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Toxicity to reproduction

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Endpoint:
fertility, other
Remarks:
90- day toxicity study with evaluation of reproductive organs
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1990-09-20 (first exposure) to 1990-12-21/22 (necropsy date)
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
Well reported study. Test procedure in accordance with generally accepted scientific standards and described in sufficient detail.
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
fertility, other
Remarks:
90- day toxicity study with evaluation of reproductive organs
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1990-09-18/19 (first exposure) to 1990-12-18/19 (necropsy date)
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
Well reported study. Test procedure in accordance with generally accepted scientific standards and described in sufficient detail.
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
Groups of 10 male and 10 female rats were exposed to particulate aerosols of vanadium pentoxide at concentrations of 0, 1, 2, 4, 8, or 16 mg/m3 (equivalent to 0, 0.56, 1.12, 2.25, 4.50, and 8.99 mg V/m³) by inhalation, 6 hours per day, 5 days per week for 3 months. Effects on fertility and sexual function of male and female rats were evaluated after 90 days of inhalation exposure.
GLP compliance:
yes
Limit test:
no
Justification for study design:
not applicable
Specific details on test material used for the study:
not specified
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system 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 (4 weeks old on receipt)
- Weight at study initiation: range of mean body weights in the exposure groups: 133-137 g (males) and 108-111 g (females)
- Housing: housed individually; stainless steel wire mesh (Lab Products, Inc., Maywood, NJ),), changed weekly
- Diet: ad libitum, except during exposure periods; NIH-07 open formula pelleted diet (Zeigler Brothers, Inc., Gardners, PA); feed was also
withheld during urine collection
- Water: ad libitum; tap water (City of Chicago municipal supply) via automatic watering system; water was withheld for the second urine collection
- Acclimation period: quarantined for 12 or 13 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
Route of administration:
inhalation: aerosol
Type of inhalation exposure (if applicable):
whole body
Mass median aerodynamic diameter (MMAD):
>= 1 - <= 1.3 µm
Remarks on MMAD:
GSD: 2.1 - 3.0
Vehicle:
air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- For the 3-month studies, vanadium pentoxide aerosol generation was based on the principle of pneumatic dispersion and consisted of two major components: a screw feeder (Model 310, Accurate, White Water, WI) that metered vanadium pentoxide powder at a constant rate and a Jet-O-Mizer jetmill (Fluid Energy Corp., Harfield, PA) that used compressed air to disperse the metered powder and form the aerosol.
- Aerosol leaving the jetmill passed through a one-stage impactor and a vertical elutriator to eliminate or deagglomerate the large particles before entering a plenum and manifold distribution system. The aerosol delivery system consisted of three holding chambers that diluted the aerosol in three stages. A metered amount of diluted aerosol was removed and mixed with conditioned air at the inlet to each exposure chamber to achieve the appropriate exposure concentration. The electrical charge buildup on the aerosol particles was neutralized by mixing the aerosol with high concentrations of bipolar ions, which were generated using a Pulse Gun (Static Control Services, Palm Springs, CA) air nozzle. For the 3-month study, a transvector air pump was installed at the aerosol inlet to each exposure chamber to provide additional control of the aerosol flow rate and improve stability of the chamber concentration.
- The stainless-steel inhalation exposure chambers (Lab Products, Inc., Maywood NJ), 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, during the first week of the 3-month study, and monthly during the 3-month study.
- For the 3-month study, a 10-stage Quartz Crystal Microbalance-based cascade impactor was used to separate the aerosol particles into sequential size ranges; the mass median aerodynamic diameter was calculated from the corresponding mass fraction of particles at each stage.

