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Administrative data

Key value for chemical safety assessment

Toxic effect type:
concentration-driven

Effects on fertility

Description of key information

Male and female fertility

An extensive literature/data search and evaluation programme on animal and human data relating to possible adverse effects of vanadium substances on reproductive toxicity has recently been conducted. As an outcome, some data are available for soluble pentavalent substances (V2O5, NH4VO3and NaVO3) via the oral and inhalation route. Two NTP studies with inhalation exposure of V2O5 in mice and rats were conducted under GLP and although not specific reproduction toxicity study they are regarded as key studies with RL1, because effects on male and female reproductive organs were evaluated. The other studies can be regarded of low reliability. The studies show supportive evidence of a potential effect of vanadium on male and female fertility.

In the two 3-months studies (NTP) with inhalation exposure of V2O5 in male and female rats and mice, animals (10/sex/group) were exposed to 0, 1, 2, 4, 8, or 16 mg V2O5/m3 (0, 0.56, 1.12, 2.25, 4.50, 8.99 mg V/m3) by whole-body inhalation for 6h/d, 5d/week for 3 months. In the highest dose (16 mg/m3), 3/10 females died during the study and necropsy body weight was statistically significantly lower. There was a statistically significantly increases in estrus cycle length at 8 and 16 mg/m3. However, this effect was highest in the lower dose group of 8 mg/m3, thus showing no clear relation to dose and treatment. In contrast, no deaths occurred in female mice. Final body weights started to be reduced in the exposure group of 4 mg/m³, but there was no statistically significant effect on estrus cycle length.

In male rats, severe toxicity was present in the high dose group with 7/10 males found dead during the study, and final body weights from animals in dose groups 4, 8 and 16 mg V2O5/m3 were significantly reduced. In male rats there was a statistically significant effect on necropsy body weight at 8 mg/m3, and no effects on male reproductive organ weights. Spermatid measurements (heads and counts) were not statistically significantly affected as well as epididymal sperm motility and concentration. Atrophy of the secondary reproductive organs in male rats at 16 mg/m3 is described, but at this dose level clear signs of general toxicity were shown. In addition, hypospermia of the testis and atypical cells of the epididymis at 16 mg/m3 are described, while 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. Thus, the findings in male rats can be considered 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. In male mice, 1/10 males died during the study in the highest dose group. Necropsy body weights were statistically significantly lower at 16 mg/m3, and there was a statistically significant effect on sperm motility at 8 and 16 mg/m3. 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.

In conclusion, the effects in female animals were observed only in a single species (rat) and not in the other species (mice). The effects in male animals were also observed only in a single species, but in mice, and not in the other species (rat). For both effects, there was no clear relation to dose and treatment and thus the findings must be regarded of low evidence for a vanadium induced effect on male and female fertility. Taking the results together, a dose level of 4 mg V2O5/m3 may be regarded as a NOEC for male and female fertility based on these studies.

 

In addition, there is one other study (Morgan A.M. and El-Tawil O. 2003) with ammonium metavanadate treatment via drinking water in rat available in which male and female animals were paired and effects on fertility evaluated. This study is only of RL3, because of limitations with respect to cohabitation, dose level (only one) and reporting. Sprague-Dawley rats were treated with ammonium metavanadate (200 ppm in drinking water). Male rats (n=10) were treated with 10 mg NH4VO3/kg bw/d corresponding to 4.35 mg V/kg bw/d (calculated with default factors according to ECHA (2012)) for 70 days, and 20 females with 11.43 mg NH4VO3/kg bw/d, (4.97 mg V/kg bw/d) for 61 days (14 days premating, during mating, till weaning of pups (21 days of age).

The results of this mechanistic study conducted only with one dose level show that mating and fertility indices were reduced in treated males mated with untreated females (group 1) and treated females mated with untreated males (group 2) (mating index: control: 100%, treated males 1: 65%; treated females: 70%; fertility index: 95%, 46.15%, 71.43%, respectively). In treated males, reduced weight of testes, epididymis, prostate gland, seminal vesicles, (p<0.05), with no reduction in body weight between control and treated males were observed. In females, the estrus cycle was disturbed, total number corpora lutea was reduced (control: 220, group 1: 54; group 2: 94), and signs of dystocia (no. of dams: 0, 1, 4) and delayed birth date (no. of dams: 0, 3, 5) were observed. No information on general is toxicity presented.

 

There is another rat study (Domingo et al. 1986) conducted with an appropriate route of exposure (oral) and different dose levels sodium vanadate compared to a control group (0, 5, 10 or 20 mg/kg bw/d sodium vanadate per os equivalent to 2.1, 4.2 and 8.4 mg vanadium/kg/day, respectively) available, but only of RL3. Females were treated for 14 days and males for 60 days before mating. Females continued to receive treatment with vanadium until study termination. Parameters evaluated on day 14 sacrifice were the nos. of corpora lutea, total implantations, living and dead fetuses and resorptions.

Fertility was not affected by treatment of male and female rats with sodium vanadate and there were no signs of maternal toxicity (no data presented). At interim sacrifice of females on day 14 of gestation, no significant adverse effects were observed on nos. of corpora lutea, total implantations, living and dead fetuses and resorptions. Thus, at 20 mg/kg bw/dsodium vanadate per os no effects could be determined on fertility of rats.

