4-methylpyridine

Carcinogenicity

Description of key information

Pyridine and pyridine derivatives are not able to classified as human carcinogens, according to IARC.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Endpoint:

carcinogenicity: oral

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: Reliable study performed by a guideline protocol under GLP.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

EPA OPP 83-5 (Combined Chronic Toxicity / Carcinogenicity)

GLP compliance:

yes

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic Farms (Germantown, NY), USA
- 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. Water consumption was measured weekly by cage for the first 13 weeks and every 4 weeks thereafter.
- Acclimation period: up to 14 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.9 -26.7
- Humidity (%): 25%-78%
- Air changes (per hr): 10/hour
- Photoperiod (hrs dark / hrs light): 12 /12

IN-LIFE DATES: From: 23 April1991. To: 13 April 1993

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle:
- Amount of vehicle (if gavage):
- Lot/batch no. (if required):
- Purity: 99%

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

102 weeks (2 years)

Frequency of treatment:

daily, in the drinking water, ad libitum

Remarks:

Doses / Concentrations:
7 mg/kg bw/d
Basis:
actual ingested
equivalent to 100 ppm in the drinking water

Remarks:

Doses / Concentrations:
14 mg/kg bw/d
Basis:
actual ingested
equivalent to 200 ppm in the drinking water

Remarks:

Doses / Concentrations:
33 mg/kg bw/d
Basis:
actual ingested
equivalent to 400 ppm in the drinking water

No. of animals per sex per dose:

50 per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

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.

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.

Other examinations:

Groups of 50 male and 50 female F344/N 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.

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).

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights of rats exposed to 400 ppm were significantly less than controls. Body weights of rats exposed to 200 ppm were generally lower than that of controls during the second year of the study.

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

Water consumption by the high dose males and females was higher than that of controls.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

High dose males: Renal tubule hyperplasia (increased number of layers of epithelium); hepatic centrilobular cytomegaly and cytoplasmic vacuolization, periportal fibrosis, and centrilobular degeneration, necrosis. High dose females: centrilobular degen.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Incidences of mononuclear cell leukemia in female rats were significantly increased in the 200 and 400 ppm groups. Neoplastic cells were found in the spleen, liver, lung, bone marrow, lymph nodes, adrenal gland, kidney.

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 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.

Relevance of carcinogenic effects / potential:

The finding of mononuclear cell leukemia is of questionable relevance to human cancer risk assessment, due to its high background incidence in the F344 female rat. This was considered to be equivocal evidence for a carcinogenic effect.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Statistically significant increase in renal carcinoma in the high dose males of 36 mg/kg bw/d or 400 ppm.

Remarks on result:

other: Effect type: carcinogenicity (migrated information)

Mononuclear cell leukemia is a common neoplasm in F344 rats. In this study, incidences of mononuclear cell leukemia were increased relative to controls in 200 and 400 ppm F344 female rats. These incidences were near the historical control range for this neoplasm. There was no supportive evidence for an increase in mononuclear cell leukemia in male rats.

Conclusions:

A 2-year cancer bioassay was undertaken on pyridine in the drinking water of male and female F344 rats, at doses of 100, 200 and 400 ppm (equivalent to 7, 14 and 33 mg/kg bw/d). 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 while 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 plus carcinoma in high dose males were significantly increased compared to controls. The severity of nephropathy in males increased slightly with exposure concentration. Incidences of mononuclear cell leukemia in female rats were significantly increased in the 200 and 400 ppm groups compared to controls. Liver lesions were observed in males and females (especially in groups exposed to 400 ppm):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. There was some evidence of carcinogenic activity in male F344 rats based on a finding of increased renal tubule neoplasms.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

7 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

adequate

Carcinogenicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

A bioassay of pyridine resulted in some tumour development in rats and mice, but IARC determined that pyridine was not able to be classified as a human carcinogen. There is insufficient evidence at this time to consider 3 -methylpyridine as a human carcinogen.

There is insufficient evidence to classify pyridine and methylpyridine derivatives as carcinogens.

Additional information

Carcinogencity was examined in a chronic bioassay with pyridine (a member of a category with 2 -, 3- and 4 -methylpyridine) in the drinking water of F344 and Wistar rats, and B6C3F1 mice. Tumors were observed in each strain of rat and mouse, but none were consistent over the 3 species. The NTP concluded that there was some evidence of carcinogenic activity of pyridine in F344 and Wistar rats, and in C3H6F1 mice. The International Agency for Cancer Research (IARC) reviewed this data and epidemiologic evidence and concluded that pyridine was not able to be classified as a human carcinogen.

A two-year cancer bioassay in rats and mice was also undertaken with 3-methylpyridine in drinking water. The conclusions of a draft report are available, with findings of “clear evidence” of carcinogenic activity in female mice based on increased incidences of alveolar/bronchiolar adenoma or carcinoma (combined) in the lung and hepatocellular carcinoma and hepatoblastoma (liver). The NOAEL is the low dose of 312.5 mg/L, equivalent to 26 mg/kg bw/d in males and 18 mg/kg bw/day in females. There was “equivocal evidence” of carcinogenic activity in male mice based on increased incidences of lung adenoma and/or carcinoma (combined).  There was “some evidence” of carcinogenic activity based on lung tumours in female rats at a rate slightly above the historical control levels, with a statistically significant trend test.

The NTP results of the chronic bioassay on 3 -methylpyridine in rats and mice is still in draft status. The increased incidences of lung tumours give rise to questions about whether the route of exposure was solely oral, or if inhalation effects could have occurred. The test substance is corrosive and irritation effects may have impacted the results. The tumours observed occur at high background frequencies and are not highly relevant for human risk assessment. There was no increase in alveolar/bronchiolar tumours in rats or mice exposed to pyridine.

There is incomplete understanding at this time of the carcinogenic potential of 3 -methylpyridine.

Justification for selection of carcinogenicity via oral route endpoint:
experimental result according to a guideline protocol

Carcinogenicity: via oral route (target organ): urogenital: kidneys