of the 125 mg/kg group.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Congestion, increase in hematopoiesis or deposits of pigment in the spleen, increase in hematopoiesis in the bone marrow, extramedullary hematopoiesis and deposits of pigment in the liver and proximal tubules of the kidney.
Histopathological findings: neoplastic:
not examined

Effect levels

Dose descriptor:
NOEL
Effect level:
5 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Heamatology, organ weights (spleen, liver) and appearance (liver, kidneys), histopathology.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Under the conditions of the present study, a NOEL of 5 mg/kg bw/day is found for both males and females .
Executive summary:

Twenty-eight-day Repeat Dose Oral Toxicity Test of N-Methylaniline in Rats

Route

:

Oral

Dosage

:

0 (vehicle), 5, 25, 125 mg/kg/day

Vehicle

:

Corn oil

Number of animals

:

Males, 5; females, 5/group

Administration period

:

Males and females, 28 days

Terminal kill

:

Males and females, days 29 to 43

GLP

:

Yes

Cyanosis were observed in both sexes receiving the 125 mg/kg dose. Hematological examination revealed anemia in both sexes given 25 and 125 mg/kg. Urinary examination revealed an increase in urine volume and yellowish-brown urine in both sexes given 125 mg/kg. Absolute and relative spleen weights were increased in both sexes receiving 125 mg/kg and females of the 25 mg/kg group. Macroscopically, enlargement of the liver and blackening of the liver and kidney in both sexes of the 25 and 125 mg/kg groups. Histopathologically, congestion, increase in hematopoiesis or deposits of pigment in the spleen, increase in hematopoiesis in the bone marrow, extramedullary hematopoiesis and deposits of pigment in the liver and proximal tubules of the kidney were observed in both sexes of all treated groups. The NOELs are considered to be 5 mg/kg/day for both males and females under the conditions of the present study.