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 3-month study, minor excursions in chamber uniformity values were observed in one or more exposure chambers, but these excursions had no impact on the 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%.
Details on mating procedure:
not specified
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- 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.
- During the 3-month study, an individual monitor was used for each exposure chamber. The voltage output of the online monitors was read and recorded, and the calibration curve was applied to the voltages measured by the RAM to convert the measured voltages to exposure chamber concentrations.
- Each RAM was calibrated daily during the 3-month study by correlating the measured voltage with vanadium pentoxide concentrations determined by gravimetric analysis of glass fiber filters.
Duration of treatment / exposure:
3 months (91-92 days)
Frequency of treatment:
6 hours per day, 5 days per week
Details on study schedule:
not specified
Dose / conc.:
1 mg/m³ air (analytical)
Remarks:
SD. 0.0; target concentration: 1 mg/m³; equivalent to 0.56 mg V/m³
Dose / conc.:
2 mg/m³ air (analytical)
Remarks:
SD. 0.0; target concentration: 2 mg/m³; equivalent to 1.12 mg V/m³
Dose / conc.:
4 mg/m³ air (analytical)
Remarks:
SD. 0.1; target concentration: 4 mg/m³; equivalent to 2.25 mg V/m³
Dose / conc.:
7.9 mg/m³ air (analytical)
Remarks:
SD. 0.2; target concentration: 8 mg/m³; equivalent to 4.50 mg V/m³
Dose / conc.:
15.9 mg/m³ air (analytical)
Remarks:
SD. 0.4; target concentration: 16 mg/m³; equivalent to 8.99 mg V/m³
No. of animals per sex per dose:
10 male and 10 female rats
Control animals:
yes
Details on study design:
- Dose selection rationale: based on decreased survival in the 32 mg/m3 males and body weight decreases in 32 mg/m3 males and females in a 16-day study, an exposure concentration of 32 mg/m3 was considered too high for use in a 3-month study. Therefore, the exposure concentrations selected for the 3-month inhalation study in rats were 0, 1, 2, 4, 8, and 16 mg/m3.
- Rationale for animal assignment (if not random): randomly into groups of approximately equal initial mean body weights.
Positive control:
not specified
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
Clinical findings were recorded weekly.

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: initially, weekly, and at the end of the study

FOOD CONSUMPTION AND COMPOUND INTAKE: No data
WATER CONSUMPTION AND COMPOUND INTAKE: No data
OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the study
- Anaesthetic used for blood collection: rats were anesthetized with a 70% CO2:30% air mixture
- Animals fasted: no data
- How many animals: mainly 8-10 per exposure group (except week 13, highest dose group: N=3 for males and 6 for females)
- Parameters checked: automated hematocrit; manual hematocrit; hemoglobin concentration; erythrocyte, reticulocyte, nucleated erythrocyte, and platelet counts; erythrocyte morphology; mean cell volume; mean cell hemoglobin; mean cell hemoglobin concentration; leukocyte count and differentials

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the study
- Animals fasted: no data
- How many animals: mainly 8-10 per exposure group (except week 13, highest dose group: N=3 for males and 6 for females)
- Parameters checked: urea nitrogen, creatinine, total protein, albumin, alanine aminotransferase, alkaline phosphatase, creatine kinase, sorbitol dehydrogenase, bile acids

URINALYSIS: Yes
- Time schedule for collection of urine: during week 12 (females) or 13 (males)
- Metabolism cages used for collection of urine: for 16 hours immediately following exposure
- Animals fasted: yes
- Parameters checked: urinalysis: volume, specific gravity; urine concentrating ability: volume, specific gravity

IMMUNOLOGY: No data

NEUROBEHAVIOURAL EXAMINATION: No data
Oestrous cyclicity (parental animals):
- At the end of the 3-month studies, samples were collected for vaginal cytology evaluations from rats exposed to 4, 8, or 16 mg/m3.
- For 12 consecutive days prior to scheduled terminal sacrifice, the vaginal vaults of the females were moistened with saline, if necessary, and samples of vaginal fluid and cells were stained. Relative numbers of leukocytes, nucleated epithelial cells, and large squamous epithelial cells were determined and used to ascertain estrous cycle stage (i.e., diestrus, proestrus, estrus, and metestrus).
Sperm parameters (parental animals):
- At the end of the 3-month studies, samples were collected for sperm motility evaluations from core study rats exposed to 0, 2, 4 and 8 mg/m3.
- Male animals were evaluated for sperm count and motility. The left testis and left epididymis were isolated and weighed. The tail of the epididymis (cauda epididymis) was then removed from the epididymal body (corpus epididymis) and weighed. Test yolk was applied to slides and a small incision was made at the distal border of the cauda epididymis. The sperm effluxing from the incision were dispersed in the buffer on the slides, and the numbers of motile and nonmotile spermatozoa were counted for five fields per slide by two observers.
- Following completion of sperm motility estimates, each left cauda epididymis was placed in buffered saline solution. Caudae were finely minced, and the tissue was incubated in the saline solution and then heat fixed at 65° C. Sperm density was then determined microscopically with the aid of a hemacytometer.
- To quantify spermatogenesis, the testicular spermatid head count was determined by removing the tunica albuginea and homogenising the left testis in phosphate-buffered saline containing 10% dimethyl sulfoxide. Homogenisation-resistant spermatid nuclei were counted with a hemacytometer.
Litter observations:
not specified
Postmortem examinations (parental animals):
GROSS NECROPSY/ ORGAN WEIGHTS
- Necropsies were performed on all animals.
- Organs weighed were the heart, right kidney, liver, lung, right testis, and thymus..

