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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
repeated dose toxicity: oral
Remarks:
combined repeated dose and carcinogenicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 November 2004-17 November 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Chronic combined oral toxicity/carcinogenicity study 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:
2012

Materials and methods

Test guideline
Qualifier:
according 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
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
3-methylpyridine
EC Number:
203-636-9
EC Name:
3-methylpyridine
Cas Number:
108-99-6
Molecular formula:
C6H7N
IUPAC Name:
3-methylpyridine
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): beta-picoline
- Physical state: clear liquid
- Analytical purity: >96% by Karl Fischer water analysis, functional group titration and gas chromatography
- Impurities (identity and concentrations):
- Composition of test material, percentage of components: 0.01 mg/ml in deionized water
- Isomers composition:
- Purity test date: > 96%
- Lot/batch No.:11108C1 from Aldrich Chemical Company
- Stability under test conditions:Stability of 0.01 mg/ml formulations was confirmed for at least 3 weeks when stored in amber bottles 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: Taconic Farms, Germantown, NY, USA
- Age at study initiation: 6 weeks
- Fasting period before study: not applicable
- Housing: Cages were polycarbonate and racks were rotated every two weeks during the study. Bedding: irradiated Sani-Chips (P.J. Murphy Forest Products Corp., Montville, NJ), changed weekly
- Diet: 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 at least twice per week
- Acclimation period: up to 12 days.
- Method of kill: carbon dioxide asphyxiation

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.2 ± 3
- Humidity (%): 50% ± 15%
- Air changes (per hr): 10/hour
- Photoperiod (hrs dark / hrs light): 12 /12

IN-LIFE DATES: From: 15 November 2004. To: 17 February 2006.

Administration / exposure

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 five times 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:
104-105 weeks
Frequency of treatment:
daily, in drinking water, available ad libitum.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
156.25 mg/L
Basis:
actual ingested
Remarks:
Doses / Concentrations:
312.5 mg/L
Basis:
actual ingested
Remarks:
Doses / Concentrations:
625 mg/L
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 F344 rats were given drinking water containing 0, 156.25, 312.5 or 625 mg/L 3-methylpyridine for 104 weeks.

All animals were observed twice daily. Clinical findings were recorded weekly, and body weights were recorded at the start of the study and weekly. Haematology and clinical chemistry values were measured in animals of the week 14 section of the study. 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 625 mg/L 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. Hematology: hematocrit; hemoglobin concentration; erythrocyte, reticulocyte, nucleated erythrocyte, and platelet counts; mean cell volume; mean cell hemoglobin; mean cell hemoglobin concentration; and leukocyte count and differentials. Clinical chemistry: urea nitrogen, creatinine, protein, albumin, alanine aminotransferase, alkaline phosphatase, creatine kinase, sorbital dehyrodrogenase, bile acids.
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 for the first 4 weeks then monthlyafterwards.

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 monthly 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. Rats were housed 2-3 per cage.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

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:
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.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Final mean body weights and body weight gains of rats (male and female) exposed to 625 mg/L were slightly less (9-10%) than those of the 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 male and female rats exposed to 625 mg/L was less than that by controls due to poor palatability.
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:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Chronic progressive nephropathy in males was increased in 625 mg/L groups. Squamous cell tumours were observed in the oral cavity.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Alveolar epithelium hyperplasia (increased Type II pneumocytes without cellular atypia) and squamous metaplasia (noncuboid epithelia) were increased, but not significantly, in all exposed groups of females compared to those of controls.
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
See Carcinogenicity Section 7.7.1
Details on results:
There was no effect of chronic exposure to 3-methylpyridine on mortality or clinical toxicity. Decreased water consumption was evident in 625 mg/L males and females compared to controls throughout the study. Body weights were slightly decreased in high dose males (10%) and females (9%)compared to controls. There was some evidence of lung tumours in female rats. Incidences of alveolar/bronchiolar adenoma in high dose females were significantly greater than those in concurrent controls, and slightly higher than in historical controls. Lung tumours (alveolar/bronchiolar adenoma and/or carcinoma) were observed in female rats at a rate slightly above the historical control levels, with a statistically significant trend test.
Alveolar epithelial hyperplasia and metaplasion, and squamous cell papilloma or carcinoma (combined) in the oral cavity were observed (1-2 per 50 animals). But due to the low incidences, the lesions are not considered to be related to the treatment.



Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
312.5 mg/L drinking water
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: equivalent to 12 mg/k bw/day in males and 14 mg/kg bw/d in females.
Dose descriptor:
LOAEL
Effect level:
625 mg/L drinking water
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: equivalent to 22 mg/kg bw/d in males and26 mg/kg bw/d in females. This dose resulted in body and liver weight changes, water consumption decreases, and nephropathy in males.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

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 chronic toxicity/carcinogenicity study (2-year) in F344 rats was undertaken with 3-methypyridine at doses of 156.25, 312.5 and 625 mg/L in the drinking water (6-7, 12-14, and 38 mg/kg bw/d, respectively in males and females) and a draft report is available for review.. There were no substance-related effects on survival or clinical findings. Body weight changes were similar in exposed animals and control animals. Consumption of water in males and females was decreased at the highest dose, with evidence of dehydration. A dose of 625 mg/L resulted in an increase in alveolar/bronchiolar adenomas and adenomas/carcinomas (combined) in female rats. The NOAEL is estimated to be 312.5 mg/L or 12-14 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.