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Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
chronic toxicity: inhalation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
1997-01-13 (first exposure) to 1999-01-11/15 (necropsy date)
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well reported study. Test procedure in accordance with generally accepted scientific standards and described in sufficient detail.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2002

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Groups of 50 male and 50 female B6C3F1 mice (approx. 6-7 weeks old) were exposed to V2O5 aerosols at concentrations of 0, 1, 2 or 4 mg/m3 by inhalation, 6 hours/d, 5 d/wk, for 104 weeks. The body weight was controlled initially and body weight and clinical finding were recorded every 4 weeks (until week 89) and every 2 weeks from week 92 on. Animals were observed twice daily. Necropsy was performed at study end.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solid
Details on test material:
- Name of test material (as cited in study report): Vanadium pentaoxide
- Substance type: technical product
- Physical state: solid
- Analytical purity: ca. 99 %
- Impurities (identity and concentrations): XRD analyses of both lots indicated the presence of vanadium pentoxide with no detectable contaminants. No impurities > 1% .
- Lot/batch No.: Lot 1210490 was used in the 16-day and 3-month studies. Lot 1210140 was used in the 16-day special studies and the 2-year studies.
- Stability under test conditions: stability was monitored throughout the study. No degradation of the bulk chemical was detected.
- Other:
Vanadium pentoxide was obtained from Shieldalloy Metallurgical Corporation (Newfield, NJ) in two lots (1210490 and 1210140).
Lot 1210490, was identified as vanadium pentoxide using X-ray diffraction (XRD) analyses and infrared and ultraviolet/visible spectroscopy and by the study laboratory using infrared spectroscopy.
Lot 1210140 was identified using infrared and ultraviolet/visible spectroscopy and by the the study laboratory using XRD analysis.
The purity of lot 1210490 was determined using elemental analyses, weight loss on drying, spark source mass spectrometry, energy-dispersive X-ray (EDX) spectroscopy, and potentiometric titration.
The purity of lot 1210140 was determined using weight loss on drying, potentiometric titration, and ICP/AES.
- No further information on test material was stated.

Test animals

Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Taconic Farms, Inc. (Germantown, NY)
- Age at study initiation: average age: 6 or 7 weeks old on the first day of the study
- Weight at study initiation: average body weight for week 1: 24.0-24.4 g (males) and 19.6-19.9 g (females)
- Housing: housed individually; stainless steel wire bottom (Hazleton Systems, Inc., Aberdeen, MD); cages and racks were rotated weekly.
- Diet: ad libitum, except during exposure periods; NTP-2000 pelleted diet (Zeigler Brothers, Inc., Gardeners, PA), changed weekly
- Water: ad libitum; tap water (Richland, WA, municipal supply water used) via automatic watering system
- Acclimation period: quarantined for 16 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): ca. 23.9± ca.2°C (75° ± 3° F)
- Humidity (%): 55% ± 15%
- Air changes (per hr): 15/hour
- Photoperiod (hrs dark / hrs light): 12 hours/day

IN-LIFE DATES: From: 1997-01-13 (first exposure) to 1999-01-11/15 (necropsy date)
- No further information on test material was stated.

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
whole body
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: MMAD = 1.0-1.3 µm, GSD = 2.3-2.8
Details on inhalation exposure:
- The generation and delivery system used in the 16-day special studies and the 2-year studies consisted of a linear dust feeder, a particle attrition chamber, and an aerosol distribution system. The linear dust feeder, a slide-bar dust-metering device, was composed of a shuttle bar, body, outlet port, and hopper. As the compressed-air-driven shuttle bar slid back and forth during generation, the metering port aligned with the hopper, which served as a reservoir for the bulk chemical, and was filled with a small amount of vanadium pentoxide powder. As the shuttle bar slid to the dispersing position, the metering port aligned with a compressed-air port in the body and a puff of air from this port dispersed the vanadium pentoxide into the particle attrition chamber. Generator output was regulated by adjusting the cadence of the shuttle bar. The particle attrition chamber used low fluid energy from an air jet tangential to the chamber to deagglomerate the vanadium pentoxide particles. After deagglomeration, the particles were swept into a classification zone where smaller particles exited to the distribution line; larger particles were thrown to the perimeter of the classifier by centrifugal force and were reentrained into the impacting air jet, and the process was repeated until the particles were sufficiently deagglomerated. The aerosol passed through the distribution lines to the exposure chambers. A pneumatic pump designed by the study laboratory was located at each chamber inlet and drew aerosol from the distribution line into the chamber inlet, where it was diluted with conditioned air to the appropriate concentration. Flow through the distribution line was controlled by Air-Vac pumps (Air-Vac Engineering, Milford, CT), and pressure was monitored by photohelic differential pressure gauges (Dwyer Instruments, Inc., Michigan City, IN).
- The Stainless steel chambers (Lab Products, Inc., Harford Systems Division, Aberdeen, MD), were designed so that uniform aerosol concentrations could be maintained throughout the chambers when catch pans were in place. The total active mixing volume of each chamber was 1.7 m³.

