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Carcinogenicity

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Description of key information

A chronic bioassay demonstrated lung and liver tumors, primarily in mice.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records
Reference
Endpoint:
carcinogenicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
9 Nov 2004 - 10 Nov 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline carcinogencity study by the U.S. National Toxicology Program
Reason / purpose for cross-reference:
reference to same study
Qualifier:
equivalent or similar to guideline
Guideline:
other: Specifications for the Conduct of Studies to Evaluate the Toxic and Carcinogenic Potential of Chemical, Biological, and Physical Agents in Laboratory Animals for the National Toxicology Program (NTP) October 2006
Principles of method if other than guideline:
The guideline "Specifications for the Conduct of Studies to Evaluate the Toxic and Carcinogenic Potential of Chemical, Biological, and Physical Agents in Laboratory Animals for the National Toxicology Program (NTP), October 2006" is similar to the OECD Combined Toxicity/Carcinogenicity Guideline 453.
GLP compliance:
yes
Species:
mouse
Strain:
B6C3F1
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: 5-6 weeks
- Fasting period before study: 11-12 days
- 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 weekly (mice).
- Diet (e.g. ad libitum): Irradiated NTP-2000 wafer feed (Zeigler Brothers, Inc., Gardners, PA), available ad libitum
- Water (e.g. ad libitum): Tap water via glass water bottles with stainless steel sipper tubes, available ad libitum, changed twice per week.
- Acclimation period: up to 12 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 50% +/-15%
- Air changes (per hr): 10/hour
- Photoperiod (hrs dark / hrs light): 12 /12

IN-LIFE DATES: From: 9 Nov (males) or 8 Nov (females) 2004. To: 10 Nov (males) or 8 Nov (females) 2006
Route of administration:
oral: drinking water
Vehicle:
water
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dosing solutions were prepared every 4 weeks for the 2-year studies by mixing the test material with tap water. The pH was adjusted if necessary with acetic acid to bring it to 6-7.5 pH units. Stability studies of 10 µl/ml formulations were performed periodically by HPLC with ultraviolet light detection and found to be stable (± 10%) under the animal room conditions. They were made and stored in sealed polyethylene bottles. The water was administered to animals in glass bottles with stainless steel sipper tubes and Teflon® seals. Analytical data indicate that little or no volatization occurred under these conditions.
Duration of treatment / exposure:
105 weeks
Frequency of treatment:
daily, 7 days per week
Remarks:
Doses / Concentrations:
312.5 mg/L
Basis:
actual ingested
Remarks:
Doses / Concentrations:
625 mg/L
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1250 mg/L
Basis:
actual ingested
No. of animals per sex per dose:
50 per sex per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
Male mice were housed 1 per cage, females were housed 5 per cage
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 3 months,
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 3 months and every 4 weeks thereafter. Female mice were housed 5 per cage, and male mice housed 1 per cage.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
Sacrificed by CO2 asphyxiation.
HISTOPATHOLOGY: Complete histopathology was performed on all mice. In addition to gross lesions and tissue masses, the following tissues were observed microscopically. The following tissues were examined: adrenal gland, bone (with marrow), brain, clitoral gland, esophagus, eyes, gallbladder, Harderian gland, heart, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, liver, lung, lymph nodes (mandibular and mesenteric), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, skin, salivary gland, spleen, stomach (forestomach and glandular), testis (with epididymis and seminal vesicle), thymus, thyroid gland, tongue, trachea, urinary bladder and uterus.
Statistics:
The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958), or Cox’s method (1972) for testing two groups for equality. Tarone’s life table test (1975) was used to identify dose-related trends. All reported P values for the survival analysis were two sided.
Survival-adjusted neoplasm rates for each group and each site-specific neoplasm are given. The survival-adjusted Poly-k test (Bailer and Portier, 1988; Portier and Bailer, 1989; Piegorsch and Bailer, 1997) was used to assess neoplasm and nonneoplastic lesion prevalence, with k= 3 as recommended by Bailer and Portier, 1988. Tests of significance included pairwise comparisons of each dosed group with controls and a test for an overall dose-related trend. Continuity-corrected Poly-3 tests were used in the analysis of lesion incidence, and reported P values are 1-sided. The significant of lower incidences or decreasing trends in lesions is represented as 1-P with a post-script “N”.
For continuous variables, organ and body weight data were analyzed with the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972). For skewed distribution values (clinical chemistry, etc.), nonparametric multiple comparison methods were used to assess the significance of the dose-related trends.
Historical control values for a given route of administration were used to assist in interpretation of control and experimental values. The relevant historical control database includes studies undertaken after 2000 when a modified diet was adopted.
Clinical signs:
no effects observed
Description (incidence and severity):
Survival rates were comparable for animals in test groups compared to controls. No clinical findings related to 3-methylpyridine were observed.
Mortality:
no mortality observed
Description (incidence):
Survival rates were comparable for animals in test groups compared to controls. No clinical findings related to 3-methylpyridine were observed.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weights of the high dose group were 10% less than controls in males and females
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 (mid and high doses) and females (high dose) was significantly decreased compared to that by controls.
Ophthalmological findings:
not examined
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Liver and lung tumours and changes in oral cavity epithelial structure were noted.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Abnormalities epithelium were noted in the alveolus and in nasal olfactory tissue.
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Liver and lung adenomas, liver carcinonomas and hepatoblastomas were documented.
Details on results:
Effect observed is an increased incidence of hepatocellular carcinoma/hepatoblastoma and combined alveolar/bronchiolar adenomas or carcinomas in females. The dose-response trend for alveolar/bronchiolar adenoma in females was statistically significant. However, alveolar/bronchiolar carcinomas in females were not significantly increased. When the incidences of alveolar/bronchiolar adenoma or carcinoma (combined) were compared with control incidences in females, statistical significance was achieved for the dose trend and at the high dose of 1250 mg/L. For alveolar/bronchiolar adenoma in females, the tumour incidences are: 5/50 in 0 mg/L, 6/50 in 312.5 mg/L, 4/49 in 625 mg/L and 11/50 of 1250 mg/L (significant dose trend). For alveolar/bronchiolar adenoma or carcinoma (combined) in females, the tumour incidences are: 11/50 in 0 mg/L, 13/50 in 312.5 mg/L, 13/49 in 625 mg/L and 21/50 of 1250 mg/L (significant at the high dose, p < 0.05). The incidence of alveolar/bronchiolar adenoma was significantly increased in mid dose males. For alveolar/bronchiolar adenoma in males, the tumour incidences are: 6/50 in 0 mg/L, 11/50 in 312.5 mg/L, 16/50 in 625 mg/L and 8/50 of 1250 mg/L.

