Tris(pentane-2,4-dionato-O,O')vanadium

Toxicity to reproduction

Administrative data

Endpoint:

fertility, other

Remarks:

Fertility Assessment Test (Sperm assessment)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

For the evaluation of DNA damage in the germ cells in COMET assay, CD-1 male mice (30-45 days old, 30-35 g, from our own stock) were housed in groups of two (experimental and control) in hanging plastic cages under controlled lighting conditions (lights on from 05:00 to 19:00 h). They were fed rat chow and water ad libitum. Working solutions of V2O5 were prepared in saline and injected i.p. in appropriate volumes containing either 5.75, 11.50, or 23 mg of V2O5/ kg body weight (1/4 of LD50. 1/2 of LD50, or LD50, determined in our laboratory for acute treatments).
Twenty-four hours after treatment, the animals were killed and the testes dissected and stored in 1 ml of RPMI medium (Sigma Chemical Co.) and minced in 2 ml cold saline. The cells were obtained and placed in 75 µL of low melting point agarose. SCG assay was performed as described by Tice et al. (1992). Briefly, after lysis at 4°C for 1 hour, slides were placed on horizontal electrophoresis unit. The DNA was allowed to unwind for 20 min in electrophoresis running buffer solution (300 mM NaOH and 1 mM Na.EDTA, pH 13). Electrophoresis was conducted for 20 min at 25 V and 300 mA. All technical steps were conducted using very dim indirect light. After electrophoresis, the slides were gently removed and alkaline pH neutralized with 0.4 M Tris, pH 7.5. Ethidium bromide (75 µL of a 20 µg/mL solution) was added to each slide and a coverglass was placed on the gel.

GLP compliance:

no

Type of assay:

mammalian comet assay

Specific details on test material used for the study:

Working solutions of V2O5 were prepared in saline

Species:

mouse

Strain:

CD-1

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: own stock (Laboratorio de Citogenética. Mutagénesis y Toxicología Reproductiva, UIBR Campo-ll, FES-Zaragoza (M.A.-L. L.A.-B), Laboratorio de Inmunología, Facultad de Medicina Veterinaria y Zootecnia (F.B -A.), and Laboratorio de Genética Toxicológica Molecular, Departamento de GTA Institutode Investigaciones Biomédicas (M V, E.R.), UNAM, México, D.F, México)
- Age at study initiation: 30-45 d
- Weight at study initiation: 30-35 g
- Assigned to test groups randomly: not specified
- Fasting period before study: not specified
- Housing: hanging plastic cages (groups of two)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): not specified
- Humidity (%): not specified
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 10/14

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: saline
- Concentration of test material in vehicle: 5.75, 11.50, or 23 mg of V2O5/kg body weight

Details on exposure:

Working solutions of V2O5 were prepared in saline and injected i.p. in appropriate volumes containing either 5.75, 11.50, or 23 mg of V2O5/kg body weight (1/4 of LD50, 1/2 of LD50, or LD50, determined in the laboratory for acute treatments)

Duration of treatment / exposure:

one treatment (injection)

Frequency of treatment:

one treatment

Post exposure period:

24 h

Dose / conc.:

5.75 other: mg/kg bw

Dose / conc.:

11.5 other: mg/kg bw

Dose / conc.:

23 other: mg/kg bw

Dose / conc.:

0 other: mg/kg bw

No. of animals per sex per dose:

1 (treatments), 3 (control)

Control animals:

yes, concurrent vehicle

Positive control(s):

not specified

Tissues and cell types examined:

testes; microscopic analysis revealed the presence of two distinct subpopulations of cells: large cells (mean diameter 67.53 µm) and small cells (mean diameter 45.8 µm)

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: 1/4 of LD50, 1/2 of LD50, or LD50, determined in the laboratory for acute treatments

