Vanadyl pyrophosphate

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January - February 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant study conducted in accordance with internationally recognised test guidelines

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: US EPA Guidelines (1985, amended 1987) and EEC Annex VMethod B.12.

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Vanadyl pyrophosphate

EC Number:

406-260-5

EC Name:

Vanadyl pyrophosphate

Cas Number:

58834-75-6

Molecular formula:

V2P2O9

IUPAC Name:

divanadium(4+) (phosphonatooxy)phosphonate dioxidandiide

Details on test material:

- Name of test material (as cited in study report): BTN/A
- Chemical name: Vanadyl pyrophosphate
- Chemical formula: (VO)2P207
- Relative molar mass: 307.8
- Physical state Fine, dark-brown powder
- Lot/batch No.:0003
- Purity: Vanadium - 30.1%, phosphorous 21.0% by weight
- Storage condition of test material: Ambient conditions, in the dark

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Ltd., Margate, Kent, UK
- Age at study initiation: 5-6 weeks
- Weight at study initiation: Males: 23.1 - 29.4 g; Females: 19.7 - 24.8 g
- Assigned to test groups randomly: yes
- Housing: Polypropylene cages with stainless steel tops
- Diet (e.g. ad libitum): SDS R&M No. 1 laboratory rodent diet available ad libitum
- Water (e.g. ad libitum): Municipal supply water available ad libitum
- Acclimation period: Minimum of 4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 23 deg C
- Humidity (%): 40 - 70% RH
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 1992-02-03 (dosing of preliminary toxicity screen) To: 1992-02-12 (Sacrifice of main assay)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

For the test substance:
- Vehicle(s)/solvent(s) used: Methyl cellulose, 1% aqueous
- Justification for choice of solvent/vehicle: Standard suspending agent used for such studies, giving acceptable properties with formulations of this substance
- Concentration of test material in vehicle: 50 - 500 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL/kg

For the positive control agent (chlorambucil):
Vehicle(s)/solvent(s) used: Ethanol, 10% aqueous
- Justification for choice of solvent/vehicle: Standard dissolution agent used for such studies, giving acceptable properties with formulations of the agent
- Concentration of test material in vehicle: 3 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL/kg

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Test substance freshly formulated using 1% (aq) methyl cellulose with mixing to maintain homogeneity.

Duration of treatment / exposure:

24 - 72 hours

Frequency of treatment:

Once only

Post exposure period:

Five male and five female mice per group were killed 24 hours after treatment; further lots of five males and five females from control and high-dose groups were killed 48 and 72 hours after treatment

Doses / concentrations

Remarks:

Doses / Concentrations:
500, 1000, 2000 mg/Kg
Basis:
nominal conc.

No. of animals per sex per dose:

15 males / 15 females control (1% (aq) methyl cellulose)
5 males / 5 females at 500 mg/kg
5 males / 5 females at 1000 mg/kg
15 males / 15 females at 2000 mg/kg

Control animals:

yes, concurrent vehicle

Positive control(s):

Chlorambucil
- Justification for choice of positive control(s): Agent with history of use giving consistent responses
- Route of administration: Oral (gavage)
- Doses / concentrations: 30 mg/kg

Examinations

Tissues and cell types examined:

bone marrow erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Preliminary toxicity screen showed unacceptable toxicity at dose levels in excess of 2500 mg/kg

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): 24, 48 and 72 hours post-dose (exposure)

DETAILS OF SLIDE PREPARATION: Femurs from each animal dissected out and epiphyses cut off to obtain access to the marrow canal. Marrow cells were flushed out with 2.5 ml foetal calf serum using a syringe and needle. The recovered cells were centrifuged at 1000 rpm for five minutes. Single drops of the cell suspension were transferred to clean, dry slides, two or three smears (for the preliminary toxicity test or main micronucleus test respectively) prepared, and the slides left to air-dry. Following fixation in methanol for ten minutes, they were stained manually, using the Schmid (May-Grunwald and Giemsa) staining technique. The slides were air-dried, cleared for five minutes in xylene and made permanent using DPX mountant

METHOD OF ANALYSIS: Slides examined under a light microscope. A total of at least 2000 erythrocytes per animal were examined and classed as polychromatic or mature. At least 1000 cells of each type were scored from each animal where possible, but where there was an appreciable deviation from unity in the ratio of polychromatic to mature erythrocytes, scoring continued until a minimum of 2000 of the predominant cell type were counted.
In the main micronucleus test, each erythrocyte scored was examined for the presence or absence of micronuclei.