Nasal olfactory epithelial metaplasia and lung alveolar epithelial hyperplasia were significantly increased at the high dose of 1250 mg/L in both males and females (p < 0.05).

In female mice, liver tumours were also observed. For hepatocellular adenoma in females, the tumour incidences are: 38/50 in 0 mg/L, 46/50 in 312.5 mg/L, 46/50 in 625 mg/L and 39/50 of 1250 mg/L (significant at low and mid doses). Hepatocellular carcinomas were significantly increased in all exposed groups of females; the tumour incidences are: 11/49 in 0 mg/L, 20/50 in 312.5 mg/L, 26/50 in 625 mg/L and 23/50 of 1250 mg/L (significant at all doses with a significant dose trend). Hepatoblastomas were increased at all doses in females, but only the dose trend was statistically significant. For hepatocellular carcinoma or hepatoblastoma (combined) in females, the tumour incidences are: 12/49 in 0 mg/L, 21/50 in 312.5 mg/L, 28/50 in 625 mg/L and 24/50 of 1250 mg/L. These tumours were not increased in males.

The conclusions of the draft report are that, under the conditions of the 2-year study:
a) There was equivocal evidence of carcinogenic activity in male mice
b) There was clear evidence of carcinogenic activity in female B6C3F1 mice (increased incidence of alveolar/bronchiolar adenoma or adenoma/carcinoma (combined), p<0.05, and hepatocellular adenoma and carcinoma or hepatoblastoma (combined), p < 0.05). The conclusion meets the NTP criteria for “clear evidence” based on a dose-related increase in malignant neoplasms or an increase of a combination of benign and malignant neoplasms.

The finding of lung tumours after oral administration gives rise to questions of whether inhalation exposure of 3-methylpyridine has occurred. Lung and liver cancer incidence in mice are not highly relevant to human risk assessment.
Relevance of carcinogenic effects / potential:
The finding of lung tumours after oral administration gives rise to questions of whether inhalation exposure of 3-methylpyridine has occurred. Lung cancer incidence in mice, which have a high background incidence, is not highly relevant to human risk assessment. Hepatic carcinoma and hepatoblastoma are not highly relevant to human risk assessment.
Dose descriptor:
NOAEL
Effect level:
< 18 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Equivalent to 26 mg/kg bw/d (males)
Remarks on result:
not determinable
Remarks:
no NOAEL identified. Effect type:carcinogenicity (migrated information)

Table 1: Nonneoplastic Pathology in the Mouth, Nose and Lung in Rats or Mice in the 2-year Bioassay

Observation

Species, sex

Incidence and Dose

Statistical Significance

Comment

Squamous cell papillomas and carcinomas, oral mucosa or tongue

Rat, male and female

1/50 in 312.5 and 1/50 in 625 mg/L

NS

occurred in less than 5% of any group, so not reported in detail.

Nasal olfactory epithelial metaplasia

Rats, females

Mice, males and females

Female Rats: 0/50 in 0, 3/50 in 156.25, 1/50 in 312.5 and 1/50 of 625 mg/L .

Male mice: 8/50 in 0, 12/50 in 312.5, 30/50 in 625, and 41/50 in 1250 mg/L.