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): 24 hours after treatment

DETAILS OF SLIDE PREPARATION: The testes were dissected and stored in 1 ml of RPMI medium (Sigma Chemical Co.) and minced in 2 ml cold saline. The cells were obtained and placed in 75 µL of low melting point agarose. SCG assay was performed as described by Tice et al. Briefly, after lysis at 4°C for 1 hour, slides were placed on horizontal electrophoresis unit. The DNA was allowed to unwind for 20 min in electrophoresis running buffer solution (300 mM NaOH and 1 mM NaEDTA, pH 13). Electrophoresis was conducted for 20 min at 25 V and 300 mA. All technical steps were conducted using very dim indirect light. After electrophoresis, the slides were gently removed and alkaline pH neutralized with 0.4 M Tris, pH 7.5. Ethidium bromide (75 µL of a 20 µg/ml solution) was added to each slide and a coverglass was placed on the gel.
The staining of the liberated DNA allowed the microscopic discrimination of cell images with and without damage.

METHOD OF ANALYSIS: The microscopic images revealed circular shapes (undamaged DNA) and "COMET" structures (damaged DNA). The extension of each image, signifying the migration distance of DNA, was determined by scaled ocular. The image length of DNA migration (in µm) was determined from 50 cells per slide and 2 slides per concentration.

Evaluation criteria:

Other criteria for evaluation were to assign the evaluated cells to classes according to their degree of DNA damage. The classes were low damage(<20%), medium damage (20—10%), high damage (41-95%), and total damage (>95%) according to Anderson et al..

Statistics:

nonparametric Wilcoxon rank-sum test was used because this method takes into account all categories of damage.

Key result

Sex:

male

Genotoxicity:

positive

Toxicity:

not examined

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

not examined

Additional information on results:

Figures 1 and 2 and table 1 represent the effect of vanadium pentoxide or saline on the DNA damage index on testicular germ cells, calculated after the measurements by COMET assay. DNA migration increased significantly depending on the dose of vanadium in both large and small cells. The degree of DNA damage in large and small cells was larger in those animals injected with the higher dose of V2O5. The results presented herein show that vanadium treatment induced SSB in DNA of testis cells.
In combination with the reproductive toxicity study (please refer to section 7.8.1), the results presented herein show that vanadium treatment induced SSB in DNA of testis cells, sperm head abnormalities and a low frequency of DLM (dominant lethal mutations). The DLM in males was measured by determining the frequency of live vs. dead and resorbed fetuses occurring after mating chemical-treated males with untreated females.

Table 1: Effects of Vanadium pentoxide on Mouse Testis Cells in the COMET Assay

	Grade of damage in large cells (%)					Grade of damage in small cells (%)
Treatment	None (<5 %)	Low (5-20 %)	Medium (21-40 %)	High (41-95 %)	Total (>95 %)	None (<5 %)	Low (5-20 %)	Medium (21-40 %)	High (41-95 %)	Total (>95 %)
Control	58	22	20	0	0	68	14	14	4	0
5.75***	16	22	16	40	6	4	4	30	56	6
11.50*	14	0	20	56	10	52	30	14	4	0
23.00***	1	0	22	76	0	0	8	26	60	6

*P<0.05 for large cells vs. control

**P<0.05 for small cells vs. control

Conclusions:

The results presented herein show that vanadium treatment induced SSB in DNA of testis cells. In combination with the reproductive toxicity study (please refer to section 7.8.1), the results presented herein show that vanadium treatment induced SSB in DNA of testis cells, sperm head abnormalities and a low frequency of DLM (dominant lethal mutations). The DLM in males was measured by determining the frequency of live vs. dead and resorbed fetuses occurring after mating chemical-treated males with untreated females. However, it should be noted that gonads contain a mixture of somatic and germ cells. For this reason, positive results in whole gonad (testis) are not necessarily reflective of germ cell damage; nevertheless, they indicate that tested chemical(s) and/or its metabolites have reached the gonad (OECD TG 489).

