Pyridine, alkyl derivs.

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Read-across from supporting substance Substance: 2-picoline CAS no.: 109-06-8 EC no.: 203-643-7 Substance category: Pyridine and pyridine derivative substances Read-across justification The American Chemistry Council and the US EPA developed a structure-based category for pyridine and pyridine derivative substances, based on the following similarities: - Closely-related structural and functional features; - Similar or predictable measured and modeled physical properties; - Similar or predictable measured and modeled biodegradability; - Similar or predictable measured and modeled environmental fate and toxicity; - Similar or predictable measured mammalian toxicity; and - Similar use and disposition patterns. All members of the Pyridine and Pyridine derivatives category are structurally-related derivatives of pyridine in that they are based on the pyridine unsaturated ring structure. Piperidine (CAS RN 110-89-4) is simply the saturated ring structure derivative of pyridine. The main category consists of three sub-categories I, II and III. Sub-category I contains the liquid pyridine and alkylpyridines with high water solubility and high vapor pressure: Pyridine (CAS no. 110-86-1), 2-picoline (CAS no. 109-06-8), 3-picoline (CAS no. 108-99-6), 4-picoline (CAS no. 108-89-4) and Pyridine, alkyl derivatives (CAS no. 68391-11-7). The human health assessment concluded that the acute toxicity of sub-category I is low via the oral (rats), high via the dermal (rabbits) and moderate via the inhalation (rats) routes of exposure. The alkyl pyridines are corrosive to rabbit skin and severely irritating to rabbit eye. Pyridine is not a skin sensitizer in guinea pigs. Oral repeated-dose studies with Pyridine in rats showed systemic toxicity ranging from changes in body weights to histopathological changes in the liver at 33 mg/kg bw/day with a NOAEL of 14 mg/kg bw/day. The studies on repeated inhalation toxicity included were a 4-day study with Pyridine (Nikula and Lewis, 1994, published), a 13-day study with 3-picoline (Chen, et al., 1984, EPA Doc. no. 878214922) and a six month inhalation study with 2-picoline (Watanabe et al., 1979, EPA Doc. no. 40-8341086), as part of the summary on repeated dose toxicity. Notably, the sub-chronic study on 2-picoline was considered most reliable, with a Klimisch rating of 1B vs. 2D and 2A for the Pyridine and the 2-picoline studies. None of the repeated dose inhalation studies allowed establishing a threshold for systemic toxicity. The study on Pyridine involved only the histopathology exam of nasal tissue after inhalation exposure, which was not according to guidelines. The studies on 3-picoline and 2-picoline showed no effects at the highest concentrations tested. Sub-category I of the Pyridine and Pyridine derivative substances group show similar physical-chemical, toxicological and ecotoxicological properties. A data matrix covering all available endpoints was previously published (US EPA Doc. No. 201-14925B1, 2009). An overall summary and detailed robust summaries are included in IUCLID section 13. The available data on the substances of the category reveals similar properties. In general, the classification and labeling of the model constituents is comparable to that of the target substance. Neither substance of the category is considered a PBT or vPvB substance. In summary, based on the data provided the read-across between 2-picoline and Pyridine, alkyl derivs., both part of the Pyridine and Pyridine derivative substances category (Sub-category I), is justified.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Report date:

1979

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.29 (Sub-Chronic Inhalation Toxicity:90-Day Study)

Deviations:

yes

Remarks:

Exposure period: 6 month

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

yes

Remarks:

Exposure period: 6 month

GLP compliance:

no

Limit test:

no

Test material

Test material information

Constituent 1

Test material form:

gas under pressure: refrigerated liquefied gas

Details on test material:

- Name of test material: Alpha picoline

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 8 weeks
- Weight at study initiation: 559 to 573 (males), 302 to 314 (females)
- Fasting before study: Yes
- Diet: Ad libitum except during exposure
- Water: Ad libitum except during exposure
- Acclimation period: 19 days prior to initiation of the study

IN-LIFE DATES:
- From: 9-14-77
- To 3-14-78 (last exposure)

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Rochester type chambers
- Source and rate of air: The vapor was swept into the inlet of the exposure chamber with compressed air where it was further diluted by tempered air to the desired concentration
- Temperature, humidity, pressure in air chamber: The a-picoline vapor was generated by metering the liquid compound at a controlled rate into a temperature regulated vaporization flask (130°C).
- Air flow rate: The chambers were operated under dynamic airflow conditions (40-50 ~/min) with temperature and humidity controlled air.

TEST ATMOSPHERE
- Brief description of analytical method used: Gas liquid chromatography using direct gas sampling loops
- Samples taken from breathing zone: Yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The gas chromatographic conditions were as follows:
Instrument: Varian Model 2400 gas chromatograph
Detection: Hydrogen flame ionization
Column: 10' x 1/8" stainless steel column packed with 10% SP-1000 on 100/120 mesh Chromo sorb WAW
Temperatures:
- Column 160 °C
- Detector 235 °C
Gas Flow Rates:
- He (carrier): 120 ml/min
- H2: 30 ml/min
- Air: 300 ml/min
Standards for the analysis were prepared by injecting known volumes of the test material into Saran bags containing a known volume of air. The target concentrations were 5, 35 and 100 ppm.
For results see table 1 below.