The reproductive toxicity of sodium metavanadate (Llobet 1993) was also studied in male Swiss mice exposed doses of 0, 20, 40, 60 and 80 mg/kg bw per day (equivalent to 8.4, 16.8, 25.2 and 33.6 mg vanadium/kg bw/day) given in the drinking water for 64 days. To evaluate the fertility of the vanadium-treated animals, males were mated with untreated females. A significant decrease in the pregnancy rate was observed at 60 and 80 mg/kg per day. However, the test substance did not reduce fertility in male mice at 20 and 40 mg/kg per day. Decreased body and epididymis weights were only observed in the 80 mg/kg per day group, while testicular weights were not altered by the treatment. Sperm count was significantly decreased at 60 and 80 mg/kg per day, but the sperm motility was unaffected. Histopathological examination revealed that testes were normal and that epididymis of treated male mice contained normal appearing sperm. In this reproduction toxicity study in mice, at 40 mg/kg bw/dsodium metavanadate per os (drinking water) no effects could be determined on fertility of rats.

Four additional references (Fortoul T.I. et al. 2007, Mussali-Galante et al. 2005, Bizarro-Nevares et al. 2016, Rodriguez-Lara V et al. 2016) are available with studies conducted with one dose of V2O5 by inhalation (1.4 mg V2O5/m3 equivalent to 0.79 mg V/m³) with a reliability of 3 conducted by the same group of researchers. The study design described in the publications is similar, but different mechanistic endpoints are addressed in the papers. The results of these publications support the evidence of a vanadium related effects on male reproductive organs at a certain exposure level. Supportive evidence might be taken also from several studies with ip injection of divanadium pentaoxide or sodium metavanadate to mice, rats or Guinea-pigs. The studies support the mechanistic evidence of a vanadium induced effect of male reproductive organs but were not taken into consideration.

Developmental Neurotoxicity

Several studies are available with exposure of suckling pups via the milk of their mothers. All studies show deficiencies with respect to methods (e.g. only one dose level, purity of the applied compound not given, no documentation of general unspecific toxicity etc.) and reporting of results and are therefore only of reliability score 3 and should thus be considered only as supportive. In these mechanistic studies, exposure of lactating mothers was conducted by IP injections in order to reach high amounts of secretion into the milk. IP injection is in general a non-relevant route of exposure, but in this case exposure of suckling pups by the milk of their mothers is the relevant route of exposure for this end point. Because of the IP administration to dams, exposure of sucklings by the milk of their mothers might be higher than under realistic exposure considerations and thus represents a worst case scenario.

These non-regulatory mechanistic studies were conducted with only one effective dose level of 3 mg NaVO3/kg body weight/day (1.25 mg V/kg bw/day) in comparison to a control group. In 6 of 7 studies conducted either in suckling pups, directly exposed pups or adult rats, effects were observed to a different degree on behaviour and motor coordination in open field tests. The effect on negative geotaxis investigated in 3 studies was not consistent and rotarod performance was affected in pups (exposed via milk or directly), but not in adult rats. In addition, histochemistry and immunohistochemistry investigations were conducted in these studies. There were occasions of a negative effect of vanadium treatment on myelin fibre density or demyelination in different brain areas in suckling pubs as well as in adult rats. Astrogliosis was observed in 4 studies in pubs with varying degree, and oligodendrocytes were shown to be sensitive against vanadium treatment with oligodendrocyte progenitor cells (in vivo and in vitro) being more sensitive to vanadium exposure than astrocytes or mature oligodendrocytes. Lipid peroxidation was shown in adult rats, but could not be detected in suckling pubs. In addition, a few other single endpoints were evaluated.

In conclusion, there is some evidence that exposure of suckling pubs to vanadium via the milk of their mothers may result in neurotoxic effects evidenced by behavioural changes, myelin damage and astrogliosis under the conditions of these studies. However, based on the available information, no conclusion can be drawn with respect to effect or no effect levels and to realistic exposure scenarios of breastfed children. 

Link to relevant study records

Referenceopen allclose all

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.
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
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 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
Specific details on test material used for the study:
not specified
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
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 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
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
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: 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
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.

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
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
Critical effects observed:
not specified
Reproductive effects observed:
not specified
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.
Endpoint:
fertility, other
Remarks:
mechanistic study to address effects on male and female fertility and reproductive performance
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
not specified
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
The study was not designed to establish a dose-response relation or a no effect level and shows deficiencies. In this study, mating period was only 5 days. In regulatory toxicity studies, the mating period is 3 weeks days and animals are placed only in the study when mating is confirmed. The study was conducted only with one dose which clearly represents an effect level and is not designed to establish a no effect level. In addition, the number of females used for investigations of effects on male mating and fertility index as well as other developmental endpoints was very low compared to guideline studies. No information on general is toxicity presented.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Sprague-Dawley rats were treated with ammonium metavanadat (200 ppm in drinking water). Male rats (n=10) were treated with 10 mg test item/kg bw/d corresponding to 4.35 mg V/kg bw/d (calculated with default factors according to ECHA (2012)) for 70 days, and 20 females with 11.43 mg NH4VO3/kg bw/d, (4.97 mg V/kg bw/d) 61 days exposure (14 days premating, during mating, till weaning of pups (21 days of age). Treated males (n=10) were mated for 5 days with untreated females (n=20), and treated females (n=20) for 5 days with untreated males (n=10). The following parameters were investigated: fertility assessment (mating, fertility index) in untreated and treated females on day 20 (n=10) of gestation or day 21 of lactation (n=10), and exposed males (n=10) analysed after the mating period for body weight, testes weight, epididymis, prostate, seminal vesicles (n=10 controls).
GLP compliance:
not specified
Remarks:
not specified
Limit test:
no
Justification for study design:
not applicable
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source of test material: Sigma Chemical Co., St. Louis, MO
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
not specified
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 180 - 200 g
- Housing: kept in cages containing 4 to 5 animals.
- Diet: ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: drinking water
Remarks on MMAD:
MMAD and GSD were not stated in the publication.
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was dissolved in drinking water at concentration of 200 ppm (de Rodriguez et al., 1998)*.