Executive summary:

Effects of vanadium pentoxide (V2O5) treatment on reproductive function and testicular DNA in male mice were investigated. These functions were evaluated with fertility rate, implants, resorptions, sperm counts, motility, and morphology. The DNA damage in individual testis cells was analyzed by single-cell gel electrophoresis technique (COMET assay). V2O5 treatment resulted in a decrease in fertility rate, implantations, live fetuses, and fetal weight, and an increase in the number of resorptions/dam. Sperm count, motility, and morphology were impaired with the advancement of treatment. Vanadium treatment induced DNA damage depending on the dose in the testis cells that was expressed and detected as DNA migration in the COMET assay. The distribution of DNA migration among cells, a function of dose, revealed that the majority of cells of treated animals expressed more DNA damage than cells from control animals. It is concluded that vanadium pentoxide was a reprotoxic and genotoxic agent in mice.

Data source

Reference

Reference Type:

publication

Title:

Reprotoxic and Genotoxic Studies of Vanadium Pentoxide in Male Mice

Author:

Altamirano-Lozano M

Year:

1996

Bibliographic source:

Teratogenesis, Carcinogenesis, and Mutagenesis 16:7-17 (1996)

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: Effects of vanadium pentoxide (V2O5) treatment on reproductive function and testicular DNA in male mice were investigated.
- Short description of test conditions: 8.5 µg/g bw V2O5 was administered to male rats intraperitoneally for 60 days. On day 61 they were subjected to a fertility assessment test
- Parameters analysed / observed: The fertility of male mice was assessed by the incidence of pregnancy in females. Other reproductive parameters determined were the number of litters, implants, resorptions, live and dead fetuses, and fetal body weight. In addition, sperm motility, sperm counts and sperm morphology was evaluated.

GLP compliance:

no

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Divanadium pentaoxide

EC Number:

215-239-8

EC Name:

Divanadium pentaoxide

Cas Number:

1314-62-1

Molecular formula:

O5V2

IUPAC Name:

dioxovanadiooxy(dioxo)vanadium

Details on test material:

Aldrich Chemical Co., Milwaukee, WI

Specific details on test material used for the study:

A working solution of vanadium pentoxide (99.6% pure, Aldrich Chemical Co., Milwaukee, WI, CAS 1314-62-1) was prepared by dissolving the compound in saline.

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: own stock (Laboratorio de Citogenética. Mutagénesis y Toxicología Reproductiva, UIBR Campo-ll, FES-Zaragoza (M.A.-L. L.A.-B), Laboratorio de Inmunología, Facultad de Medicina Veterinaria y Zootecnia (F.B -A.), and Laboratorio de Genética Toxicológica Molecular, Departamento de GTA Institutode Investigaciones Biomédicas (M V, E.R.), UNAM, México, D.F, México)
- Age at study initiation: 45 d
- Weight at study initiation: (P) Males: 26-29 g;

- Housing: hanging plastic cages
- Diet (e.g. ad libitum): ad libitum, Purina chow
- Water (e.g. ad libitum): ad libitum
- Acclimation period: not specified
ENVIRONMENTAL CONDITIONS
- Temperature (°C): not specified
- Humidity (%): not specified
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 10/14

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

water

Remarks:

saline

Details on exposure:

A working solution of vanadium pentoxide was prepared by dissolving the compound in saline and injected intraperitoneally (i.p.) in an appropriate volume containing 8.5 µg/g body weight. Controls were treated with saline.

Details on mating procedure:

- M/F ratio per cage: 1/2
- Length of cohabitation: 5 overnight matings
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

60 days

Frequency of treatment:

every third day

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 other: µg/g bw/0.3 d

Doses / concentrations 2

Dose / conc.:

8.5 other: µg/g bw/0.3 d

No. of animals per sex per dose:

Group A (vehicle control): 20 males
Group B: 15 animals were injected with V2O5 every 3rd day during 60 days (fertility assessment)
Groups C-H: 30 animals were injected with V2O5 every 3rd day. Groups of 5 animals were sacrificed every 10 days after the beginning of the treatment (sperm assessment)

Control animals:

yes, concurrent vehicle

Details on study design:

Sixty-five males that proved fertile were allotted randomly to the following experimental groups. Group A: controls (20 animals) were injected with vehicle every 3rd day during 60 days. Beginning on day 61, they were subjected to a fertility assessment test and sacrificed 5 days later. Group B: 15 animals were injected with V2O5 every 3rd day during 60 days. On day 61 they were subjected to a fertility assessment test and sacrificed 5 days later. Groups C-H: 30 animals were injected with V2O5 every 3rd day and groups of 5 animals were sacrificed every 10 days after the beginning of the treatment.
- Dose selection rationale: 8.5 µg/g bw corresponds to 1/2 of the LD50 determined in the same laboratory for subchronic treatments.