Duration of treatment / exposure:

Approximately 6 months (males 124 exposures in 182 days, females 125 exposures in 183 days)

Frequency of treatment:

6 hours/day, 5 days/week

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0, 5, 35, or 100 ppm
Basis:
nominal conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0, 5.1 ± 0.5, 35.4 ± 1.6 and 99.8 ± 6.4 ppm
Basis:
analytical conc.

No. of animals per sex per dose:

20 rats (10/sex) per dose

Control animals:

yes, concurrent no treatment

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule for examinations: Prior to exposure and at monthly intervals thereafter
- Parameters: observations of respiration, piloerection, exophthalmos/palpebral closure and tremor

BODY WEIGHT: Yes
- Time schedule for examinations: Twice weekly for the first 4 weeks of exposure and weekly thereafter.

ORGAN WEIGHT: Yes
- Organ weights for liver, kidneys, brain, heart, and testes (male) were obtained from all animals at necropsy, and organ/body weight ratios were calculated.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the termination of the exposure (6 months)
- Anaesthetic used for blood collection: Yes (identity) / No / No data
- Animals fasted: Yes
- How many animals: 10
- Parameters: Hematological parameters included red blood cell counts, white blood cell counts (Coulter Counter, MOdel ZBI, Coulter Electronics, Hialeah, FL) and white blood cell differential counts, hemoglobin concentration (Hemoglobinometer, Coulter Electronics, Hialeah, FL) and packed cell volume.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the termination of the exposure (6 months)
- Animals fasted: Yes / No / No data
- How many animals: 10
- Parameters: Clinical chemistry studies included the determination of blood urea nitrogen (BUN), serum glutamic pyruvic transaminase (SGPT), serum alkaline phosphatase, and serum glucose levels (Centrifichem Methods, Union Carbide Co., Rye, NY).

URINALYSIS: Yes
- Time schedule for collection of urine: At the termination of the exposure (6 months)
- Animals fasted: Yes
- How many animals: 10
- Parameters: Urinalysis included determination of pH, sugar, ketones, bilirubin, occult blood, urobilinogen, protein (Ames Multilabstix, Ames Co., Elkhart, IN) and specific gravity (TS Meter, AO Optical, Buffalo, NY).

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Prior to exposure and at monthly intervals thereafter
- Dose groups that were examined: Five rats per sex per group
- Battery of functions tested: 3 simple tasks for evaluating sensory stimuli and motor function responses were conducted. For the visual placing test, the animal is lifted by the tail to a height of about 20 cm above a bench top and lowered to the bench top to elicit the visual placing response which is usually characterized by an extension of both fore and hind limbs before contact. The wire maneuver test is conducted by allowing the animal to grasp a horizontal wire with its forelimbs (hanging in midair) and subsequently observing the ability of the animal to lift its hind limbs to the wire. The hind limb. grasp test consists of placing the animal head down on a vertical, cylindrical post and observing the ability of the hind limbs to grasp the post, thus preventing the animal from immediately falling.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Gross necropsies were conducted on all animals.

HISTOPATHOLOGY: Yes
- Representative specimens of organs and tissues were taken from all animals and fixed in 10% neutral buffered formalin. Histopathologic examination of all gross lesions, liver, kidney, lung, brain and spinal cord were conducted on all rats of the control and 100 ppm exposure groups. The above tissues were processed by conventional methods, embedded in paraffin blocks, sectioned (6-8 um), stained with hematoxylin and eosin and evaluated by light microscopy. Particular attention was given to examination of nervous tissue. The brain was fixed in toto and the spinal cord was removed intact within the vertebral column. After fixation, the brain was serially crosssectioned at 5 different levels, starting in the rostral portion of the cerebrum and proceeding caudally to include the components of the brain stem and cerebellum. The spinal cord was removed from the vertebral canal before preparing both longitudinal and cross-sectional specimens from the cervical, thoracic and lumbosacral regions. The sections of brain and spinal cord were stained with Margolis' and Pickettts Luxol Fast Blue-Periodic Acid-Schiff Hematoxylin Method (Putt, 1972). All sections were examined by light microscopy.

Statistics:

Hematology, clinical chemistry, organ and body weight data were analyzed using an analysis of variance and Dunnett's test (Steel and Torrie, 1960). The level of significance in all cases was p <0.05. The sensory and motor function test results were analyzed using Fisher's exact probability test at a significance level of p <0.05, (Siegel, 1956).

