Nicotinonitrile

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Reliable according to guidelines from Japan.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Nicotinonitrile

EC Number:

202-863-0

EC Name:

Nicotinonitrile

Cas Number:

100-54-9

Molecular formula:

C6H4N2

IUPAC Name:

pyridine-3-carbonitrile

Constituent 2

Reference substance name:

Pyridine-3-carbonitrile

IUPAC Name:

Pyridine-3-carbonitrile

Details on test material:

- Name of test material (as cited in study report): 3-Cyanopyridine
- Molecular formula (if other than submission substance):
- Molecular weight (if other than submission substance):
- Smiles notation (if other than submission substance):
- InChl (if other than submission substance):
- Structural formula attached as image file (if other than submission substance): see Fig.
- Substance type: white solid
- Physical state: solid
- Analytical purity: 99.9%
- Impurities (identity and concentrations):
- Composition of test material, percentage of components:
- Isomers composition:
- Purity test date:
- Lot/batch No.: 709S4067
- Expiration date of the lot/batch:
- Radiochemical purity (if radiolabelling):
- Specific activity (if radiolabelling):
- Locations of the label (if radiolabelling):
- Expiration date of radiochemical substance (if radiolabelling):
- Stability under test conditions: stable
- Storage condition of test material:
- Other: melting point of 50 degrees C and has a solubility in water at 20 degrees C of 12.3%

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: CD, Japan Charles River K.K.
- Age at study initiation: four weeks old
- Weight at study initiation: males: 213.1-245.8 g, females: 158.8-180.5 g
- Fasting period before study:
- Housing: three to a cage during quarantine and tabing and individually following division into groups in stainless steel hanger cages with a barrier system
- Diet (e.g. ad libitum): solid feed (MF Oriental Kobo Kogyo K.K.) sterilized by high-pressure steam treatment
- Water (e.g. ad libitum): sodium hypochlorite (about 2 ppm) added to water
- Acclimation period: 15 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-26 degrees C
- Humidity (%): 45-65 %
- Air changes (per hr): 13 times/hour
- Photoperiod (hrs dark / hrs light): illumination period of 7-19 hours

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

Administration was oral, with forced administration being conducted once a day repeatedly for 28 days using a stomach tube. The administered volume was 5 mmL/kg calculated based on the most recent body weight of each individual.

Frequency of treatment:

Once daily for 28 days

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0 mg/kg
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
5 mg/kg
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
30 mg/kg
Basis:
actual ingested

Doses / concentrations 4

Remarks:

Doses / Concentrations:
180 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

Six males and six females in each of four test groups comprising the above three dose groups and a control group administered injection-use water.

Control animals:

yes

Details on study design:

The doses were set based on the results of two-week repeat dose toxicity preliminary tests. In these tests, death was observed with the administration of 600 mg/kg, inhibited increase in body weight was seen at 200 mg/kg and above, and reduced feed consumption was seen at 60 mg/kg and above. Further, in blood chemical analysis, an increase in phospholipids, an increase in liver weight, and a decrease in heart weight were observed at 60 mg/kg and above. Accordingly, the high dosage in the present test was set to 180 mg/kg, and below that the middle and low dosages in the present test were set to 30 and 5 by dividing by a common ratio of 6.

Examinations

Observations and examinations performed and frequency:

General condition and presence or absence of dead animals: twice daily during the administration period, once before and once after administration, and twice daily during the recovery period, once in the morning and once in the afternoon.
Body weight and consumption levels: twice weekly during administration and recovery periods.
Urinalysis: during fourth week of administration and second week of recovery, in a metabolic cage, fresh urine collected between 8:00 and 12:00 under conditions of no food, but with water
Hematological analysis: at the end of the administration period and at the end of the recovery period, after depriving the animals of food for 18 hours
Blood Chemical analysis: after letting blood collected after hematological analysis stand for about 60 minutes at room temperature
Measurement of Organ Weight, Dissection and Histopathological examination: after collecting blood, during dissection

Sacrifice and pathology:

After collecting blood, the animal was bled to death and dissected. The outer intestines, bone, and veins were cut away. The brain, heart, lungs (including bronchia), thymus, submaxillary glands (including the sublingual gland), liver, spleen, kidneys, adrenals, testes, and ovaries were extracted and the (absolute) weights of these organs were measured. Further, based on the body weight on the day of dissection, the ratio of the organ weight to body weight (relative weight) was calculated. The pituitary gland, spinal cord, eyeballs, subaural gland, thyroid gland (including epithelial corpuscles), pancreas, stomach, bladder, femur (including marrow), and other visibly abnormal parts were collected and fixed with a 10 percent neutral buffer formalin solution (the eyeballs were prefixed with glutaldehyde solution and the testes with Buan solution). Paraffin sections were prepared from the saliva glands, heart, liver, kidneys, spleen, adrenals, testes, ovaries, and bladder, and any visibly abnormal tissue of the control group and high-dose group at the end of the administration period. Hematoxylin-eoisin (HE) stain was applied and the sections were examined by optical microscope. Since changes were observed in the liver, spleen, kidneys, bladder, testes, and adrenals, the middle dose group was also examined at the end of the administration period; even the liver and kidneys of the low dose group were examined. These organs were also examined in the control group and high doese group at the end of the recovery period. Some of the organs were observed using special dyes.

Statistics:

The average value and the standard deviation of each group were calculated for body weight, feed consumption, urinalysis (qualitative reactions excluded), hematological examination, blood chemical examination, organ weight, and the organ weight to body weight ratio. The uniformity of dispersion was calibrated by the Bartlett method. When dispersion was uniform, the Dunnett multiple comparison calibration was employed, and when nonuniform, Steel multiple comparison calibration was employed for comparison with the control group. Further, in the histopathological examination, Mann-Whitney U calibration was employed. A level of significance of 5 percent was employed in all cases.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

salivation immediately following administration in males and females at 180 mg/kg from middle of administration period

Mortality:

mortality observed, treatment-related

Description (incidence):

salivation immediately following administration in males and females at 180 mg/kg from middle of administration period

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

inhibited body weight increase in males at 180 mg/kg throughout administration period

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

reduced feed consumption in males and females at 180 mg/kg during initial period of administration

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

increase or tendency toward an increase in leukocyte count, a reduction in erythrocyte count, an increase in reticulocyte count, and an increase in MCV and MCH in males and females at 180 mg/kg, increased eosinophils + decreased lymphosites in 180 mg/kg m

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

increased total protein, albumin, A/G ratio, GPT, total cholesterol and phospholipids for males/females at 180 mg/kg. reduced triglycerides in 180 mg/kg males, reduced cholinesterase and acetylcholinesterase in 180 mg/kg females

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

increase in the quantity of urine, reduction in osmotic pressure and specific gravity, a paling of the color of urine, and a tendency toward a drop in pH were seen in males and females of the 180 mg/kg group

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

An increase, or a tendency to increase in the absolute and relative liver and kidney weights were observed in the males and females in the 180 mg/kg/day group, and the absolute and relative adrenal weights were increased in the males of this same group.

Details on results:

In histological analysis, centrilobular hepatocytic hypertrophy was seen in the livers of males and females in the 30 mg/kg and higher groups; the occurance of hyalin droplets in the proximal tubule epithelium of the kidneys was observed in males in the 30 mg/kg and higher groups; hypertrophy of the zona fasciculata was observed in the adrenals of males and females of the 180 mg/kg group; extramedullary hematopoiesis and hemosiderin deposits in the red pulp were observed in the spleens of males and females of the 180 mgkg group; and necrosis of spermatocytes and spermatids (round), decreased spermatids (elongate), and vacuolation of Sertoli cells were observed in the testes in the 180 mg/kg group. Urocystitis and neutrophil cellular infiltration of the renal pelvis were observed in one female of the 180 mg/kg group. Recovery from the above changes took place, or tended to take place, by the 14-day recovery test.

Effect levels

Dose descriptor:

NOAEL

Effect level:

5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Liver weight and centrilobular hypertrophy in males and females at 30 mg/kg bw/d and higher. Hyaline deposits in the kidneys of males at 30 mg/kg bw/d and higher. Overt toxicity in multiples systems at the high dose of 180 mg/kg bw/d.

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

3-Cyanopyridine was administered by oral gavage to CD rats at doses of 5, 30 and 180 mg/kg bw/day, once daily for 28 days, followed by a 14 day recovery period. Major findings after 28 days included decrease body weight gain in the 180 mg/kg group, increased absolute and relative organ weights (liver, kidney and adrenal in 180 mg/kg males and females), centrilobular hepatocytic hypertrophy in the livers of males and females in the 30 mg/kg and higher groups, and vacuolation of Sertoli cells in the testes of 180 mg/kg males, accompanied by decreased spermatocytes and spermatids. The NOAEL of 3-cyanopyridine under the conditions of this test was 5 mg/kg/day.