Positive control:

no positive control

Examinations

Parental animals: Observations and examinations:

Health indicators recorded in adult male mice were general appearance, mortality rate, and body weight (initial vs. final).

Oestrous cyclicity (parental animals):

not examined

Sperm parameters (parental animals):

Parameters examined in [P] male parental generations: testis weight, sperm count in vas deferens, sperm motility, sperm morphology

Litter observations:

number of litters, live and dead fetuses, and fetal body weight.

Postmortem examinations (parental animals):

At autopsy the testes were dissected and weighed with the aid of an analytical balance. Sperm parameters were assessed.

Postmortem examinations (offspring):

not examined

Statistics:

The results of percent of fertility, sperm motility, and abnormal sperm were analyzed using the "z"-test. The frequency of implants, resorptions, live and dead fetuses, body and testis weight, and sperm count were analyzed using the Student's t-test.

Reproductive indices:

incidence of pregnancy in females, number of litters, implants, resorptions, live and dead fetuses, and fetal body weight

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The final body weight of V2O5-treated animals during 60 days was lower than controls, while differences were not observed in those animals sacrificed at days 10, 20, 30, 40, or 50 after the beginning of the treatment

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

The sperm count diminished significantly in V2O5-treated animals during 20 days or longer. A marked reduction in sperm motility was observed with the advancement of treatment in mice treated with V2O5.
A significant increase of the percentage of morphologic abnormalities in spermatozoa obtained from vanadium-treated animals was observed after 50 and 60 days of treatment.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

The fertility rate was significantly lower in V2O5-treated mice than in controls (10/30; 33 % vs. 34/40; 85 %, P < 0.05, "z"-test). The number of implants and pups was lower in females mating with V2O5-treated males, while the incidence of resorptions was increased in these females. The body weight of fetuses born from dams impregnated by V2O5-treated mice was lower than controls.

Details on results (P0)

please refer to "Any other information on results incl. tables"

Effect levels (P0)

1

Dose descriptor:

dose level: applied every third day during 60 days

Effect level:

8 other: µg/g bw/0.3 d

Based on:

test mat.

Sex:

male

Basis for effect level:

organ weights and organ / body weight ratios

reproductive function (sperm measures)

reproductive performance

2

Key result

Dose descriptor:

dose level: decrease in fertility rate, implantations, live fetuses, and fetal weight, and an increase in the number of resorptions/dam. Sperm count, motility, and morphology were impaired with the advancement of treatment

Effect level:

2.7 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

organ weights and organ / body weight ratios

reproductive function (sperm measures)

reproductive performance

Target system / organ toxicity (P0)

Critical effects observed:

yes

Lowest effective dose / conc.:

8 other: µg/g bw/0.3 d

System:

male reproductive system

Organ:

germ cells

testes

Treatment related:

yes

Dose response relationship:

no

Relevant for humans:

yes

Results: P1 (second parental generation)

Effect levels (P1)

Remarks on result:

not measured/tested

Results: F1 generation

General toxicity (F1)

Clinical signs:

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

the number of live fetuses of V2O5-treated males is significantly reduced when compared to the controls

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weight of fetuses bom from dams impregnated by V2O5 treated mice was lower than controls.