HISTOPATHOLOGY: Yes.
- Complete histopathology was performed on control, 8 and 16 mg/m3 rats. 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, 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.
- The lung and nose of rats in all remaining exposure groups were also examined.
Postmortem examinations (offspring):
not specified
Statistics:
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)
Reproductive indices:
not specified
Offspring viability indices:
not specified
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
- Abnormal breathing, thinness, lethargy, abnormal posture, and ruffled fur were observed in rats exposed to 16 mg/m3. Abnormal breathing, marked by shallow, rapid respiration, was first observed during and immediately following exposure periods; this was observed in all 16 mg/m3 rats by week 2 and in all 8 mg/m3 rats by week 4. By week 9, abnormal breathing was also observed in 16 mg/m3 rats during non exposure periods. Some rats in the 16 mg/m3 groups had diarrhea and nasal/eye discharge. Abnormal posture was observed in two males exposed to 8 mg/m3, and one of these rats was thin with ruffled fur and nasal/eye discharge.
Dermal irritation (if dermal study):
not specified
Mortality:
mortality observed, treatment-related
Description (incidence):
- 7 males and 3 females exposed to 16 mg/m3 died during the study

Please also refer to the field "Attached background material" below.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- Final mean body weights and body weight gains of males exposed to 4 mg/m3 or greater and of females exposed to 16 mg/m3 were significantly less than those of the chamber controls
- in males exposed to vanadium pentoxide, there was a statistically significant effect on necropsy body weight at 8 mg/m³
- in females exposed to vanadium pentoxide, necropsy body weight was statistically significantly lower at 16 mg/m3

Please also refer to the field "Attached background material" below
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:
effects observed, treatment-related
Description (incidence and severity):
- Microcytic erytrocytosis was observed in males and females
- Exposure of rats to vanadium pentoxide affected the circulating red cell mass
- In general, the erythrocyte counts reflected similar and proportional, but not significant, decreases.
- There were exposure concentration-related decreases in the mean cell volumes and mean cell hemoglobin values on day 23, which are consistent with a developing erythrocyte microcytosis.
- The mean cell hemoglobin concentration was minimally decreased in 16 mg/m3 females
- Microscopic evaluation of the red blood cell morphology detected increased polychromasia, anisochromia, and hypochromia in the 16 mg/m3 groups
- At week 13, there was evidence of an exposure-related leukopenia, demonstrated by decreases in leukocyte counts in 16 mg/m3 males and females; leukopenia appeared to be related to decreased lymphocyte counts. This alteration in lymphocyte counts suggests a physiological response and would be consistent with a stress-related corticosteroid- induced lymphopenia. Rats are considered a steroid-sensitive species and corticosteroid-induced lymphopenia may be related to lympholysis in blood and altered distribution (Jain, 1986).
- The 16 mg/m3 males and females demonstrated microscopic evidence of lymphoid depletion in numerous lymphoid organs. Since there was an exceptional effect on body weights of 16 mg/m3 rats, it is possible that these animals were under significant stress that then lead to increased endogenous corticosteroid concentrations and subsequent decreases in tissue and blood lymphocytes.
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Lungs:
- Incidence if several nonneoplastic lesions of the lung and nose were increased in animals exposed to 2 mg/m3 or greater
- There were significant increases in the incidences of epithelial hyperplasia of the lung in males and females exposed to 2 mg/m3 or greater.The epithelial hyperplasia occurred in the distal airways and associated alveolar ducts and alveoli. In the distal airways, the epithelium was composed of ciliated cuboidal to columnar cells crowded together in multiple layers, sometimes forming folds and small papillary projections. More distal and including the alveolar ducts and alveoli, the epithelium tended to be a single layer. The epithelial cells were larger and rounded and appeared morphologically similar to bronchioles. Goblet cells sometimes occurred within the epithelium of some of the smaller airways. Alveoli often contained one or two cells that were very large and occasionally binucleate.
- The incidences of inflammation or fibrosis were significantly increased in males exposed to 2 mg/m3 or greater and females exposed to 4 mg/m3 or greater.
- In one female exposed to 16 mg/m3, a single focus of squamous metaplasia was observed within an area of hyperplasia.
- Exudate of the bronchiole was diagnosed in many males and females exposed to 8 or 16 mg/m3 and was characterized by a mucoid material with admixed inflammatory cells within the lumens of some of the hyperplastic airways. The inflammation was characterized by accumulations of alveolar macrophages with abundant foamy cytoplasm in the alveoli adjacent to airways lined by hyperplastic epithelium. Mononuclear cells were also observed within the interstitium.
- Foci of fibrosis were found in the same areas and sometimes extended into surrounding alveoli. These foci were irregularly shaped and consisted of loosely arranged, delicate collagen fibers, fibroblasts, and mononuclear cells.

