Pyridine

Repeated dose toxicity: oral

Administrative data

Endpoint:

chronic toxicity: oral

Remarks:

combined repeated dose and carcinogenicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: 2-year Cancer Bioassay conducted according to guidelines by the U.S. National Toxicology Program

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EPA OTS 798.3260 (Chronic Toxicity)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Pyridine

EC Number:

203-809-9

EC Name:

Pyridine

Cas Number:

110-86-1

Molecular formula:

C5H5N

IUPAC Name:

pyridine

Details on test material:

- Name of test material (as cited in study report):
- 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:
- Physical state: clear colorless liquid
- Analytical purity: >99% by elemental analysis, Karl Fischer water analysis, functional group titration and gas chromatography
- Impurities (identity and concentrations): no impurity over 0.1% composition
- Composition of test material, percentage of components: 0.01 mg/ml in deionized water
- Isomers composition:
- Purity test date: > 99%
- Lot/batch No.:00103BV from Aldrich Chemical Company
- 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:Stability of 0.01 mg/ml formulations were performed for at lease 3 weeks when stored in the dark at room temperature.
- Storage condition of test material: stored in the dark at 1-8 degrees C.
- Other:

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Portage, MI
- Age at study initiation: 7-8 weeks
- Weight at study initiation:
- Fasting period before study:
- Housing: Cages and racks were rotated every two weeks during the study. See-Through Systems polycarbonate, solid bottom (Lab Products,
Inc., Rochelle Park, NJ), changed twice per week. Heat-treated hardwood chips (P.J. Murphy Forest Products, Montville, NJ), changed three times per week (male rats), twice per week (female rats), or weekly (mice).
- Diet (e.g. ad libitum): NIH-07 open formula pelleted diet (Zeigler Brothers, Inc., Gardners, PA), available ad libitum
- Water (e.g. ad libitum): Deionized water via glass water bottles with stainless steel sipper tubes, available ad libitum, changed twice per week
- Acclimation period: up to 14 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.4 -234.4
- Humidity (%): 24%-71%
- Air changes (per hr): 10/hour
- Photoperiod (hrs dark / hrs light): 12 /12

IN-LIFE DATES: From: 14 May 1991. To: 4 May 1993

Administration / exposure

Route of administration:

oral: drinking water

Vehicle:

water

Details on oral exposure:

Doses administered were 100, 200 and 400 ppm per day in the drinking water.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Periodic analyses of the dose formulations of pyridine were conducted at the study laboratory and analytical chemistry laboratory using high-performance liquid chromatography. During the 2-year studies, dose formulations were analyzed approximately every 6 to 10 weeks. All dose formulations analyzed and used during the 13-week studies were within 10% of the target concentration. Results of periodic referee analyses performed by the analytical chemistry laboratory during the 13-week studies agreed with the results obtained by the study laboratory.

Duration of treatment / exposure:

102 weeks (2 years)

Frequency of treatment:

daily, in drinking water, available ad libitum.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
8 mg/kg bw/d
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
17 mg/kg bw/d
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
36 mg/kg bw/d
Basis:
actual ingested

No. of animals per sex per dose:

50 per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

Groups of 50 male and 50 female Wistar rats were given drinking water containing 0, 100, 200 or 400 ppm pyridine for 2 years. All animals were observed twice daily. Clinical findings were recorded weekly, and body weights were recorded at the start of the study and weekly. A complete necropsy and microscopic examination were performed. All major tissues were fixed and preserved in 10% neutral buffered formalin, processed, sectioned, and stained with hematoxylin and eosin for microscopic examination. Necropsy was performed on all core (main) study animals. Organs weighed were heart, right kidney, liver, lung, right testis, and thymus. Complete histopathology were completed on the 0 and 1000 ppm dose groups. The following tissues were examined: adrenal gland, bone (with marrow), brain, clitoral gland, esophagus, heart, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, liver, lung, lymph nodes (mandibular and mesenteric), mammary gland (with adjacent skin), nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, spleen, stomach, testis (with epididymis and seminal vesicle), thymus, thyroid gland, trachea, urinary bladder, uterus. Gross lesions and tissue masses were recorded and analyzed. The kidney of male rats and the liver of all rats were also examined in all other exposure groups.

