Registration Dossier

Toxicological information

Repeated dose toxicity: inhalation

Currently viewing:

Administrative data

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
2009-05-07 to 2010-04-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented paper
Cross-reference
Reason / purpose:
reference to same study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010
Reference Type:
publication
Title:
First steps towards an understanding of a mode of carcinogenic action for vanadium pentoxide
Author:
Schuler, D. et al.
Year:
2011
Bibliographic source:
J Toxicol Pathol 24(3): 149 -162.

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Divanadium pentaoxide was administered by nose-only inhalation to female mice for a period of 16 days (6 h/day) at target concentrations of 0.25, 1.0 and 4.0 mg/m3 test item in air. Female B6C3F1/Hsd mice were allocated to 4 groups of 48 mice each. The mice of group 1 served as air controls. The mice of each group were further allocated in seven subgroups to evaluate a range of specific toxicological end-points in the lungs and to obtain data on the concentration of vanadium in blood and lungs.
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 pentoxide
- Molecular weight: 182 g/mol
- Physical state: solid, yellow-orange
- Storage condition of test material: at room temperature (range of 20 ± 5 °C) under dry conditions.

Test animals

Species:
mouse
Strain:
B6C3F1
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories Ltd., 5190 Dominion Drive, Dublin, VA24084, U.S.A.
- Age at study initiation: 8 to 9 weeks
- Weight at study initiation (at acclimatisation): 16.9 to 23.2 g (± 17%)
- Housing: individually in Makrolon® type-2 cages with wire mesh tops and standard softwood bedding (‘Lignocel’ J. Rettenmaier & Söhne GmbH & Co, KG, 73494 Rosenberg / Germany, imported by Provimi Kliba AG, 4303 Kaiseraugst / Switzerland).
- Diet (ad libitum): pelleted standard Kliba Nafag 3433 (batch no. 76/08) mouse maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland)
- Water (ad libitum): community tap-water from Itingen.
- Acclimation period: at least 7 days under laboratory conditions, after clinical health examination. Only mice without any visible signs of illness were used for the study. The mice of groups 1 to 4 were accustomed to the restraint tubes and the exposure conditions for 3 daily periods of approximately 1, 3 and 5 hours, respectively.

ENVIRONMENTAL CONDITIONS
Optimal Hygienic Conditions behind a barriersystem in an air-conditioned facility.
- Temperature: 22 ± 3 °C
- Relative humidity: 30 - 70%
- Air changes: approximately 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12 (Music during the light cycle.)

Administration / exposure

Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
Vehicle:
clean air
Remarks on MMAD:
MMAD / GSD: Gravimetric determination of MMAD (GSD):
Group 3: 1.22 - 1.26 µm (1.89 - 1.92)
Group 4: 1.24 - 1.43 µm (1.89 - 1.92)
Determination of particle size distribution in group 2 was not feasible due to the low aerosol concentration. However, the MMAD for group 2 was considered to be similar to the other groups because one single aerosol generation system was used for all three treatment groups. The MMADs were at the lower limit of the target range of 1 to 3 μm, therefore deposition of the particles can be assumed to have occurred mainly in the lower but also in the upper respiratory tract.
Chemical determination of MMAD (GSD):
Group 3: 1.27 - 1.34 µm (1.77 - 1.86)
Group 4: 1.33 - 1.41 µm (1.75 - 1.82)
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus/Method of holding animals in test chamber: inhalation exposure was performed using a system similar to that originally described by Sachsse et al. ( 1976). The mice are confined separately in restraint tubes which are positioned radially around the flow-past, nose-only exposure chamber described by Cannon et al. (1983). The design of this chamber is based upon the fluid dynamic modeling of the test aerosol flow.
The exposure system ensured a uniform distribution and provided a constant flow of test material to each exposure tube. The flow of air at each tube was 0.5 L/min, which was sufficient to minimize re-breathing of the test aerosol as it was more than twice the respiratory minute volume of a mouse.
Before commencement of the exposure of the group(s), technical trials (procedures based on GLP) were conducted (without mice) using the inhalation system foreseen for the study.

- System of generating particulates/aerosols: a dust aerosol was generated from the test item using a rotating brush aerosol generator connected to a micronizing jet mill. The aerosol generated was then discharged into the exposure chamber through a 63Ni charge neutraliser. An airvacuum dilution system was used to achieve the target aerosol concentration for groups 2 and 3. Air control mice (group 1) were exposed to air under the same conditions as mice treated with the test item.
The air for aerosol generation was delivered by a model 'Atlas Copco ZT30-8' oil-free, rotating gear-wheel compressor system'. Additionally, there was an air-cleaning filter system mounted at the entry in the exposure room.

- Temperature, humidity, pressure in air chamber: oxygen concentrations in the chamber were measured continuously during each exposure using a calibrated device. The oxygen concentration was maintained above 19% during the exposure period. The results were reported three times during each daily exposure.
The relative humidity and temperature in the chamber were measured continuously during each exposure using a calibrated device. The results were reported three times during each daily exposure.