Nose:
- The incidences of hyperplasia and metaplasia of the nasal respiratory epithelium were significantly increased in males exposed to 8 or 16 mg/m3 and in females exposed to 4 mg/m3 or greater. There were significantly increased incidences of inflammation of the nose in males and females exposed to 16 mg/m3. The hyperplasia and metaplasia involved the respiratory epithelium covering the ventral portion of the nasal septum, the vomeronasal organ, and, to a lesser extent, the ventral lateral walls of the anterior portion of the nasal cavity. There were cellular crowding and epithelium thickening (hyperplasia) in some areas with loss of goblet cells and one or more layers of flattened nonkeratinizing squamous epithelium (metaplasia) in others.
- Inflammation was characterized by an infiltrate primarily of lymphocytes and lesser numbers of neutrophils within the affected mucosa and subjacent submucosa. Occasionally, neutrophils predominated, and an exudate was present.

Other:
- Depletion of lymphocytes in the spleen, thymus, and lymph nodes, atrophy of metaphyseal bone of the femur, and atrophy of the secondary reproductive organs were observed in 16 mg/m3 males and females and hypospermia of the testis and atypical cells of the epididymis were observed in 16 mg/m3 males. These lesions may have been associated with the marked body weight loss and general debilitation of these rats.
Histopathological findings: neoplastic:
not specified
Other effects:
effects observed, treatment-related
Description (incidence and severity):
- Significant exposure-related changes in pulmonary function were observed in male and female rats exposed to 4, 8, or 16 mg/m3; these results indicate that more restrictive lesions were present in groups exposed to 4 mg/m3 or greater, evidenced by reduced lung compliance, changes in breathing measurements, impaired capacity to diffuse carbon monoxide, reduced static and dynamic lung volumes, and exaggerated flows.
- Pulmonary function changes indicate an obstructive disease in the 16 mg/m3 groups, evidenced by changes in breathing mechanics, static lung volumes, and forced expiratory maneuvers. Expiratory resistance, an indicator of bronchoconstriction, and end expiratory and residual volume were increased, while dynamic lung volume was decreased.
- Together, the pulmonary function changes indicate that a restrictive disease was present in male and female rats exposed to 4 mg/m3 or greater, while an obstructive lung disease may have been present only in the 16 mg/m3 groups.
- The pulmonary lavage data indicate an inflammatory response in the lungs of exposed rats. Exposure concentration-related increases were observed in the total numbers of cells, lymphocytes, neutrophils, and protein recovered in pulmonary lavage fluid from rats exposed to vanadium pentoxide at concentrations up to 8 mg/m3.
Reproductive function: oestrous cycle:
effects observed, treatment-related
Description (incidence and severity):
- there was an increases in oestrus cycle length at 8 (statistically significant) and 16 mg/m3 (not statistically significant). However, this effect was highest in the mid dose group (8 mg/m3), thus showing no clear relation to dose and treatment.
- the number of cycling females in the 16 mg/m3 group was reduced.

