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Description of key information

Repeated dose oral toxicity has been well studied for pyridine and 3-methylpyridine, representative members of a chemical category, and a reliable study exists for repeated dose inhalation toxicity for 3-methylpyridine.   The oral toxicity studies are two year bioassay data in rats (F344 and Wistar) and mice (B6C3F1).  NOAELs are established.   

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
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:
supporting 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
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
EPA OTS 798.3260 (Chronic Toxicity)
GLP compliance:
yes
Limit test:
no
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
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.
Remarks:
Doses / Concentrations:
8 mg/kg bw/d
Basis:
actual ingested
Remarks:
Doses / Concentrations:
17 mg/kg bw/d
Basis:
actual ingested
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
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).
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.
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)
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)
Critical effects observed:
not specified

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). 

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.
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.
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

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Reliable with restrictions; acceptable, well- documented study report which meets basic scientific principles.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
yes
Remarks:
14 days of exposure, 14 days of recovery
GLP compliance:
not specified
Limit test:
yes
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals or test system and environmental conditions:
Rats were eight weeks old and weighing between 211 to 240 g.
Route of administration:
inhalation: vapour
Type of inhalation exposure:
nose only
Vehicle:
air
Details on inhalation exposure:
One group was exposed nose-only, six hours/day, five days/week for two weeks at a concentration of 290 ppm. One group was exposed simultaneously to air only. Five rats per group were sacrificed after the tenth exposure for pathologic examination and five rats per group were allowed to recover for 13 days post exposure.
Duration of treatment / exposure:
6 hours/day, 5 days/week for 14 days.
Frequency of treatment:
Once daily, 6 hours/day, 5 days/week
Remarks:
Doses / Concentrations:
290 ppm
Basis:
nominal conc.
No. of animals per sex per dose:
10 per sex per group.
Control animals:
yes, concurrent vehicle
Details on study design:
290 ppm is equivalent to 1105 mg/m3 of 3-methylpyridine, with a molecular weight of 93.
Observations and examinations performed and frequency:
Rats were weighed and observed daily throughout the exposure and recovery periods, weekends excluded
Sacrifice and pathology:
Organs and tissues examined were the heart, lungs, nasal cavities, trachea, liver, pancreas, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, kidneys, urinary bladder, bone marrow (sternal), spleen, thymus, mesenteric lymph nodes, thyroid, testes, epididymides, adrenal glands, brain and eyes. Mean organ weights and organ-to-body ratios were calculated for the heart, lungs, liver, spleen, kidneys, testes and thymus.
Other examinations:
At the end of the exposure period, blood and urine samples were collected for clinical analysis. Urine samples were collected overnight from ten rats per group prior to the first exposure and after the ninth exposure and from five rats per group on the twelfth day of recovery. Samples were analyzed for volume, osmolality, pH, blood, sugar, protein, bilirubin, urobilinogen and ketone. Each specimen was noted for color and transparency and the sediment from each sample was examined microscopically. Tail blood samples were collected from ten rats per group prior to the first exposure and after the tenth exposure and from five rats per group on the thirteenth day of recovery. Samples were analyzed for erythrocyte count, hemoglobin concentration, mean corpuscular volume, platelet count, leukocyte count and relative numbers of neutrophils, band neutrophils, lymphocytes, atypical lymphocytes, eosinophils, monocytes and basophils. Hematocrit, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration were calculated from the erythrocyte data. In addition, serum activities of alkaline phosphatase, alanine aminotransferase and aspartate aminotransferase and serum concentrations of urea nitrogen, creatinine, total protein and cholesterol were measured.
Statistics:
Least Significant Difference test and Dunnett’s test
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Rats exposed to 290 ppm of the test substance had elevated mean liver weights and liver-to-body weight ratios compared to controls. This change was absent after 13 days of recovery.
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
The only significant finding after exposure of male rats to 290 ppm of the test substance was elevated mean liver weights and liver-to-body weight ratios compared to controls. This effect resolved after 13 days of recovery. There were no significant findings on body weight, food consumption, haematology, clinical chemistry, gross or histopathology.
Dose descriptor:
NOAEC
Effect level:
> 290 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
other: see 'Remark'
Critical effects observed:
not specified