Results on temperature/humidity/oxygen concentration:
Group 1: 22.7 ± 0.3 °C; 1.0 ± 0.3 %; 20.7 ± 0.0%
Group 2: 22.1 ± 0.1 °C; 3.8 ± 0.1 %; 20.6 ± 0.0 %
Group 3: 21.8 ± 0.1 °C; 1.9 ± 0.8 %; 20.6 ± 0.0 %
Group 4: 21.2 ± 0.1 °C; 0.7 ± 0.6 %; 20.4 ± 0.0 %

- Air flow rate: measured for the collection of samples for the determination of test item concentration and particle size using a dry-test meter (‘Schlumberger Industries SA’, City of Geneva) and/or a pressure gauge (Timeus & Co., Zürich), calibrated with a reference dry-test meter.
The exposure air flow rate was adjusted as appropriate before the start of the exposure using calibrated flow-meters and / or pressure gauges. The actual airflow rates were recorded three times during each daily exposure.

The aerosol concentrations of the test item determined gravimetrically and chemically, particle size distribution determined gravimetrically and chemically, relative humidity, temperature and oxygen concentration, were measured on test aerosol samples collected directly from the delivery tube in the breathing zone of the mice, at an empty port of the exposure chamber.

- Method of particle size determination: distribution of particle size in the generated aerosol was measured by gravimetry twice during the 16-day treatment period in each of groups 3 and 4 using a cascade impactor. In group 3, sampling was performed over a period of 4 consecutive days. Following gravimetric determination of the particulate material on each impactor screen, the impactor samples were analyzed chemically. The samples were analyzed for vanadium using an AAS method and reported as vanadium pentoxide after recalculation.

TEST ATMOSPHERE
- Brief description of analytical method used:
1.) Gravimetrical determination: once daily for groups 2 to 4 using Millipore® durapore filters, Type HVLP, (Polyvinylidenedifluoride membrane, pore size 0.45 μm) loaded in a 47 mm in-line stainless steel filter sampling device (Gelman Science Inc., Ann Arbor, Michigan / U.S.A.).
2.) Chemical determination: vanadium pentoxide concentrations in the generated aerosol were performed once daily for groups 2 to 4 using the filters collected for gravimetric determinations. After weighing, filter samples and a blank filter were transferred to appropriately labeled vials. The samples were analyzed for vanadium using an atomic absorption spectroscopy (AAS) method and reported as vanadium pentoxide after recalculation.

NOMINAL DETERMINATION OF AEROSOL CONCENTRATION
The test item usage was measured by weighing the generator cylinder containing the test item before and after exposure to determine the quantity of test item used. The weight used was then divided by the total air-flow volume to give the nominal concentration.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Please refer to "Details on exposure" above
Duration of treatment / exposure:
16 consecutive days (except for the mice investigated for cell proliferation (day 7))
Frequency of treatment:
6 hours daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/m^3 (group 1)
Basis:

Remarks:
Doses / Concentrations:
0.231 ± 0.070 mg/m^3 (group 2)
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
1.03 ± 0.12 mg/m^3 (group 3)
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
4.02 ± 0.45 mg/m^3 (group 4)
Basis:
analytical conc.
No. of animals per sex per dose:
48 female mice
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the target aerosol concentrations were proposed by the Sponsor and are based on the results obtained within the previous National Toxicology Program (Toxicology and Carcinogenesis Studies of Vanadium Pentoxide (CAS No. 1314-62-1) in F344/N Rats and B6C3F1 Mice (Inhalation Studies)). NTP Technical Report Series No. 507. NIH Publication No. 03-4441. U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, NTP, Research Triangle Park, NC.

Examinations

Observations and examinations performed and frequency:
VIABILITY/MORTALITY
- Twice daily, prior to and following exposure and at least once daily during the quarantine and the acclimatization periods.

CLINICAL SIGNS
- Recorded once weekly during the acclimatization period.
- During 16-day treatment period, at least once daily before exposure. On day 8 of exposure, only the clinical signs of subgroup B were recorded.

BODY WEIGHTS
- Each mouse was weighed approximately weekly during the acclimatization and treatment periods (generally before exposure/dosing, with the exception of subgroups D and E of groups 3 and 4 where the body weights were recorded after exposure on day 9 of exposure).

VANADIUM IN WHOLE BLOOD AND LUNGS (examination performed in subgroup A mice):
- Whole blood: within 5 minutes after the end of the last exposure, blood samples were collected from the vena cava of mice.
- Lungs: removed and weighed.
- All samples were analyzed for vanadium using an atomic absorption spectroscopy (AAS) method validated under Harlan Laboratories Study B34154 and reported as Vanadium.