Please also refer to the field "Attached background material" below.
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
not specified
CLINICAL CHEMISTRY
- At all time points, evidence of a hepatocellular effect was demonstrated by increases in serum alanine aminotransferase activities in 8 and 16 mg/m3 males and females. However, sorbitol dehydrogenase activities, another marker of hepatocellular injury or leakage, were not affected, and there were no histopathologic liver changes consistent with hepatocellular injury. This suggests that the increased alanine aminotransferase activities were not related to hepatocellular injury. Studies have shown that corticosteroids can induce increases in liver alanine animotransferase activities (Rosen, 1959; Rosen et al., 1959). Thus, stress-related corticosteroid-induced increases in liver alanine aminotransferase activities may, in part, help explain increased serum alanine aminotransferase activities.
- At all time points, there were generally exposure concentration-related decreases in serum alkaline phosphatase activities and total protein and albumin concentrations in males and females. At week 13, urea nitrogen concentrations were increased in 16 mg/m3 males and females, suggesting a possible effect on renal clearance. However, creatinine concentrations, another marker of renal clearance, were minimally decreased on day 23 in 8 mg/m3 males and at week 13 in 16 mg/m3 males and females; these decreases would be consistent with the decreased body weights observed in these groups. Since alkaline phosphatase activity and total protein, albumin, and urea nitrogen concentrations can be affected by altered nutritional status, the changes in these variables may have been related secondarily to body weight decreases and altered food intake.
- Significant increases and decreases in bile acid concentrations and creatine kinase activities in various exposure groups at various time points were not considered to be toxicologically relevant.

URINALYSIS
- After 12 (females) or 13 (males) weeks of exposure, the baseline (water-replete) overnight urine collection demonstrated decreased urine volumes and increased urine specific gravities in the 8 mg/m3 male and 16 mg/m3 female groups, suggesting these animals were able to concentrate their urine; the 16 mg/m3 male group was not tested. These findings also suggest that these exposure groups may have been in a partially dehydrated state prior to the water deprivation studies.
- The 4-hour urine collection following a 16-hour water deprivation period demonstrated no differences in urine volume or specific gravity. This suggests that animals in all exposed groups maintained their ability to concentrate urine.
- For baseline and water deprivation test samples, microscopic evaluation of the urine demonstrated slight increases in formed elements in 8 mg/m3 males (baseline: casts, epithelial cells, erythrocytes, and leukocytes; water-deprived: casts) and 16 mg/m3 females (baseline and water deprived: leukocytes) (data not presented). While changes in formed urine elements can be indicative of various renal effects, the alterations in this study were not excessive and possibly reflected the hydration status of the animals; they were not considered to be toxicologically relevant.

ORGAN WEIGHTS
- Other organ weight differences were considered to be related to body weight decreases

GROSS PATHOLOGY
- The carcasses of males and females exposed to 16 mg/m3 were very thin, and the spleens and thymuses appeared disproportionately small
- Lungs of 4 mg/m3 or greater males and 8 and 16 mg/m3 females varied from red to pale or mottled.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
- in males exposed to vanadium pentoxide there was a statistically significant effect on necropsy body weight at 8 mg/m³ and no effect on male reporductive organ weights. Spermatid measurements (heads and counts) were not statistically significant effected as well as epididymal sperm motility and concentration.
- atrophy of the secondary reproductive organs in male rats at 16 mg/m3 is described in the study.
- a statistically significant reduction of absolute testes weight, which was not reflected in the relative testis weights, is given for the 16 mg/m3 group, and may thus be related most probably to the lower necropsy body weight. At 8 mg/m3, no such effects were observed on absolute and relative testes weights which were in the same order of magnitude as in control animals and the other treatment groups.
- a hypospermia of the testis and atypical cells of the epididymis at 16 mg/m3 is described in the study (data for 16 mg/m3 not shown) where only a very mild effects without statistical significance is evident in the 8 mg/m3 group, and is most probably an unspecific effect related to general toxicity at this dose level.

Please also refer to the field "Attached background material" below.

Dose descriptor:
NOAEC
Remarks:
reproductive toxicity
Effect level:
4 mg/m³ air
Based on:
test mat.
Sex:
female
Basis for effect level:
other: increased oestus cycle at and above 8 mg/m3 and decreased no. of cycling females at 16 mg/m3
Dose descriptor:
NOAEC
Remarks:
reproductive toxicity
Effect level:
> 16 mg/m³ air
Based on:
test mat.
Sex:
male
Basis for effect level:
other: male fertility parameters were not affected at the highest dose level
Critical effects observed:
not specified
Clinical signs:
not specified
Dermal irritation (if dermal study):
not specified
Mortality / viability:
not specified
Body weight and weight changes:
not specified
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 specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Sexual maturation:
not specified
Anogenital distance (AGD):
not specified
Nipple retention in male pups:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Histopathological findings:
not specified
Other effects:
not specified
Behaviour (functional findings):
not specified
Developmental immunotoxicity:
not specified
not specified
Remarks on result:
not measured/tested
Critical effects observed:
not specified
Reproductive effects observed:
not specified
Conclusions:
Groups of 10 male and 10 female F344/N rats were exposed to V2O5 aerosols (whole body exposure) at concentrations of 0, 1, 2, 4, 8 or 16 mg/m3 (equivalent to 0, 0.56, 1.12, 2.25, 4.50, and 8.99 mg V/m³) by inhalation, 6 hours/d, 5 d/wk, for 3 months (91-92 days). Effects on fertility and sexual function of male and female rats were evaluated after inhalation exposure.
The results did not indicate effects on male sexual functions. In female rats exposed to 8 mg/m3, oestrous cycles were significantly longer than in animals of the chamber control group, and the number of cycling females in the 16 mg/m3 group was reduced.