Positive control:

none

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical findings were recorded weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded at the start of the study, weekly for the first 13 weeks,
and then once every 2 weeks until study termination.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water consumption was measured weekly by cage for the first 13 weeks and every 4 weeks thereafter. Rats were housed 5 per cage.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: no

CLINICAL CHEMISTRY: no

URINALYSIS: Yes / No / No data
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Yes / No / No data
- Animals fasted: Yes / No / No data
- Parameters checked in table [No.?] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes / No / No data
- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other:

OTHER: At the end of the 13-week studies, blood was collected from the retroorbital sinus of all rats just before sacrifice for plasma pyridine
concentration measurements.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: A necropsy was performed on all animals, in which organs and tissues were examined for grossly visible lesions and all major tissues were observed microscopically. The following tissues were examined: adrenal gland, bone (with marrow), brain, clitoral gland, esophagus, heart, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, liver, lung, lymph nodes (mandibular and mesenteric), mammary gland (with adjacent skin), nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, spleen, stomach, testis (with epididymis and seminal vesicle), thymus, thyroid gland, trachea, urinary bladder, uterus and gross lesions and tissue masses.

Statistics:

Product-limit procedure of Kaplan and Meier (1958); Cox’s (1972) method for testing two groups of equality; Tarone’s (1975) life table test to identify dose-related trends. All reported P values for the survival analyses are two sided. Organ and body weight data, which have approximately
normal distributions, were analyzed with the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972). Other endpoints which have skewed distributions, were analyzed using the nonparametric multiple comparison methods of Shirley (1977) and Dunn (1964).
Jonckheere’s test (Jonckheere, 1954) was used to assess the significance of the dose-related trends and to determine whether a trend-sensitive test (Williams’ or Shirley’s test) was more appropriate for pairwise comparisons than a test that does not assume a monotonic dose-related trend (Dunnett’s or Dunn’s test).

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Final mean body weights of 400 ppm animals, and second year body weights for the 200 ppm animals were significantly less than controls. Mean body weight gains of males and females exposed to the two highest doses were significantly less than controls.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

Water consumption by males and females exposed to 200 or 400 ppm generally was greater than that by controls.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Increased incidence of renal adenomas and hypertrophy occurred in high dose males. Hepatic centrilobular degeneration, hypertrophy, pigmentation and chronic inflammation occurred in males and females ingesting 400 ppm pyridine.

Details on results:

Survival of male and female rats was not significantly different from controls. Mean body weights of 400 ppm males and females generally were less than those of controls throughout the study and those of 200 ppm males and females were less than those of controls during the second year of the study. Water consumption by males and females exposed to 200 or 400 ppm generally was greater than that by controls. Incidences of renal tubule adenoma and renal tubule adenoma or carcinoma (combined) in male rats exposed to 400 ppm were significantly increased compared to controls and exceeded the historical control ranges. The findings from an extended evaluation (step section) of the kidneys did not reveal additional carcinomas, but additional adenomas were observed in each group of males. In the standard evaluation, an increased incidence of renal tubule hyperplasia was observed in 400 ppm males compared to controls. The severity of nephropathy in males increased slightly with exposure concentration. Incidences of mononuclear cell leukemia in female rats (but not in male rats) were significantly increased in the 200 and 400 ppm groups compared to controls and the incidence in the 400 ppm group exceeded the historical control range. Exposure concentration-related nonneoplastic liver lesions were observed in males and females and the incidences were generally increased in groups exposed to 400 ppm. These included centrilobular cytomegaly, cytoplasmic vacuolization, periportal fibrosis, fibrosis, centrilobular degeneration and necrosis and pigmentation. Bile duct hyperplasia occurred more often in exposed females than in controls.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: 100 ppm (7 mg/kg/day)

2

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: 200 ppm (14 mg/kg/day)

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

A 2 year carcinogenicity toxicity study in F344 rats was undertaken with pyridine at doses from 100-400 ppm in the drinking water (7 to 33 mg/kg bw/d). Consumption of water in males and females was increased over control levels, but body weights were significantly lower in the 400 ppm dose group compared with controls. Doses of 400 ppm resulted in a statistically significant increase in renal adenomas and renal tubule hyperplasia, but not renal carcinomas, in males. Females (but not males) displayed a statistically significant increase in mononuclear cell leukemia. Animals of both sexes displayed an increased incidence of non-neoplastic hepatocellular injury and glandular stomach mineralization. The NOAEL was 100 ppm (7 mg/kg bw/d). The NTP concluded that there was some evidence of carcinogenic activity in male F344 rats based on renal adenomas, and equivocal evidence in female F344 rats of carcinogenic activity of pyridine based on mononuclear cell leukemia.