*Reference:
- Faria de Rodriguez C, Villalobos H, Nava de leal C. Teratogenic effects of ammonium metavanadate on the CNS of the offspring of albino rats. A histological and histochemical study. Invest Clin 1998;39(Suppl 1):55–85.
Details on mating procedure:
- M/F ratio per cage:1 male (untreated or exposed) / 2 untreated females or 1 untreated male / 2 females (untreated or exposed)
- Length of cohabitation: 5 days
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of gestation
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
- Males: 70 days
- Females: 61 days (14 days premating, during mating, and throughout the whole length of gestation and lactation periods till weaning (21 days after births) (Manson & Kang, 1989)*)

*Reference:
- Manson JM, Kang YJ. Test methods for assessment of female reproductive and developmental toxicology. In: Hayes AW, editor. Principles and methods of toxicology. New York: Raven Press; 1989, p. 311–61.
Frequency of treatment:
daily
Details on study schedule:
not specified
Dose / conc.:
200 ppm
Remarks:
males: 10 mg test item/kg bw/d corresponding to 4.35 mg V/kg bw/d (calculated with default factors according to ECHA (2012))
females: 11.43 mg test item/kg bw/d, corresponding to 4.97 mg V/kg bw/d
No. of animals per sex per dose:
10 male and 20 female rats
Control animals:
yes
Details on study design:
Fertility of males was investigated by mating of 10 exposed males and 10 control males with virgin untreated females (ratio: 1 male / 2 untreated females). The untreated females were investigated to evaluate the effects of ammonium metavanadate exposure on fertility.

Fertility of females was estimated in 20 adult exposed female rats and in another 20 control female counterparts. After 14 days of ammonium metavanadate exposure, each two females were placed in an individual cage with one adult untreated male of proven fertility
Positive control:
not specified
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: No data
DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations:
females: at the end of gestation period
males: at sacrifice

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE: No data

FURTHER OBSERVATIONS:
- number of dams showing delayed birth date and signs of dystocia were recorded.
Oestrous cyclicity (parental animals):
The control and ammonium metavanadate exposed females were examined for oestrous cycle regularity during the premating period.
Sperm parameters (parental animals):
After sacrifice of treated and control males, which were mated with untreated females, the following measurements were recorded: body weight and weight of testes, epididymis, prostate, and seminal vesicles.
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- dead and live foetuses, foetal body weight at birth and at days 4, 7, 14 and 21 after birth, foetal survival and viability indices during lactation period.
- pups from all groups were obtained at day 20 of gestation and at day 21 of lactation and examined for presence of any behavioral defects (especially in the offspring obtained during lactation).
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: males were removed after the mating period and sacrificed
- Maternal animals: half of the untreated females were sacrificed with their foetuses at 20th day of gestation, while the other half was sacrificed with their pups at day 21 of lactation to record the fertility end points.

GROSS NECROPSY / ORGAN WEIGHTS:
- Number of corpora lutea, implantation sites, resorptions, dead and live foetuses.
- gravid uterine and placental weights.

Postmortem examinations (offspring):
SACRIFICE
Pups from all groups were obtained at day 20 of gestation and at day 21 of lactation.

GROSS NECROPSY
Pups were examined for presence of any gross malformation. One third of these foetuses were examined for presence of any visceral anomalies and the remaining two thirds of the foetuses were examined for presence of any skeletal anomalies.
Statistics:
The data of treated male and female groups were compared to the control group. The data presented as percentage were analyzed using Chi-square, however, other data were analyzed using either one-way ANOVA or Student’s t-tests. The differences in the data were considered statistically significant at probability of P <0.05.
Reproductive indices:
Mating and fertility indices
Offspring viability indices:
Foetal surivival and viability indices during the lactation period
Clinical signs:
not specified
Dermal irritation (if dermal study):
not specified
Mortality:
not specified
Body weight and weight changes:
no effects observed
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:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Reproductive function: oestrous cycle:
effects observed, treatment-related
Description (incidence and severity):
The oestrous cycle regularity was disturbed in the ammonium metavanadate exposed females. Number of females with a regular oestrous cycle were 20/20 and 12/20 in the control group and treatment group, respectively.