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
The animals were observed daily throughout the study and there were no signs of toxicity observed at any treatment level at any time. There were no mortalities throughout the study. The monthly observations made during the sensory/motor function testing which includes observations on respiration, piloerection, exophthalmos/palpebral closure, tremor and the visual placing test were normal for all animals tested throughout the study. Based on results for the wire maneuver and hind limb clamping tests there were no statistically significant differences noted which indicated any deficit in motor function performance due to treatment. The sporadic statistically significant findings all suggested that the treatment groups performed better than controls. These findings, however, did not demonstrate any strong dose or time response relationship and were thus attributed to normal variation. It was observed that after the second or third months on study it was very difficult to judge the male rats on the wire maneuver test because they had gained so much weight that none of the rats, including the controls, were able to perform as they had done earlier in the study.
Therefore, the wire maneuver results for the latter half of the study are not very meaningful. While not statistically significant the wire maneuver results for male rats in the 5th and 6th month seem to suggest there may have been a deficit in response at the highest exposure level. However, for the reasons mentioned above and the lack of any histopathologic evidence suggesting damage to the nervous system these results are likely due to animal variation and have little or no toxicologic significance.

BODY WEIGHT AND WEIGHT GAIN
Body weight data showed no differences between control and rats exposed to the test item. The relative weights of heart and liver in males exposed to 5 and 100 ppm respectively were the only values which were statistically increased when compared to control values. The increase in the relative heart weight was barely significant statistically and did not demonstrate a dose-response relationship. Since there were no other indications of toxicity, this observation was considered to be of no toxicologic significance. The increase in the relative liver weight in males exposed to 100 ppm test item was associated 'with an increased absolute liver weight. Similarly, in females exposed to 100 ppm test item there was a suggestion that both the absolute and relative liver weight was increased with no dose response relationship observed. Histopathologic examination did not reveal any treatment related effects in either males or females exposed to 100 ppm attest item. Thus the suggestion of an increased liver weight mayor may not be of toxicologic significance.

HAEMATOLOGY, CLINICAL CHEMISTRY, URINALYSIS
The only statistically significant difference observed was a decrease in the red blood cell (RBC) count in male and female rats exposed to higher concentrations of test item. To confirm whether this was real, the RBC count for males was repeated. Since the effect was not reproducible, it was concluded that the initial observation was an artifact. Female rats exposed to 100 ppm test item also showed a statistically significant decreased RBC count. However, the PCV was not different from controls. Furthermore, the RBG value, while statistically significant, was within the normal range (within ± 1 S.D.) of historical control rats of similar age and sex. Therefore this observation was not judged to be toxicologically significant. No differences were observed in group means for control and exposed animals.

PATHOLOGY
All observations were considered to be spontaneous in nature and were typical of those commonly observed in rats of this strain and age. Thus, no grossly visible lesions could be attributed to exposure to the test item. Histopathologic examination was conducted on all rats in the control and 100 ppm exposure groups. There were no histopathologic observations considered to be related to test item exposure. All observations were considered to be spontaneous in nature and were typical of those commonly observed in rats of this strain and age.

Effect levels

Dose descriptor:

NOEC

Effect level:

> 100 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Male and female Sprague-Dawley rats were exposed to 0, 5, 35 or 100 ppm test item vapor 5 days per week for 6 months. There were no effects judged to be related to treatment by the parameters measured. The parameters included urinalysis, clinical chemistry, hematology, body weights, organ weights, sensory/motor function performance and gross pathology. Furthermore, histopathologic examination with special emphasis on the central nervous (CNS) conducted on the control and 100 ppm exposure groups did not indicate any effects attributable to exposure to the test item.

Executive summary:

Determination of acute inhalation toxicity was carried out with 2-picoline (CAS no.: 109-06-8, EC no.: 203-643-7). The read-across between 2-picoline and Pyridine, alkyl derivs., both part of the Pyridine and pyridine derivative substances category (sub-category I), is justified.

A sub-chronic repeated dose toxicity study (6 month) was carried out equivalent or similar to EU Method B.29 and OECD Guideline 413 (Subchronic Inhalation Toxicity). Male and female Sprague-Dawley rats were exposed to 0, 5, 35 or 100 ppm test item vapor 5 days per week for 6 months. Nominal concentrations correspond to 0, 5.1 ± 0.5, 35.4 ± 1.6 and 99.8 ± 6.4 ppm measured. Parameters assessed included urinalysis, clinical chemistry, hematology, body weights, organ weights, sensory/motor function performance and gross pathology. Furthermore, histopathologic examination with special emphasis on the central nervous (CNS) was conducted on the control and 100 ppm exposure groups.

No signs of toxicity were observed at any treatment level at any time. There were no statistically significant differences noted that indicated any deficit in motor function performance due to treatment. There were no statistically significant differences in the body weights of those rats in any treatment group when compared to the control. There were no statistically significant differences between organs of treatment and control groups that were considered to be treatment-related. Histopathologic examination did not reveal any treatment-related effects in ether males or females exposed to 100 ppm of the test substance. There were no statistically significant differences between hematological parameters of treatment and control groups that were considered to be treatment-related. No grossly visible lesions could be attributed to test substance exposure.

No threshold for systemic toxicity was established. The NOAEC (NOEC) was determined to be 100 ppm equivalent to 0.38 mg/L.