Evaluation criteria:

Main micronucleus test: The frequencies of micronucleated cells per 1000 erythrocytes were calculated. The ratio ofpolychromatic to mature cells was also determined; a decrease in this ratio possibly indicating inhibition of cell division following treatment. The incidence of micronuclei in the mature cell population 24 hours after treatment was regarded as reflecting the pretreatment situation, since most of these cells were produced before treatment. The frequency of micronuclei in polychromatic cells provided an index of induced genetic damage.

Statistics:

The frequencies of micronucleated cells per 1000 polychromaticerythrocytes scored were subjected to the Mann-Whitney Uprocedure (Mann and Whitney, 1942). Significance was determined by reference to tabulated values of RI. Where the RI-value obtained equalled the lower or upper limit given in the reference tables, exact p-values were calculated using the Wilcoxon Rank Sum Test. Data from males and females within each group were compared using a two-tailed test. Where there was no significant difference within the group, the sexes were pooled for further analysis. Where a significant difference was apparent between males and females of a group, comparisons to control data were made on a sex-by-sex basis. For each sampling time (24, 48 or 72 hours), each treated group was compared with concurrent vehicle controls using a one-tailed test.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 625, 1250, 2500 and 5000 mg/kg
- Solubility: Not soluble, administered as a suspension
- Clinical signs of toxicity in test animals: Approximately 21 hours after treatment, both male animals dosed at 5000 mg/kg, and one male dosed at 2500 mg/kg, showed signs including reduced activity, hunched/prone posture, slow respiration, ungroomed fur at the base of the tail and bleeding in the anal region: these animals were killed in extremis at this time. Approximately 28 hours after treatment, both female animals dosed at 5000 mg/kg showed signs of reduced activity and hunched posture, and both had also lost approximately 2 g in weight since dosing: these animals were killed in extremis at this time. No other animals showed any reactions to treatment and all survived. Incidences of weight loss were noted in surviving animals of all groups, but these were small and not dose-related.
- Evidence of cytotoxicity in tissue analyzed: No real indication of bone marrow toxicity (i.e. reduced cell proliferation, evidenced by a reduction in the ratio of polychromatic:mature erythrocytes) was seen in animals treated at 625 or 1250 mg/kg although the polychromatic:mature cell ratio in a single animal in each group was reduced. Evidence of bone-marrow toxicity was apparent in all animals killed in extremis. No such effect was seen in surviving animals treated at 2500 mg/kg.
- Rationale for exposure: 2500 mg/kg selected as highest tolerable dose
- Harvest times: 24, 48 and 72 hours post-dose

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): Frequencies of micronucleated polychromatic erythrocytes in animals killed 24, 48 or 72 hours after administration of BTN/A were similar to those in concurrent vehicle controls.
- Ratio of PCE/NCE (for Micronucleus assay): Among mice killed 24 hours after treatment, the mean PCE/NCE ratio was 0.8 for the vehicle control group. Corresponding values for animals dosed at 500, 1000 or 2000 mg/kg were similar: 0.8, 0.9 or 0.8 respectively. The ratio in animals treated with the positive control agent was 0.6. Among mice killed after 48 or 72 hours, the mean ratio in vehicle Control animals was 1.0 at both time points. Corresponding values for animals dosed with the substance at 2000 mg/kg were 1.1 at the 48 hour time point and 1.0 at the 72 hour time point.
- Statistical evaluation: Statistical analysis indicated a significant increase in micronucleated polychromatic cells over control values in male animals dosed at 500 mg/kg. However, as values for individual animals were all within the historical range for vehicle control animals at the testing laboratory, and as no such effect was apparent at higher BTN/A dosages, this was considered not to be biologically significant. Biologically and statistically significant increases over controls were seen in animals of the positive control group given chlorambucil.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
It is concluded that, under the conditions of test, there was no evidence of induced chromosomal or other damage leading to micronucleus formation in polychromatic erythrocytes of treated mice 24, 48 or 72 hours after oral administration of BTN/A. The test procedure was highly sensitive to the chromosome-damaging action of chlorambucil

Executive summary:

An in-vivo assessment of clastogenic action has been undertaken by the performance of a bone marrow erythrocyt micronucleus test in the mice follwing methods described by OECD TG 474 / EC B12. There was no evidence of induced chromosomal or other damage leading to micronucleus formation in polychromatic erythrocytes of treated mice 24, 48 or 72 hours after oral administration of the substance.