CHAMBER ATMOSPHERE CHARACTERIZATION
- The particle size distribution in each chamber was determined prior to the start of all studies, during the first week of the 16-day and 3-month studies, during the first 2 weeks of the 2-year studies, and monthly during the 3-month and 2-year studies.
- For the 16-day special studies and the 2-year studies, a Mercer-style seven-stage impactor was used. The stages (glass coverslips lightly sprayed with silicon) were analyzed by ICP/AES, and the relative mass collected on each stage was analyzed by probit analysis.

OTHER
- The uniformity of aerosol concentration in the inhalation exposure chambers without animals was evaluated before each of the studies began; concentration uniformity with animals present in the chambers was also measured. During the 16-day and 3-month studies, minor excursions in chamber uniformity values were observed in one or more exposure chambers, but these excursions had no impact on the studies. Chamber concentration uniformity was acceptable throughout the 16-day special studies and 2-year studies.
- The stability of vanadium pentoxide in the exposure system was tested with XRD analysis. XRD analyses indicated no detectable build-up of degradation products at a detection limit of approximately 1%.



Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- During all studies, chamber aerosol concentrations were monitored with real-time aerosol monitors (RAMs) that used a pulsed-light-emitting diode in combination with a silicon detector to sense light scattered over a forward angular range of 45° to 95° by particles traversing the sensing volume. The instruments respond to particles 0.1 to 20 μm in diameter.
- For the 16-day special studies and the 2-year studies, the sampling system consisted of a valve that multiplexed each RAM to two or three exposure chambers and to a HEPA filter and/or the control chamber or room; selection of sampling streams and data acquisition from each RAM was remotely controlled by a computer. Equations for calibration curves were stored in the computers and were used to convert the measured voltages to exposure concentrations.
- Each RAM was calibrated daily during the 16-day and 3-month studies by correlating the measured voltage with vanadium pentoxide concentrations determined by gravimetric analysis of glass fiber filters and one to two times per week during the 2-year studies by ICP/AES or ICP/mass spectrometry analysis of Pallflex® TX40H120WW glass fiber filters.
Duration of treatment / exposure:
104 weeks
Frequency of treatment:
6 hours per day, 5 days per week
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
1 mg/m³ V2O5
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
2 mg/m³ V2O5
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
4 mg/m³ V2O5
Basis:
nominal conc.
No. of animals per sex per dose:
core study: 50 males and 50 females per dose
tissue burden analyses: 40 female mice per exposed group; separate control group of 15 female mice was used as chamber controls
Control animals:
yes
Details on study design:
- Dose selection rationale: the highest exposure concentration (4 mg/m3) selected for mice was based on reduced body weight gains of 8 and 16 mg/m3 males and females, increased lung weights of 4 mg/m3 or greater males and females, increased incidences of epithelial hyperplasia, and increased incidences and severities of inflammatory lesions of the lungs in mice exposed to 8 mg/m3 versus those exposed to 4 mg/m3 in the 90-day study.
- Rationale for animal assignment (if not random): randomly into groups of approximately equal initial mean body weights.
- No further information on test material was stated.
Positive control:
not stated

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Clinical findings were recorded every 4 weeks from week 5 through 89 and every 2 weeks from week 92 to the end of the study

BODY WEIGHT: Yes
- Time schedule for examinations: animals were weighed at the beginning of the studies. Body weights were recorded every 4 weeks from week 5 through 89 and every 2 weeks from week 92 to the end of the study

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:
Tissue Burden Studies:
- Lungs and whole blood were collected from five mice in each exposure group sacrificed on day 1, 5, 12, 26, 54, 171, 362, or 535. Groups of five chamber control animals were bled at each of these time points and returned to their chambers and used for subsequent bleedings. The lungs and associated tissues were removed from exposed animals; after the left and right lung were prepared for histopathologic examination, and the right lung was digested to measure vanadium concentration. Vanadium concentrations in blood and right lung nitric acid digests were measured. Calculations of lung deposition and clearance parameters were performed. Blood vanadium concentration was measured in all animals at all time points after day 12.
Sacrifice and pathology:
Method of Sacrifice: CO2 asphyxiation.