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

please refer to "Any other information on results incl. tables"

Effect levels (F1)

1

Dose descriptor:

dose level: decrease in live fetuses, and fetal weight

Generation:

F1

Effect level:

8.5 other: µg/g bw/0.3 d

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

body weight and weight gain

2

Key result

Dose descriptor:

dose level: decrease in live fetuses, and fetal weight

Generation:

F1

Effect level:

2.7 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

body weight and weight gain

Target system / organ toxicity (F1)

Critical effects observed:

no

Results: F2 generation

Effect levels (F2)

Remarks on result:

not measured/tested

Overall reproductive toxicity

Reproductive effects observed:

yes

Lowest effective dose / conc.:

2.7 mg/kg bw/day

Treatment related:

yes

Relation to other toxic effects:

reproductive effects in the absence of other toxic effects

Dose response relationship:

no

Relevant for humans:

yes

Any other information on results incl. tables

Table 1: Effects of Vanadium Pentoxide on Reproductive Function of CD-1 Male Mice

	Control	Vanadium pentoxide (8.5µg/g)
Males (n)	20	13
Mated females (n)	40	Id
Pregnant (n)	34	10
Fertility (%)^a	85	33
Implantation sites^b	10.88 ± 1 60	5.80 ± 1.33**
Resorptions^b	0.24 ± 0.42	2.00 ± 1.67*
Live fetuses^b	10.53 ± 1.42	3.40 ± 0.49**
Dead fetuses^b	0.12 ± 0.32	0.40±0.49
Fetal weight (mg)^b	145 ± 4.0	121± 7.0*

^a(Pregnant/females mated) x 100

^bMean ± SD

* P < 0.05

** P<0.01

Table 2: Effects of Vanadium Pentoxide on Body and Testis Weight, Sperm Count, Sperm Motility and Sperm Morphology (Mean ± SD)

Vanadium pentoxide (8.5 µg/g)
	Control	10 days	20 days	30 days	40 days	50 days	60 days
Males (n)	20	5	5	5	5	5	20
Initial weight(g)	28.04 ± 0.61	26.74 ± 0.71	27 19 ±0.34	27.33 ± 0.51	28.00 ± 0.25	28.14 ± 0.30	27.44 ± 0.49
Final weight(g)	31.20 ± 1.09	27.14 ± 0.71	27.00 ± 0.43	20.83 ± 0.66	27.14 ± 0.90	26.81 ± 1.03	24.64 ± 1.34*
Testis weight (mg)	135.90 ± 16.80	131.80 ± 24.30	134.10 ± 21.90	129.70 ± 23.30	130.10 ±20.10	124.00 ± 26.00*	118.72 ± 22.33**
Sperm count (x 10^6/ml)	29.19 ± 2.43	23.50 ± 7.53	19.80 ± 5.31**	16.67 ± 3.68**	16.67 ± 2.68**	19.91 ± 1.28**	7.27 ± 2.31**
Motility (%)	73.10 ± 19.40	40.60 ± 10.10**	35.50 ± 11.70**	18.10 ± 13.40**	14.20 ± 9.30**	4.30 ± 8.80**	4.01 ± 2.91**
Abnormalsperm (%)	6.40 ± 1.80	5.50 ± 3.90	3.80 ± 4.30*	4.70 ± 5.10	6.90 ± 5.10	8.10 ± 4.10*	10.83 ± 3.70**

* P < 0.05

** P < 0.01

Applicant's summary and conclusion

Conclusions:

V2O5 treatment resulted in a decrease in fertility rate, implantations, live fetuses, and fetal weight, and an increase in the number of resorptions/dam. Sperm count, motility, and morphology were impaired with the advancement of treatment.

Executive summary:

Effects of vanadium pentoxide (V2O5) treatment on reproductive function in male mice were investigated. V2O5 was administered intraperitoneally at a dose of 8.5 µg/g bw every three days (ca. 2.7 mg/kg bw/day) for 60 days. Reproductive functions were evaluated with fertility rate, implants, resorptions, sperm counts, motility, and morphology. V2O5 treatment resulted in a decrease in fertility rate, implantations, live fetuses, and fetal weight, and an increase in the number of resorptions/dam. Sperm count, motility, and morphology were impaired with the advancement of treatment.