Please also refer to the field "Attached background material" below.
Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
Testes (control group: 3.88 ± 0.19; treatment group: 2.75 ± 0.35; p < 0.05), epididymis (control group: 0.61 ± 0.06; treatment group: 0.42 ± 0.08; p < 0.05), prostate (control group: 0.54 ± 0.01; treatment group: 0.44 ± 0.03; p < 0.05), and seminal vesicles weights (control group: 1.18 ± 0.25; treatment group: 0.86 ± 0.29; p < 0.05) were statistically significant decreased in treated males compared with the control ones.

Please also refer to the field "Attached background material" below.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
- fertility index was significantly reduced in untreated female group impregnated with treated males (control group: 95 %; treatment group: 46.15 %) and in treated female group impregnated with untreated males (control group: 95 %; treatment group: 71.43 %).
- mating index was significantly reduced in untreated female group impregnated with treated males (control group: 100 %; treatment group: 65 %) and in treated female group impregnated with untreated males (control group: 100 %; treatment group: 70 %).
- 50% of the treated females impregnated with untreated males showed delayed birth date and 40% of them showed signs of dystocia compared to 50
and 16.6%, respectively, for the untreated females impregnated with treated males.
- the number of implantation sites were statistically significant (p < 0.05) reduced in pregnant females of both treated groups compared with the control group; the number of resorptions (postimplanatation deaths), dead foetuses, and pre- and postimplantation losses were statistically significant (p < 0.05) increased in pregnant females of both treated groups; the incidence of resorptions was statistically significant (p < 0.05) increased in treated female group impregnated with untreated males in comparison with untreated female group impregnated with treated males.
- number of corpora lutea was reduced in both treated groups (control: 220, treated males: 54; treated females: 94).
- signs of dystocia (no. of dams: control: 0, treated males:1, treated females: 4) and delayed birth date (no. of dams: control: 0, treated males: 3, treated females: 5) were observed.
- placental and gravid uterine weights were statistically significantly decreased (p 0.05) in both treated groups compared with control group.

Please also refer to the field "Attached background material" below.
BODY WEIGHT AND WEIGHT CHANGES:
- body weight of treated males was not affected compared to the controls.

REPRODUCTIVE PERFORMANCE:
- no signs of delayed birth date or dystocia detected in the control females.

Please also refer to the field "Attached background material" below.
Remarks on result:
other: see "Remarks"
Remarks:
The results show that mating and fertility indices were reduced in treated males and treated females (mating index: Control: 100%, treated males: 65%; treated females: 70%; Fertility index: 95%, 46.15%, 71.43%, respectively). Reduced weight of testes, epididymis, prostate gland, seminal vesicles, (p<0.05), with no reduction in body weight between control and treated males were observed. The oestrous cycle was disturbed in treated females and total number corpora lutea was reduced (Control: 220, treated males: 54; treated females: 94). Furthermore, signs of dystocia (no. of dams: control: 0, treated males:1, treated females: 4) and delayed birth date (no. of dams: control: 0, treated males: 3, treated females: 5) were observed.
Critical effects observed:
not specified
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
During lactation, the pups behavioral responses (such as learning and memory responses) were decreased in both treated groups. The incidence of these effects was more pronounced in the treated female group impregnated with untreated males compared with untreated female group impregnated with treated males.
Dermal irritation (if dermal study):
not specified
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
The number of viable foetuses were statistically significant reduced in pregnant females of both treated groups compared with the control group, and the number of dead foetuses were statistically significant increased in pregnant females of both treated groups.
During lactation, the foetal survival and viability indices were decreased in both treated groups. The incidence of these effects was more pronounced in the treated female group impregnated with untreated males (survival index: control group: 100 %; treatment group: 85.71 %; viability: control group: 99.07 %; treatment group: 74.28 %) compared with untreated female group impregnated with treated males (survival index: control group: 100 %; treatment group: 90 %; viability: control group: 99.07 %; treatment group: 85 %).

Please also refer to the field "Attached background material" below.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The body weight of foetuses obtained from treated females impregnated with untreated males was significantly different (at birth and at the end of lactation period) from those obtained from untreated females impregnated with treated males.

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:
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:
effects observed, treatment-related
Description (incidence and severity):
Gross examination of the foetuses of both treated groups obtained at 20th day of gestation and after weaning revealed statistically significant (p < 0.05) increase in the percentage of stunted growth, subcutaneous hemorrhages, and micrognathia. The incidence of these foetal anomalies was pronounced in the foetuses of treated female group than in the foetuses of treated male group.

Ammonium metavanadate statistically significant (p < 0.05) increased the incidence of visceral anomalies in the foetuses of both treated groups compared with control foetuses. These anomalies were in the form of dilated brain ventricles (hydrocephaly), dilated nares, olfactory bulb and cerebral hemisphere hypoplasia, micro- and anophthalmia, intrathoracic hemorrhage, heart and lung hypoplasia, hydroureter, renal hypoplasia, and dilated renal pelvis. The incidence of these visceral anomalies was greater in the foetuses of treated female group compared with foetuses of treated male group.

The incidence of skeletal anomalies in the foetuses of both treated groups was statistically significant (p < 0.05) increased compared with the control foetuses. The recorded skeletal anomalies were in the form of wide separation of parietal bones, incomplete ossification of parietal and/or interparietal bones, incomplete ossification of sternbrae, reduced sternbrae number, wavy and extra ribs, absence of carpal and metacarpal, tarsal and metatarsal bones, and absence of caudal bones and phalanges. The incidence of these skeletal anomalies was pronounced in foetuses of treated female group than in the foetuses of treated male group.