Female mice: 2/49 in 0, 2/44 in 312.5, 7/49 in 625, 14/47 in 1250 mg/L

NS

 

 

S at mid and high dose; p < 0.01

 

 

S at high dose; p < 0.01

 

Nasal olfactory epithelial atrophy

Mice, males and females

Male mice: 3/50 in 0, 4/50 in 312.5, 8/50 in 625, and 7/50 in 1250 mg/L.

Female mice: 1/49 in 0, 2/44 in 312.5, 2/49 in 625, 7/47 in 1250 mg/L

NS

 

S at high dose; p < 0.01

 

Lung alveolar epithelial hyperplasia

Mice, males and females

Male mice: 4/50 in 0, 6/50 in 312.5, 6/49 in 625, and 7/50 in 1250 mg/L.

Female mice: 2/50 in 0, 4/50 in 312.5, 3/49 in 625, 8/50 in 1250 mg/L

NS

 

S at high dose; p < 0.01

Females in 625 mg/L group also had 3 bronchiolar hyperplasias, not included.

S: Statistically significant, p < 0.05               

NS: Not statistically significant

Table 2: Incidence of Alveolar/Bronchiolar Tumors (Lung) and Liver in Rats and Mice Exposed to 3 -Methylpyridine

 

Observation

Species, sex

Incidence and Dose

Statistical Significance

Comment

Alveolar/bronchiolar adenoma, carcinomas or combined

Rat, male 

No difference from controls

NS

 

Alveolar/bronchiolar adenoma

 

Alveolar/bronchiolar

Carcinoma

 

Alveolar/bronchiolar adenoma or carcinoma (combined)

Rats, females

 

0/50 in 0, 3/50 in 156.25, 2/50 in 312.5 and 5/50 of 625 mg/L .

 

No difference from controls

 

0.50 in 0, 4/50 in 156.25, 2/50 in 312.5, and 5/50 in 625 mg/L

S, p < 0.05

 

 

NS

 

 

S, p < 0.05

 

 

 

 

Alveolar/bronchiolar adenoma

 

Alveolar/bronchiolar

Carcinoma

 

Carcinomas or combined

Mice, males

6/50 in 0, 11/50 in 312.5, 16/50 in 625, and 8/50 in 1250 mg/L.

 

No difference from controls

 

 No difference from controls

S at mid dose; p < 0.05

 

 

NS

 

 

NS

 

Alveolar/bronchiolar adenoma

 

Alveolar/bronchiolar Carcinoma

 

Alveolar/bronchiolar adenoma or carcinoma (combined)

Mice, females

5/50 in 0, 6/50 in 312.5, 4/49 in 625, 11/50 in 1250 mg/L

 

No difference from controls

 

11/50 in 0, 13/50 in 312.5, 13/49 in 625, 21/50 in 1250 mg/L

 

S dose trend, p < 0.05

 

 

NS

 

 

S at high dose; p < 0.05

 

Hepatocellular adenoma

 

 

Hepatocellular carcinoma

 

 

Hepatocellular carcinoma or hepatoblastoma

 

Hepatocellular adenoma or carcinoma (combined)

 

Mice, females

38/49 in 0, 46/50 in 312.5, 46/50 in 625, 39/50 in 1250 mg/L

 

11/49 in 0, 20/50 in 312.5, 26/50 in 625, 23/50 in 1250 mg/L

 

12/49 in 0, 21/50 in 312.5, 28/50 in 625, 24/50 in 1250 mg/L

 

No data provided

S at low and mid dose, p < 0.05

 

S, all doses and dose trend, p < 0.05

 

S, all doses and dose trend, p < 0.05

 

 

 

S: Statistically significant, p < 0.05               

NS: Not statistically significant

 

Conclusions:
A two-year cancer bioassay in B6C3F1 mice was undertaken with 3-methylpyridine in drinking water. The conclusions of a draft report are available, with findings of “equivocal evidence” of carcinogenic activity in male mice based on increased incidences of alveolar/bronchiolar adenoma and alveolar/bronchiolar adenoma or carcinoma (combined). There was “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 less than 312.5 mg/L, equivalent to 26 mg/kg bw/d in males and 18 mg/kg bw/day in females.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
18 mg/kg bw/day
Study duration:
chronic
Species:
mouse
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 3 -methylpyridine in rats and mice showed lung and liver tumours primarily in mice, which have low relevance for cancer risk in humans. Methodologic issues with the study design (dehydration, potential for inhalation exposure) further complicate interpretation of results. 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 to classify pyridine and methylpyridine derivatives as carcinogens.

Additional information

A two-year cancer bioassay in rats and mice was 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. There is incomplete understanding at this time of the carcinogenic potential of 3 -methylpyridine.

Carcinogencity was also examined in a chronic bioassay with pyridine (a category member with 2 -,3- and 4 -methylpyridine)

in the drinking water of F344 and Wistar rats, and B6C3F1 mice. Because of the development of alpha-2-microglobulinemia in the kidney, a second strain of rat, the Wistar, was used in a second bioassay. 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.

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

Carcinogenicity: via oral route (target organ): respiratory: lung