Sacrifice and pathology:
PATHOLOGY:
- Mice were anesthetized by intraperitoneal injection of pentobarbitone killed by exsanguination and examined for macroscopical findings.
- All surviving mice of allocation B and C were weighed and necropsied after 7 days and 16 days of exposure, respectively.
- All mice surviving to the end of the observation period were and killed.
- Lung weight was recorded at the scheduled dates of necropsy.
- Samples of the lungs were collected and fixed in neutral phosphate buffered 4% formaldehyde solution. The lungs were instilled with the fixative.
- Lungs from all allocation B and C mice were processed, embedded in paraffin wax, cut at a nominal thickness of 4 micrometers. The lung slides were stained for the two endogenous proliferation markers PCNA and Ki67.
- Lung slides of allocation C mice were stained with hematoxylin and eosin.

HISTOPATHOLOGY
- Slides stained with hematoxylin and eosin of all lungs of allocation C mice (16 days of exposure) were examined. Attempts were made to correlate gross observations with microscopic findings.
Statistics:
Statistical analysis:
The following statistical methods were used to analyze body weighs and organ weights:
- Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups.
- Fisher's exact-test was used to analyze macroscopical findings.
- Group means and standard deviations were calculated for continuous data.

Results and discussion

Results of examinations

Details on results:
VIABILITY AND MORTALITY
- One mouse of group 3 (subgroup C) was found dead shortly after the end of exposure during the second week of treatment.
- This death was considered as incidental and not related to treatment.
- There were no further unscheduled deaths.

CLINICAL SIGNS
- No clinical signs were noted during the course of this study.

BODY WEIGHTS
- Marginal body weight loss was noted in groups 3 and 4 in week 1 (statistically significance in group 4).
- At the end of the treatment period these animals recovered and body weights were similar across all groups.
- There were no effects on the body weight development in group 2.

VANADIUM IN WHOLE BLOOD AND LUNGS
- The analyses were for the concentrations of vanadium in blood and lung tissue and are reported as such, rather than being converted to vanadium pentoxide. Although most of the vanadium in lung will probably have remained as vanadium pentoxide, there are likely changes in oxidation state within this tissue during absorption that were not identified. It is unlikely that any of the vanadium in blood will be present as the pentoxide.
- The concentration of vanadium in the lungs increased dose-proportionally from group 2 to group 3.
- Vanadium values for blood and lung samples in group 4 were higher when compared with group 3, but less than dose proportionally; please refer for results to table 1. in "Any other information on results incl. tables" below.

ORGAN WEIGHTS
- After 16 days of vanadium pentoxide exposure, lung weights and lung weight/body weight ratios showed dose-related increases in groups 3 and 4 (subgroups A, C, D, E); differences to controls were statistically significant.
- At the interim kill after 7 days (subgroup B), there were no effects on the lung weights in group 3 and the statistically significant increased lung weight in group 4 was less pronounced.
- There were no effects on the lung weight in group 2 that were considered to be related to treatment.
- The statistically significant increases in the weight of the right lung lobe, but not the left lung lobe, and in the lung weight/body weight ratios of subgroup D animals were considered to be incidental in the absence of corresponding histopathological findings in this group, and the absence of significant weight changes in the other subgroups that had been simultaneously exposed.

PATHOLOGY
- At necropsy of subgroups A, B, C, D and E animals, no gross findings of the lung were recorded that distinguished test item exposed mice from controls.
- A reddish discolored lung was noted in the animal of group C which prematurely died. This kind of lesion is a common alteration in decedents and, therefore, was considered to be related to a certain degree of tissue autolysis due to delayed necropsy of this animal and not to represent of a primary treatment-related effect.

HISTOPATHOLOGY
- Multifocal/diffuse alveolar histiocytosis and multifocal subacute alveolitis was noted with a dose-related increase in severity in almost all mice of subgroup C in groups 3 and 4.
- In addition, multifocal granulocytic infiltration was noted in 4 out of 6 mice of group 3 and 5 out of 6 mice of group 4.
- Focal alveolar histiocytosis of minimal degree which was noted in 1 mouse of group 2. This finding was considered to be incidental.

Effect levels

open allclose all
Dose descriptor:
LOAEC
Effect level:
1.03 mg/L air (analytical)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Data suggest a sub-acute LOAEC of 1 mg/m3 based on histological changes in the lungs of mice and a NOAEC of 0.25 mg/m3.
Dose descriptor:
NOAEC
Effect level:
0.231 mg/L air (analytical)
Based on:
test mat.
Sex:
female

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1.

Group

Vanadium in whole blood [μg/L]

Vanadium in lung samples [μg/g]

2

- *

8.02 ± 0.84

3

51.15 ± 6.02

31.01 ± 2.22

4

160.13 ± 16.48

64.35 ± 7.45

* below lowest calibration point (13μg/L)

Applicant's summary and conclusion

Conclusions:
Data suggest a sub-acute LOAEC of 1 mg/m3 based on histological changes in the lungs of mice and a NOAEC of 0.25 mg/m3.