Please also refer to the field "Attached background material" below.
Histopathological findings:
not specified
Other effects:
not specified
Behaviour (functional findings):
not specified
Developmental immunotoxicity:
not specified
not specified
Remarks on result:
other: see "Remarks"
Remarks:
The number of viable foetuses were statistically significant reduced in pregnant females of both treated groups compared with the control group. The behavioural responses as well as foetal survival and viability indices were decreased in both treated groups during the lactation period. The incidence of these effects was more pronounced in the treated female group. The morphological, visceral, and skeletal anomalies were recorded statstically significant increased in foetuses of both treated groups, with more pronounced effects on foetuses of treated females.
Critical effects observed:
not specified
Reproductive effects observed:
not specified
Conclusions:
In this study, Sprague-Dawley rats of both sexes were treated with ammonium metavanadat (200 ppm in drinking water; males: 10 mg test item/kg bw/d corresponding to 4.35 mg V/kg bw/d (calculated with default factors according to ECHA (2012); females: 11.43 mg test item/kg bw/d, corresponding to 4.97 mg V/kg bw/d). Male and female rats were treated with the substance for 70 days and 61 days (14 days premating, during mating, till weaning of pups (21 days of age), respectively. Treated males were mated for 5 days with untreated females, and treated females for 5 days with untreated males. The results showed that mating and fertility indices were reduced in treated males and treated females (mating index: control: 100%, treated males: 65%; treated females: 70%; fertility index: 95%, 46.15%, 71.43%, respectively). In addition, reduced weight of testes, epididymis, prostate gland, seminal vesicles, (p<0.05), with no reduction in body weight between control and treated males were observed. Furthermore, the oestrous cycle was disturbed in treated females and total number corpora lutea was reduced (control: 220, treated males: 54; treated females: 94). Signs of dystocia (no. of dams: control: 0, treated males:1, treated females: 4) and delayed birth date (no. of dams: control: 0, treated males: 3, treated females: 5) were observed.

The number of viable foetuses were statistically significant reduced in pregnant females of both treated groups compared with the control group. The behavioural responses as well as foetal survival and viability indices were decreased in both treated groups during the lactation period. The incidence of these effects was more pronounced in the treated female group. The morphological, visceral, and skeletal anomalies were recorded statstically significant increased in foetuses of both treated groups, with more pronounced effects on foetuses of treated females.

In conclusion, this non-regulatory study was not designed to establish a dose-response relation or a no effect level and shows deficiencies. In this study, mating period was only 5 days. In regulatory toxicity studies, the mating period is 3 weeks days and animals are placed only in the study when mating is confirmed. The study was conducted only with one dose which clearly represents an effect level. In addition, the number of females used for investigations of effects on male mating and fertility index as well as other developmental endpoints was very low compared to guideline studies. No information on general is toxicity presented. Therefore, the study is with RL3 only of limited value for regulatory purposes.
Endpoint:
fertility, other
Remarks:
investigation of male fertility
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
no specified
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
Materials and methods are described only very briefely and only a summary of results is presented and no individual data. Only a limited number of endpoints were adressed, and the number of animals per group (n=8) for the mating trial was very low resulting only in a limited number of animals available for evaluation.
Qualifier:
no guideline followed
Principles of method if other than guideline:
In this study, the effects of sodium metavanadate on the fertility of male mice was investigated. Groups of 24 male Swiss mice were exposed to sodium metavanadate at doses of 0, 20, 40, 60, and 80 mg/kg bw/day (equivalent to 8.4, 16.8, 25.2 and 33.6 mg vanadium/kg bw/day) via drinking water for 64 days. A control group was run concurrently. At the end of the exposure period, each group was divided into two subgroups: 8 animals for mating trial and 16 animals for pathology and sperm examinations. All male mice were examined for clinical signs and body weight. During the mating trials, males were mated with untreated females for 4 days and the following parameters were investigated in females after gestation days 10 to 14: number of pregnant females, total number of implantations, number of early/late resorptions as well as number of dead and alive foetuses The males of the second subgroup were investigated for the following parameters: testis weight, epididymis weight, sperm movement, sperm morphology, sperm count (testis and epididymis) and histopathology (testis only).
GLP compliance:
not specified
Remarks:
not specified in the publication
Limit test:
no
Justification for study design:
not applicable
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source of test material: Sigma Chemical Co. (St. Louis, MO, USA)
Species:
mouse
Strain:
Swiss
Details on species / strain selection:
not specified
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Panlab (Barcelona, Spain)
- Weight at study initiation: 28 - 30 g
- Diet (ad libitum): Panlab rodent chow (Panlab, Barcelona)
- Water(ad libitum): tap water
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 21 - 23 °C
- Relative humidity: 40 - 60 %
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
oral: drinking water
Vehicle:
unchanged (no vehicle)
Details on exposure:
not specified
Details on mating procedure:
- M/F ratio per cage: 1 treated male: 2 untreated females
- Length of cohabitation: 4 days (time for completion of one oestrous cycle in mice)
- Females were previously observed to confirm normal oestrus cyclicity
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
64 days
Frequency of treatment:
daily
Details on study schedule:
not specified
Dose / conc.:
20 mg/kg bw/day (nominal)
Remarks:
equivalent to 8.4 mg vanadium/kg bw/day
Dose / conc.:
40 mg/kg bw/day (nominal)
Remarks:
equivalent to 16.8 mg vanadium/kg bw/day
Dose / conc.:
60 mg/kg bw/day (nominal)
Remarks:
equivalent to 25.2 mg vanadium/kg bw/day
Dose / conc.:
80 mg/kg bw/day (nominal)
Remarks:
equivalent to 33.6 mg vanadium/kg bw/day
No. of animals per sex per dose:
24 male mice
Control animals:
yes
Details on study design:
The concentrations of sodium metavanadate required to deliver the desired dose during the period of exposure were adjusted twice a week based on the measured daily fluid intake and body weight. At the end of the exposure period, each group was divided into two subgroups: 8 animals for the mating trial and 16 animals for the pathology and sperm examinations.