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 10 male and 10 female mice were exposed to particulate aerosols of vanadium pentoxide at concentrations of 0, 1, 2, 4, 8, or 16 mg/m3 (equivalent to 0, 0.56, 1.12, 2.25, 4.50, and 8.99 mg V/m³) by inhalation, 6 hours per day, 5 days per week for 3 months. Effects on fertility and sexual function of male and female mice were evaluated after 90 days of inhalation exposure.
GLP compliance:
yes
Limit test:
no
Justification for study design:
not applicable

Test material

Constituent 1
Chemical structure
Reference substance name:
Divanadium pentaoxide
EC Number:
215-239-8
EC Name:
Divanadium pentaoxide
Cas Number:
1314-62-1
Molecular formula:
V2O5
IUPAC Name:
divanadium pentaoxide
Test material form:
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.
Specific details on test material used for the study:
not specified

Test animals

Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals or test system 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 (4 weeks old on receipt)
- Weight at study initiation: range of mean body weights in the exposure groups: 25-26 g (males) and 20-21 g (females)
- Housing: housed individually; stainless steel wire mesh (Lab Products, Inc., Maywood, NJ), changed weekly
- Diet: ad libitum, except during exposure periods; NIH-07 open formula pelleted diet (Zeigler Brothers, Inc., Gardners, PA)
- Water: ad libitum; tap water (City of Chicago municipal supply) via automatic watering system
- Acclimation period: quarantined for 10 or 14 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

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure (if applicable):
whole body
Mass median aerodynamic diameter (MMAD):
>= 1 - <= 1.3 µm
Remarks on MMAD:
GSD: 2.1 - 3.0
Vehicle:
air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- For the 3-month studies, vanadium pentoxide aerosol generation was based on the principle of pneumatic dispersion and consisted of two major components: a screw feeder (Model 310, Accurate, White Water, WI) that metered vanadium pentoxide powder at a constant rate and a Jet-O-Mizer jetmill (Fluid Energy Corp., Harfield, PA) that used compressed air to disperse the metered powder and form the aerosol.
- Aerosol leaving the jetmill passed through a one-stage impactor and a vertical elutriator to eliminate or deagglomerate the large particles before entering a plenum and manifold distribution system. The aerosol delivery system consisted of three holding chambers that diluted the aerosol in three stages. A metered amount of diluted aerosol was removed and mixed with conditioned air at the inlet to each exposure chamber to achieve the appropriate exposure concentration. The electrical charge buildup on the aerosol particles was neutralized by mixing the aerosol with high concentrations of bipolar ions, which were generated using a Pulse Gun (Static Control Services, Palm Springs, CA) air nozzle. For the 3-month study, a transvector air pump was installed at the aerosol inlet to each exposure chamber to provide additional control of the aerosol flow rate and improve stability of the chamber concentration.
- The stainless-steel inhalation exposure chambers (Lab Products, Inc., Maywood NJ), 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, during the first week of the 3-month study, and monthly during the 3-month study.
- For the 3-month study, a 10-stage Quartz Crystal Microbalance-based cascade impactor was used to separate the aerosol particles into sequential size ranges; the mass median aerodynamic diameter was calculated from the corresponding mass fraction of particles at each stage.