PATHOLOGY: Yes
- Complete necropsies were performed on all core study mice.
- All organs and tissues were examined for grossly visible lesions, and all major tissues were prepared for microscopic examination.

HISTOPATHOLOGY: Yes
- Complete histopathology was performed on all core study animals. In addition to gross lesions and tissue masses, the following tissues were examined: adrenal gland, bone with marrow, brain, clitoral gland, esophagus, gallbladder (mice only), heart and aorta, large intestine (cecum, colon, and rectum), small intestine (duodenum, jejunum, and ileum), kidney, larynx, liver, lung and mainstem bronchi, lymph nodes (mandibular, mediastinal, mesenteric, and bronchial), mammary gland (except male mice), nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, skin, spleen, stomach (forestomach and glandular), testis (with epididymis and seminal vesicle), thymus, thyroid gland, trachea, urinary bladder and uterus.
Other examinations:
Five male and five female mice were randomly selected for parasite evaluation and gross observation of disease.
Statistics:
Survival Analyses:
The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958) and is presented in the form of graphs. Animals found dead of other than natural causes were censored from the survival analyses; animals dying from natural causes were not censored. Statistical analyses for possible doserelated effects on survival used Cox’s (1972) method for testing two groups for equality and Tarone’s (1975) life table test to identify dose-related trends. All reported P values for the survival analyses are two sided.

Analysis of Continuous Variables:
Two approaches were employed to assess the significance of pairwise comparisons between exposed and control groups in the analysis of continuous variables. Organ and body weight data, which historically have approximately normal distributions, were analyzed with the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972). Hematology, clinical chemistry, urinalysis, urine concentrating ability, cardiopulmonary, immunotoxicologic, cell proliferation, tissue concentrations, spermatid, and epididymal spermatozoal data, which have typically 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). Average severity values were analyzed for significance with the Mann-Whitney U test (Hollander and Wolfe, 1973). Treatment effects were investigated by applying a multivariate analysis of variance (Morrison, 1976) to the transformed data to test for simultaneous equality of measurements across exposure concentrations. (for more information see publication)

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
effects observed, treatment-related
Details on results:
CLINICAL SIGNS AND MORTALITY
- Survival of the highest dosed males (4 mg/m³) was significantly less than controls
- Abnormal breathing was observed particularly those exposed to 2 or 4 mg/m³
- Many mice exposed to vanadium pentoxide were thin

BODY WEIGHT AND WEIGHT GAIN
-Mean body weight of all females and highest dosed males was below the controls; and mean body weights of males exposed to 2 mg/m3 were less from week 85 to the end of the study

HISTOPATHOLOGY: NON-NEOPLASTIC
- Non-neoplastic lesions occurred in respiratory system (lung, larynx, and nose) of males and females, and the severities of these lesions generally increased with increasing exposure concentration.
-Bronchial lymph node hyperplasia was present in most of the exposed females.