To evaluate the fertility of the vanadate-treated males, each mouse was mated with two vanadate-untreated females during the mating trials (please refer for further information to the field "Details on mating procedure" above)
Positive control:
not specified
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: daily and at the end of the experiment

FOOD CONSUMPTION: No data

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: daily

Oestrous cyclicity (parental animals):
not specified
Sperm parameters (parental animals):
PATHOLOGY AND SPERM EXAMINATION TRIAL
Parameters examined in males:
- testis weight and epididymis weight were recorded
- sperm movement and percentage of motile cells were determined
- sperm was investigated for morphological abnormalities and the classification of these abnormalites was made according to Wyrobek and Bruce (1975)*. The percentage of abnormal forms and the relative percentage of each kind of abnormal shape were calculated.
- spermatozoa in the right epididymis were counted
- number of spermatids in the right testicle was counted

*Reference:
- A.J. Wyrobek and W.R. Bruce, Chemical induction of sperm abnormalities in mice. Proc. Natl. Acad. Sci. USA, 72 (1975) 4425.
Litter observations:
MATING TRIALS:
Ten days after the end of the mating period (days 10 - 14 of gestation), females were sacrificed and their litters examined. The number of dead and live foetuses was recorded.
Postmortem examinations (parental animals):
SACRIFICE:
- mating trial: ten days after the end of the mating period (days 10 - 14 of gestation), females were sacrificed and their litters examined. The following measurements were recorded: number of pregnant animals, total implantations, early and late resorptions, and dead and live foetuses.
- pathology and sperm examination trial: male mice were sacrificed at the end of the experiment.

PATHOLOGY AND SPERM EXAMINATION TRIAL
Histopathology / Organ weights:
- testes and epididymis weights were recorded.
- left testicle was fixed for 24 hours and embedded in paraffin. Sections of 4 µm were stained with hematoxylin-eosin and observed microscopically. The diameters of 50 tubules were measured and then the tubular diameter was calculated. The tubule was evaluated for the existence of complete spermatogenesis and for focal or diffuse atrophy, depending on the number of tubules affected. Sertoli cells were classified as normal, atrophic, or with cytoplasmic vacuolization. The existence of multinucleated cells, within the tubular lumen or among the spermiogenic cells, was assessed. The possible existence of degenerative changes in interstitial Leydig cells was also studied (e.g., nuclear atrophy or cytoplasmic vacuolization).
Postmortem examinations (offspring):
not specified
Statistics:
Homogeneity of variances was analysed employing Bartlett's test. If variances were homogenous, ANOVA was used to test all dose groups simultaneously. The Kruskal-Wallis test was used when variances were not homogenous. Differences between control and metavanadate-treated groups were analysed using either Student's t-test or modified t-test.
Reproductive indices:
not specified
Offspring viability indices:
not specified
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not specified
Mortality:
not specified
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- 80 mg/kg bw/day: a statistically significant reduction in body weight gain was seen (control: 41.4 ± 2.3 g: 80 mg/kg bw/day: 36.2 ± 3.9 g; p < 0.05).

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:
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:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
- 80 mg/kg bw/day: a statistically significant decrease in epididymis weight occurred in animals treated with sodium metavanadate (control: 95.5 ± 8.2 mg; 80 mg/kg bw/day: 84.4 ± 8.8 mg; p < 0.01).
- 60 and 80 mg/kg bw/day: epididymal sperm counts were statistically signifcant decreased (control: 0.80 ± 0.25 x10E6/epididymis/g animal; 60 mg/kg bw/day: 0.45 ± 0.96 x10E6/epididymis/g animal; 80 mg/kg bw/day: 0.55 ± 0.20 x10E6/epididymis/g animal; p < 0.05).
- 80 mg/kg bw/day: testicular sperm counts were statistically signifcant decreased (control: 0.30 ± 0.10 x10E6/testis/g animal; 80 mg/kg bw/day: 0.20 ± 0.08 x10E6/testis/g animal; p < 0.05).

Please also refer to the field "Attached background material" below.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
- 60 and 80 mg/kg bw/day: fertility was statistically significant decreased to 43.8% and 62.5% at the 60 and 80 mg/kg bw/day dose levels, respectively (control: 81.3 %; p < 0.001 or p < 0.01, respectively)).