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 3-month study, minor excursions in chamber uniformity values were observed in one or more exposure chambers, but these excursions had no impact on the 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%.
Details on mating procedure:
not specified
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- 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.
- During the 3-month study, an individual monitor was used for each exposure chamber. The voltage output of the online monitors was read and recorded, and the calibration curve was applied to the voltages measured by the RAM to convert the measured voltages to exposure chamber concentrations.
- Each RAM was calibrated daily during the 3-month study by correlating the measured voltage with vanadium pentoxide concentrations determined by gravimetric analysis of glass fiber filters.
Duration of treatment / exposure:
3 months (91-92 days)
Frequency of treatment:
6 hours per day, 5 days per week
Details on study schedule:
not specified
Doses / concentrationsopen allclose all
Dose / conc.:
1 mg/m³ air (analytical)
Remarks:
SD. 0.0; target concentration: 1 mg/m³; equivalent to 0.56 mg V/m³
Dose / conc.:
2 mg/m³ air (analytical)
Remarks:
SD. 0.0; target concentration: 2 mg/m³; equivalent to 1.12 mg V/m³
Dose / conc.:
4 mg/m³ air (analytical)
Remarks:
SD. 0.1; target concentration: 4 mg/m³; equivalent to 2.25 mg V/m³
Dose / conc.:
7.9 mg/m³ air (analytical)
Remarks:
SD. 0.2; target concentration: 8 mg/m³; equivalent to 4.50 mg V/m³
Dose / conc.:
15.9 mg/m³ air (analytical)
Remarks:
SD. 0.4; target concentration: 16 mg/m³; equivalent to 8.99 mg V/m³
No. of animals per sex per dose:
10 male and 10 female animals
Control animals:
yes
Details on study design:
- Dose selection rationale: based on decreased survival in the 32 mg/m3 males and body weight decreases in 32 mg/m3 males and females in a 16-day study, an exposure concentration of 32 mg/m3 was considered too high for use in a 3-month study. Therefore, the exposure concentrations selected for the 3-month inhalation study in rats were 0, 1, 2, 4, 8, and 16 mg/m3.
- Rationale for animal assignment (if not random): randomly into groups of approximately equal initial mean body weights.
Positive control:
not specified

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
Clinical findings were recorded weekly.

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: initially, weekly, and at the end of the studies.

FOOD CONSUMPTION AND COMPOUND INTAKE: No data
WATER CONSUMPTION AND COMPOUND INTAKE: No data
OPHTHALMOSCOPIC EXAMINATION: No data
HAEMATOLOGY: No data
CLINICAL CHEMISTRY: No data
URINALYSIS: No data
NEUROBEHAVIOURAL EXAMINATION: No data
IMMUNOLOGY: No data



Oestrous cyclicity (parental animals):
- At the end of the 3-month study, samples were collected for vaginal cytology evaluations from female mice exposed to 4, 8, or 16 mg/m3.
- For 12 consecutive days prior to scheduled terminal sacrifice, the vaginal vaults of the females were moistened with saline, if necessary, and samples of vaginal fluid and cells were stained. Relative numbers of leukocytes, nucleated epithelial cells, and large squamous epithelial cells were determined and used to ascertain oestrous cycle stage (i.e., diestrus, proestrus, estrus, and metestrus).
Sperm parameters (parental animals):
- At the end of the 3-month study, samples were collected for sperm motility evaluations from male mice exposed to 4, 8, or 16 mg/m3.
- Male animals were evaluated for sperm count and motility. The left testis and left epididymis were isolated and weighed. The tail of the epididymis (cauda epididymis) was then removed from the epididymal body (corpus epididymis) and weighed. Modified Tyrode’s buffer was applied to slides and a small incision was made at the distal border of the cauda epididymis. The sperm effluxing from the incision were dispersed in the buffer on the slides, and the numbers of motile and nonmotile spermatozoa were counted for five fields per slide by two observers.
- Following completion of sperm motility estimates, each left cauda epididymis was placed in buffered saline solution. Caudae were finely minced, and the tissue was incubated in the saline solution and then heat fixed at 65° C. Sperm density was then determined microscopically with the aid of a hemacytometer.
- To quantify spermatogenesis, the testicular spermatid head count was determined by removing the tunica albuginea and homogenising the left testis in phosphate-buffered saline containing 10% dimethyl sulfoxide. Homogenisation-resistant spermatid nuclei were counted with a hemacytometer.
Litter observations:
not specified
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes
- Necropsy was performed at study end on all animals.
- Organs weighed were the heart, right kidney, liver, lung, right testis, and thymus.

HISTOPATHOLOGY: Yes
- Complete histopathology was performed on control, and 16 mg/m3 mice. 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, 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.
- The lungs of mice in all remaining exposure groups and the thymus in 8 mg/m3 mice were also examined.
Postmortem examinations (offspring):
not specified
Statistics:
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)
Reproductive indices:
not specified
Offspring viability indices:
not specified

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not specified
Mortality:
mortality observed, non-treatment-related
Description (incidence):
- One male exposed to 16 mg/m3 died before the end of the study.