LUNG:
- There were significantly increased incidences of alveolar epithelial hyperplasia and bronchiolar epithelial hyperplasia in the lungs of exposed male and female mice. Incidences of chronic inflammation and histiocytic cellular infiltrate were significantly increased in exposed groups of mice, and the incidences of interstitial fibrosis were increased in mice exposed to 2 or 4 mg/m3.
- Males: alveolar epithelium, hyperplasia (3/50, 41/50, 49/50, 50/50); bronchiole, epithelium, hyperplasia (0/50, 15/50, 37/50, 46/50); inflammation chronic (6/50, 42/50, 45/50, 47/50); alveolus, infiltration cellular, histiocyte (10/50, 36/50, 45/50, 49/50); interstitial fibrosis (1/50, 6/50, 9/50, 12/50)
- Females: alveolar epithelium, hyperplasia (0/50, 31/50, 38/50, 50/50); bronchiole, epithelium, hyperplasia (0/50, 12/50, 34/50, 48/50); inflammation chronic (4/50, 37/50, 39/50, 49/50); alveolus, infiltration cellular, histiocyte (0/50, 34/50, 35/50, 45/50); interstitial fibrosis (0/50, 1/50, 4/50,
8/50)
- The hyperplasia was essentially a diffuse change with proliferation of epithelium in the distal terminal bronchioles and immediately associated alveolar ducts and alveoli. Normally flattened epithelium was replaced with cuboidal epithelium. The hyperplasia of the alveolar epithelium was pronounced and increased in severity with increasing exposure concentration, while the hyperplasia of the distal bronchioles was minimal to mild with slight increases in severity in mice exposed to 4 mg/m3. The changes in mice were similar to those observed in rats but were not as pronounced.
- The inflammatory lesions were primarily minimal to mild and consisted of interstitial, perivascular, and peribronchiolar infiltrates of mostly mononuclear inflammatory cells (mostly lymphocytes) that were occasionally present within alveoli. The most prominent histiocytic infiltrate occurred within alveoli in close proximity to alveolar/bronchiolar neoplasms, particularly carcinomas.

LARYNX:
- There were significantly increased incidences of minimal squamous metaplasia of the respiratory epithelium of the epiglottis in exposed groups.
- Males: respiratory epithelium, epiglottis, metaplasia, squamous (2/49, 45/50, 41/48, 41/50)
- Females: respiratory epithelium, epiglottis,metaplasia, squamous (0/50, 39/50, 45/49, 44/50)
- Squamous metaplasia was diagnosed when the ciliated cells were replaced by one to three layers of flattened squamous epithelium. This change commonly occurs in mice in NTP inhalation studies and represents a common response to laryngeal injury.

NOSE:
- There were increased incidences of minimal to mild suppurative inflammation of the nose in males and females exposed to 2 or 4 mg/m3.
- Males: inflammation suppurative (16/50, 11/50, 32/50, 23/50); olfactory epithelium, degeneration, hyaline (1/50, 7/50, 23/50, 30/50); respiratoryepithelium, degeneration, hyaline (8/50, 22/50, 38/50, 41/50); respiratory epithelium, metaplasia, squamous (0/50, 6/50, 6/50, 2/50)
- Females: inflammation suppurative (19/50, 14/50, 32/50, 30/50); olfactory epithelium, atrophy (2/50, 8/50, 5/50, 14/50); olfactory epithelium, degeneration, hyaline (11/50, 23/50, 34/50, 48/50); respiratory epithelium, degeneration, hyaline (35/50, 39/50, 46/50, 50/50); respiratory epithelium, metaplasia, squamous (0/50, 3/50, 7/50, 8/50); respiratory epithelium, necrosis (0/50, 0/50, 1/50, 7/50)
- The inflammation consisted of focal aggregates of few to moderate numbers of neutrophils generally subjacent to the epithelium of the turbinates, septum, or lateral wall of the anterior nose. In the more severe cases (predominantly in females exposed to 4 mg/m3), a short segment of the overlying epithelium was ulcerated (necrosis). Similarly, in some males and females, the overlying respiratory epithelium was replaced by one or more layers of flattened epithelium (squamous metaplasia), and the incidences were marginally increased in some exposed groups of mice.
- The majority of the olfactory epithelium covers the turbinates in the distal portion of the nose. There were marginal but significant increases in the incidences of atrophy of this epithelium in females exposed to 1 or 4 mg/m3.

BRONCIAL LYMPH NODE
- There were significant increases in the incidences of hyperplasia of the bronchial lymph node in exposed groups of females, and while not significant, a positive trend in the incidences of this lesion also occurred in males.
- Females: hyperplasia (3/39, 13/40, 14/45, 20/41)

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
LUNG:
- The incidences of alveolar/bronchiolar carcinoma and alveolar/bronchiolar adenoma or carcinoma (combined) were significantly increased in all groups of exposed male and female mice compared to controls
- Males: alveolar/bronchiolar adenoma (13/50, 16/50, 26/50, 15/50); alveolar/ bronchiolar carcinoma (12/50, 29/50, 30/50, 35/50); alveolar/ bronchiolar adenoma or carcinoma (22/50, 42/50, 43/50, 43/50)
- Females: alveolar/bronchiolar adenoma (1/50, 17/50, 23/50, 19/50); alveolar/ bronchiolar carcinoma (0/50, 23/50, 18/50, 22/50); alveolar/ bronchiolar adenoma or carcinoma (1/50, 32/50, 35/50, 32/50)
- The incidences of alveolar/bronchiolar adenoma were significantly increased in males exposed to 2 mg/m3 and in all groups of exposed females.
- These increased incidences exceeded the historical ranges for controls.