Please also refer to the field "Attached background material" below.
CLINICAL SIGNS (PARENTAL ANIMALS)
- No clinical signs of toxicity were observed.

BODY WEIGHT (PARENTAL ANIMALS)
- 20, 40 and 60 mg/kg bw/day no adverse effects on body weight gain was observed at lower dose levels.

ORGAN WEIGHTS (PARENTAL ANIMALS)
- 20, 40, 60 and 80 mg/kg bw/day: no effects on the weights of the testes weights were observed at any dose.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
- 20, 40, 60 and 80 mg/kg bw/day: the sperm motility was unaffected and the percentage of morphological normal spermatozoa in all mice treated with vanadate were comparable to the values in control mice.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- 20 and 40 mg/kg bw/day: fertility was not statstically significant different from controls (control: 81.3 %; 20 and 40 mg/kg bw/day: 75.0 %)
- 20, 40, 60 and 80 mg/kg bw/day: the average number of total implantations and resorptions in the vanadate-treated groups were not statstically significant different from controls.

HISTOPATHOLOGY (PARENTAL ANIMALS)
- 20, 40, 60 and 80 mg/kg bw/day: histologic study of the testis and epididymis revealed no changes at any dose of metavanadate. No gross lesions (degenerated or atrophic seminiferous tubules, Leydig cells hyperplasia or hypoplasia) were evident in the vanadate-treated animals, whereas examination of the epididymal epithelium revealed normal cellular structures. The tubular diameters were also unaffected.

Please also refer to the field "Attached background material" below.
Remarks on result:
not determinable because of methodological limitations
Critical effects observed:
not specified
Clinical signs:
not specified
Dermal irritation (if dermal study):
not specified
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
- 20, 40, 60 and 80 mg/kg bw/day: the average number of dead and live foetuses in the vanadate-treated groups were not statstically significanty different from controls.

Please also refer to the field "Attached background material" below
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 determinable because of methodological limitations
Critical effects observed:
not specified
Reproductive effects observed:
not specified
Conclusions:
Llobet et al. (1993) investigated the effects of sodium metavanadate on the fertility of male mice. Groups of 24 male Swiss mice were exposed to sodium metavanadate at doses of 0, 20, 40, 60, and 80 mg/kg bw/day (equivalent to 8.4, 16.8, 25.2 and 33.6 mg vanadium/kg bw/day) via drinking water for 64 days. At the end of the exposure period, a mating trial in a small number of animals was conducted and sperm parameters were evaluated.

Sodium vanadate treatment produced no clinical signs of toxicity at any dose level or adverse effects on body weight gain at the dose levels of 20, 40 and 60 mg/kg bw/day. However, a statsically significant decrease in body weight gain was seen in animals treated with 80 mg/kg bw/day (p < 0.05). Furthermore, testes weights were not effected at dose levels of 20, 40, 60 and 80 mg/kg bw/day, while a statistically significant decrease in epididymis weight was observed at 80 mg/kg bw/day (p <.001). Male fertility was not affected at 20 and 40 mg/kg bw/d but dropped statistically significant at 60 and 80 mg/kg bw/day (p <0.001 or 0.01, respectively). The average number of total implantations, resorptions and dead/live foetuses were not affected by vanadium treatment at any dose level. Epididymal sperm counts were statistically signifcant decreased at the 60 and 80 mg/kg bw/day dose levels (p < 0.05), while the testicular sperm counts were statistically signifcant decreased only at the dose level of 80 mg/kg bw/day. At the 20, 40, 60 and 80 mg/kg bw/day dose levels, sperm motility was unaffectd and the percentage of morphological normal spermatozoa were comparable among groups. Histologic study of the testis and epididymis revealed no changes at any dose of sodium metavanadate. No gross lesions (degenerated or atrophic seminiferous tubules, Leydig cells hyperplasia or hypoplasia) were evident in the vanadate-treated animals and examination of the epididymal epithelium revealed normal cellular structures. The tubular diameters were also unaffected.

In conclusion, this non-guideline study in albino mice treated with different doses of sodium vanadate in drinking water revealed effects on male reproductive performance at 60 and 80 mg/kg bw/d by a reduction of the percentage of pregnant females only, although the study was limited in the way it was conducted and presented. Further parameters evaluated show only an effect on epididymal weights and sperm count, but not on sperm parameters. Based on the results of this study, aa dose level of 40 mg/kg bw/d (equivalent to 16.8 mg vanadium/kg bw/day) given by the oral route represnets a NOEL.
Endpoint:
fertility, other
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
not stated
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
Materials and methods are described only very briefly and only a summary of results is presented and no individual data. Only a limited no. of endpoints were adressed and the no. of animals per group was very low resulting only in a limited no. of animals available per study endpoint.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
Male and female Sprague-Dawley rats per group received 0, 5, 10 or 20 mg/kg bw/d sodium vanadate (equivalent to 2.1, 4.2 and 8.4 mg vanadium/kg/day, respectively) per os. Females were treated for 14 days and males for 60 days before mating. Females continued to receive treatment with the test item until study termination on postnatal day 21. Females were allowed to give birth and to rear their offspring until postnatal day 21. The following parameters were investigated in the offspring on postnatal days 1, 4 and 21: mortality, body weight gain, general symptoms, body and tail lenghts. Lastly, the relative organ weights of heart, lungs, spleen, liver, kidneys and testes of the offspring were determined on lactation day 21.
GLP compliance:
not specified
Remarks:
not specified in the publication
Limit test:
no
Justification for study design:
not applicable
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source of test material: Merck Company (Darmstadt, FRG)
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
not specified
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Biocentre (Barcelona, Spain)
- Weight at study initiation (females only): 240 - 280 g
- Diet (ad libitum): high protein rat diet (Panlab, Barcelona, Spain)
- Water (ad libitum)