Please also refer to the field "Attached background material" below.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- final mean body weights and body weight gains of 8 and 16 mg/m3 males and 4 mg/m3 or greater females were significantly less than those of the chamber controls.
- in male mice, necropsy body weights were statistically significantly lower at 16 mg/m3.
- in female mice, necropsy body weight was statistically significantly lower in all treatment groups.

Please also refer to the field "Attached background material" below (file: "Reproductive funtion sperm measures and oestrus cycle.pdf")
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 specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The incidences of inflammation of the lung were increased in mice exposed to 2 mg/m3 or greater. Mice exposed to 2 mg/m3 or greater had epithelial hyperplasia of the lung. The severities of these lesions generally increased with increasing exposure concentration. Inflammation was characterized by multiple foci of a mixed cellular infiltrate oriented around blood vessels and bronchioles. The infiltrate was composed primarily of macrophages with abundant cytoplasm and fewer lymphocytes and neutrophils. The infiltrate extended into the surrounding perivascular interstitium and often filled adjacent alveoli. Hyperplasia involved alveolar and, to a lesser extent, bronchiolar epithelium. This change involved the distal airways and associated alveolar ducts and alveoli. Normally flattened epithelium was replaced with larger cuboidal cells.
Histopathological findings: neoplastic:
not specified
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
The epididymal spermatozoal motility of males exposed to 8 or 16 mg/m3 was statistically significant decreased. However, the effect on sperm motility was not very pronounced without a clear dose-response relationship. The lowest value was observed in the 8 mg/m3 group (mid dose) and was less pronounced in the high dose group.

Please also refer to the field "Attached background material" below.
Reproductive performance:
not specified

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
- The mouse that died early appeared thin.
- There were no other clinical findings related to vanadium pentoxide exposure.

ORGAN WEIGHTS (PARENTAL ANIMALS)
- Other organ weight changes were considered related to body weight decreases.

HISTOPATHOLOGY - NON-NEOPLASTIC
Lymphoid depletion of the thymus was observed in some males and females in the 16 mg/m3 groups (males: chamber control, 0/9; 8 mg/m3, 0/8; 16 mg/m3, 2/7; females: 0/9, 0/9, 1/10). This lesion may have been associated with the body weight decreases and debilitation
of the mice.

REPRODUCTIVE FUNCTION: OESTROUS CYCLE (PARENTAL ANIMALS)
No significant differences were noted in oestrous cycle parameters between exposed and chamber control females.


Atrophy of the secondary reproductive organs observed at 16 mg/m3 is also described (no supporting data provided). These findings are most probably secondary to the observed general toxicological and body weights effect in the high dose group and can thus not be attributed to a direct vanadium induced toxicological finding.

Please also refer to the field "Attached background material" below.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEC
Remarks:
reproductive toxicity
Effect level:
> 16 mg/m³ air
Based on:
test mat.
Sex:
female
Basis for effect level:
other: no effects on estrus cycle parameters determined
Dose descriptor:
NOAEC
Remarks:
reproductive toxicity
Effect level:
4 mg/m³ air
Based on:
test mat.
Sex:
male
Basis for effect level:
other: decreased epididymal spermatozoal motility at an above 8 mg/m3

Target system / organ toxicity (P0)

Critical effects observed:
not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:
not specified
Dermal irritation (if dermal study):
not specified
Mortality / viability:
not specified
Body weight and weight changes:
not specified
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 specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Sexual maturation:
not specified
Anogenital distance (AGD):
not specified
Nipple retention in male pups:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Histopathological findings:
not specified
Other effects:
not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not specified

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not specified

Details on results (F1)

not specified

Effect levels (F1)

Remarks on result:
not measured/tested

Target system / organ toxicity (F1)

Critical effects observed:
not specified

Results: F2 generation

Target system / organ toxicity (F2)

Critical effects observed:
not specified

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Groups of 10 male and 10 female B6C3F1 mice were exposed to V2O5 aerosols (whole body exposure) at concentrations of 0, 1, 2, 4, 8 or 16 mg/m3 (equivalent to 0, 0.56, 1.12, 2.25, 4.50, and 8.99 mg V/m³) by inhalation, 6 hours/d, 5 d/wk, for 3 months (91-92 days). Effects on fertility and sexual function of male and female mice were evaluated after inhalation exposure.
The results showed that epididymal spermatozoal motility was significantly decreased in males exposed to 8 and 16 mg/m3. Female oestrus cycle parameters were not affected.

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