SPLEEN:
- There was a positive trend in the incidences of hemangiosarcoma of the spleen in male mice (chamber control, 0/50; 1 mg/m3, 0/50; 2 mg/m3, 0/50; 4 mg/m3, 3/50).
- However, the incidence in the 4 mg/m3 group was within the historical range in controls.
- In addition, the incidences of hemangioma or hemangiosarcoma (combined) in all organs were not significant. The marginal increase in the incidence of hemangiosarcoma of the spleen was not considered related to vanadium pentoxide exposure.

OTHER FINDINGS
TISSUE BURDEN STUDIES:
- Histopathology: lung lesions were identified early, and were more severe in females exposed to higher concentrations and progressed with time. Minimal bronchiolar epithelial hyperplasia and interstitial inflammation were initially observed in the 4 mg/m3 group on day 5. Bronchiolar epithelial hyperplasia was characterized by slight piling up of cells, some nuclear pleomorphism, and occasional karyorrhectic cells. On day 12, exposure-related lesions were evident in 2 mg/m3 females, while alveolar epithelial hyperplasia was more prominent in the 4 mg/m3 group. Lesions were first observed in the 1 mg/m3 group on day 54, and lesion severities in the 2 and 4 mg/m3 groups appeared to be increased on day 54. On day 362, lesion severity in lungs of females exposed to 4 mg/m3 was noticeably increased, and bronchiolar hyperplasia included more nuclear pleomorphism. On day 535, mice exposed to 2 mg/m3 had hyperplastic alveolar epithelial lesions and, similar to females exposed to 4 mg/m3, the hyperplasia was oriented around the terminal bronchiolar/alveolar duct. Also on day 535, lung nodules/masses were observed. While some were confirmed as neoplasms, many were in the right lung lobe and thus were not confirmed histopathologically. Similar lesions, although more severe, were observed in the core group of animals at 2 years.
- Lung burden data appeared proportional to exposure concentration in mice. Rats removed vanadium from the lungs much slower than mice, with clearance half-times of vanadium lung burdens approximately six to nine fold longer in rats than in mice at comparable exposure concentrations. In rats and mice, lung clearance half-times were considerably longer than those observed in the 16-day special studies.
- The maximum lung burdens occurred between days 26-54 in mice. The lung burdens appeared to reach steady state at the lowest exposure concentrations (1 mg/m3). A decline in lung burdens was observed. The retention of vanadium in the lungs at 18 months was 2% to 3% in mice. The total lung doses for mice exposed to 1, 2, or 4 mg/m3 were estimated to be 153, 162, and 225 μg vanadium, respectively. There was little difference in the total lung dose for mice, especially between the 1 and 2 mg/m3 groups.

Effect levels

Dose descriptor:
NOAEL
Remarks:
local
Effect level:
1 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: non-neoplastic changes (epithelial hyperplasia, squamous metaplasia, chronic inflammation, degeneration) in the respiratory system (lung, larynx, and nose) of male and female mice

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Chamber concentration uniformity was acceptable throughout the 16-day special studies and 2-year studies.

Applicant's summary and conclusion

Conclusions:
The survival rate of male mice exposed to 4 mg/m3 was less than that of chamber controls, and mean body weights of male mice exposed to 4 mg/m3 and all exposed groups of female mice were generally less than those of the chamber controls throughout the study. As in the 3-month studies, the respiratory tract was the primary site of toxicity.
Under the conditions of this 2-year inhalation study there was clear evidence of carcinogenic activity of vanadium pentoxide in male and female B6C3F1 mice based on increased incidences of alveolar/bronchiolar neoplasms. Exposure to vanadium pentoxide caused a spectrum of non-neoplastic lesions in the respiratory tract (nose, larynx, and lung) including alveolar and bronchiolar epithelial hyperplasia, inflammation, fibrosis, and alveolar histiocytosis of the lung in male and female mice. Hyperplasia of the bronchial lymph node occurred in female mice. The lowest concentration tested (1 mg/m3) represents a LOAEC for local effects in the respiratory tract.