An additional inhalation study of pyridine toxicity (Watanabe, et.al, 1979) is known to exist as part of the U.S.Voluntary HPV submission package on the Pyridine and Pyridine Derivatives Category (See: http://www.epa.gov/chemrtk/hpvis/hazchar/Category%20Pyr%20and%20Pyr%20Derivs_Sept2009.pdf.)  However, legal access to refer to the findings of this study was not able to be arranged. A publically-available robust summary of the study is provided as an attachment here. The conclusion of this 1979 subchronic study is that the NOAEC in CD rats after a 6-month inhalation study (6 hr/day, 5 days/week) is >100 ppm or 323 mg/m3, the highest concentration tested. This is consistent with the NOAEC seen herein, in the shorter Chen and Krauss study, of 290 ppm or 1105 mg/m3.

This assessment from read-across from 3 -Methylpyridine (the registered substance), applies to all members of the chemical category. The 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). 

Conclusions:
The effect of 14-days of inhalatory exposure (nose-only) to 290 ppm (1105 mg/m3) of 3-methylpyridine in male rats was investigated. The only finding was an elevated mean liver weights and liver-to-body weight ratios compared to controls, which resolved after 13 days of recovery. The NOEC was greater than 1105 mg/m3.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
1 105 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
adequate

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The U.S. National Toxicology Program (NTP) has undertaken 2 -year carcinogenicity/chronic toxicity assays of pyridine, as well as of 3 -methylpyridine, in drinking water of rats and mice. These substances comprise a chemical category along with 2 -methylpyridine and 4 -methylpyridine. For pyridine and 3 -methylpyridine, F344 rats displayed a strain-specific nephrotoxicity involving alpha-2 -microglobulinemia. Humans do not develop kidney toxicity by an alpha-2 -microglobulin-mediated mechanism of action; hence, this toxicity is not relevant to humans.

A second strain of rat which is not susceptible to this toxicity, Wistar rats, were added as a supplemental group in the dosing of pyridine. Actual doses from consuming drinking water were comparable between the two strains, at 7, 14 and 33 mg/kg bw/d for F344, and 8, 17 and 36 mg/kg bw/d in Wistars. Nontumorigenic findings in the F344 study were nephrotoxicity due to alpha-2 -microglobulinemia, and in Wistar rats, hepatic centrilobular liver degeneration/necrosis and fibrosis in male rats. The NOAELs were 7 and 8 mg/kg bw/d for F344 and Wistar rats, respectively. 3 -Methylpyridine also resulted in nephrotoxicity due to alpha-2 -microglobulinemia in F344 rats. For pyridine, 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. Consumption of 3 -methylpyridine resulted in lung adenomas and carcinomas, primarily in mice, and liver tumours in female mice. The NOAELs are higher for 3 -methylpyridine, at 12 to 77 mg/kg bw/d. The NOAELs of pyridine were used for development of DNELs for the chemical category. The methylpyridines have been investigated in subchronic and subacute studies in rats. The NOAEC was determined to be greater than 380 mg/m3.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
experimental study according to a guideline method

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
experimental study using a valid protocal

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: liver; urogenital: kidneys

Repeated dose toxicity: inhalation - systemic effects (target organ) digestive: liver

Justification for classification or non-classification

No specific target organ toxicity is proposed for pyridine and methylpyridines as a result of repeated dose exposure. The toxicity observed in chronic studies of pyridine in rats is either strain specific and not relevant to humans, or is a reflection of gastroenterologic (liver) adaptation. The toxicity observed in chronic studies of 3 -methylpydine is tumour development (lung, after oral exposure) or liver, not relevant to humans. There is a question about whether inhalation of orally-consumed 3 -methylpyridine may have contributed to the development of lung tumors. There is insufficient evidence to classify for specific target organ toxicity.