4-methylpyridine

Repeated dose toxicity: oral

Administrative data

Endpoint:

chronic toxicity: oral

Remarks:

combined repeated dose and carcinogenicity

Type of information:

migrated information: read-across based on grouping of substances (category approach)

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:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboraties, 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 May1993

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 all treated animals 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):

no effects observed

Description (incidence and severity):

Water consumption by treated males and females was similar to 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):

The incidence of testicular adenoma in rats exposed to 400 ppm was significantly increased compared to controls. Severity of nephropathy was marked in all groups and additional evidence of kidney disease, including mineralization in the glandular stomach

Histopathological findings: neoplastic:

no effects observed

Details on results:

Survival was singificantly decreased in treated rats at the 200 and 400 ppm levels. Mean body weight were significantly lower in all treated rats, compared to control values. There was no significant difference in water consumption of the treated compared to control groups. The incidence of testicular adenoma in rats exposed to 400 ppm was significantly increased compared to controls. The appearance of interstitial cells was similar in both hyperplasia and adenoma and the diagnoses were based on size. Some interstitial cell neoplasms nearly replaced normal tissue. Hyperplasia was defined as a proliferation no larger than the diameter of a seminiferous tubule, and interstitial cell adenoma was larger. Incidences of interstitial cell hyperplasia were observed in control and exposed groups and were slightly, but not significantly, increased in rats exposed to 200 or 400 ppm. Severity of nephropathy was marked in all groups and additional evidence of kidney disease, including mineralization in the glandular stomach, parathyroid gland hyperplasia and fibrous osteodystrophy, was observed in 100 and 200 ppm rats. Relative to the controls, the incidences of hepatic centrilobular degeneration and necrosis, fibrosis, periportal fibrosis and/or pigmentation were increased in exposed males primarily at the 200 and 400 ppm level. The NOAEL was 100 ppm (8 mg/kg bw/d) and the LOAEL was 200 ppm (17 mg/kg bw/d).

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: 100 ppm (8 mg/kg/day)

2

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: 200 ppm (17 mg/kg/day)

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

The NTP does not have a historical database for neoplasms in Wistar rats. In an outside study analyzing neoplasm rates in 1,370 control Wistar rats, a control rate of 3.9% (range, 0% - 22%) was reported for interstitial cell neoplasms of the testis (Walsh and Poteracki, 1994). The rate for interstitial cell adenomas in the NTP study of Wistar rats exposed to 400 ppm pyridine was only marginally outside this historical range, and incidences of this neoplasm were not increased relative to controls in the 100 or 200 ppm groups. This was considered to be equivocal evidence for a carcinogenic effect.

A category of pyridine and methyl pyridine derivatives is comprised of: pyridine, 2-methylpyridine, 3-methylpyridine and 4-methylpyridine. The basis of the category is structural similarity (based on the pyridine unsaturated ring structure) and similar physical properties, environmental fate and ecotoxicity, and mammalian toxicity. Similar toxicological properties derive from similar physical-chemical properties and common pathways of metabolism and elimination among all members of the category. This category is accepted by the U.S. Environmental Protection Agency (EPA). 

Applicant's summary and conclusion

Conclusions:

A 2 year carcinogenicity/ toxicity study in Wistar rats was undertaken with pyridine at doses from 100-400 ppm in the drinking water (8 to 36 mg/kg bw/d). Consumption of water in males and females was comparable to control levels, but body weights were significantly lower in all dose group compared with controls. Significant results include a significant increase in the incidence of testicular adenoma in males at the 400 ppm level. Hyperplasia occurred at the 200 and 400 ppm level, but at levels comparable to that of controls. Hepatic centrilobular toxicity occurred at the 200 and 400 ppm dose levels in males. Nephropathy was not significantly increased due to treatment. The NOAEL was 100 ppm (8 mg/kg bw/d) and the LOAEL was 200 ppm (17 mg/kg bw/d).
Three members of the pyridine and methyl pyridine derivatives category are listed in Regulation (EC) No. 1272/2008, Annex VI: Pyridine (Index #613-002-00-7, 2-methylpyridine (Index # 613-036-00-2) and 4-methylpyridine (Index # 613-037-00-8). The classifications are similar within the category, with the methyl derivatives classified as more corrosive than pyridine. The application of a chemical category is adequate for classification and labelling.