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
Route of administration:
oral: gavage
Vehicle:
not specified
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was given as a solution to the animals.
Details on mating procedure:
- Proof of pregnancy: daily vaginal smears were taken. Finding sperm indicated copulation and designated day 0
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
female rats: starting 14 days before mating and up to postnatal day 21
male rats: 60 days before mating
Frequency of treatment:
daily
Details on study schedule:
not specified
Dose / conc.:
5 mg/kg bw/day (nominal)
Remarks:
equivalent to 2.1 mg vanadium/kg/day
Dose / conc.:
10 mg/kg bw/day (nominal)
Remarks:
equivalent to 4.2 mg vanadium/kg/day
Dose / conc.:
20 mg/kg bw/day (nominal)
Remarks:
equivalent to 8.4 mg vanadium/kg/day
No. of animals per sex per dose:
about 10 males / 10 females
Control animals:
yes
Details on study design:
not specified
Positive control:
not specified
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: No data
BODY WEIGHT: No data

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE: No data

Oestrous cyclicity (parental animals):
not specified
Sperm parameters (parental animals):
not specified
Litter observations:
PARAMETERS EXAMINED
The offspring were observed for mortality, normal body weight gain, and general symptomatology after 1, 4 and 21 days of nursing. Also, body and tail lengths were measured on the same days.
Postmortem examinations (parental animals):
not specified
Postmortem examinations (offspring):
SACRIFICE
- after 21 days, all offspring were sacrificed.

HISTOPATHOLOGY / ORGAN WEIGTHS
The heart, lungs, spleen, liver, kidneys and testicles (in males) were removed and weighed, and the relative organ weights were calculated.
Statistics:
The results were evaluated by means of the distribution-free ranking test according to Wilcoxon in the modified version of Mann and Whitney (Mann and Whitney, 1947)*, and using χ² test.

* H.B. Mann and D.R. Whitney, Ann. MAth. Stat. 18, 50 - 60 (1947).
Reproductive indices:
not specified
Offspring viability indices:
not specified
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not specified
Mortality:
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
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed
CLINICAL SIGNS:
There were no signs of maternal toxicity during the study.

REPRODUCITVE PERFORMANCE:
Sodium metavanadate given orally to rats did not show recognizable adverse effects with respect to the fertility, which was not reduced.

Please also refer to the field "Attached background material" below.
Remarks on result:
not determinable because of methodological limitations
Critical effects observed:
not specified
Clinical signs:
not specified
Dermal irritation (if dermal study):
not specified
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
The number of litters, living and dead young per litter in treated rats did not show any significant differences with the control group on days 1 and 4 of nursing.
Statistically significantly decreases or increases on living young per litter (control: 11.0 ± 1.9; 10 mg/kg bw/day: 4.6 ± 2.6; p < 0.01) and dead young per litter (control: 1.0 ± 06; 10 mg/kg bw/day: 4.7 ± 2.1; p < 0.01) could be observed on day 21 of nursing.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The average body weight per litter did not show any significant differences with the control group on days 1 and 4 of nursing, except for a statistically significantly decrease in the average body weight per litter for the highest dose tested on day 4 of lactation (control: 138.8 g ± 23.8; 20 mg/kg bw/day: 87.5 ± 33.7; p < 0.05)
Statistically significantly decreases on average body weight per litter could be observed on day 21 of nursing (control: 466.9 ± 95.2; 10 mg/kg bw/day: 252.7 ± 150.9; p < 0.01).

Body weight of animals in the treated groups showed a statistically significantly decrease in both males and females during the period of lactation, when compared with the control group (P < 0.001).
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:
effects observed, treatment-related
Description (incidence and severity):
There were statistically significantly decreases in the relative organ weights of spleen (males: 20 mg/kg/day (P<0.05); females: 5 and 20 mg/kg/day (P<0.05 and 0.01, resepctively)), liver (males and females: 5, 10 and 20 mg/kg/day (P < 0.05 or 0.01)) and kidneys (males: 5 mg/kg/day (P< 0.05); females: 5, 10 and 20 mg/kg/day (P< 0.05 or 0.01)). A dose-response may be induced.
Gross pathological findings:
not specified
Histopathological findings:
not specified
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Body length and tail length of animals in the treated groups showed a statistically significantly decrease in both males and females during the period of lactation, when compared with the control group (P < 0.05 or P < 0.01 or P < 0.001).
Behaviour (functional findings):
not specified
Developmental immunotoxicity:
not specified
ORGAN WEIGHTS:
A statistically significantly decrease was observed in the relative heart weight of male pups in the highest dose (P < 0.05).
No significant differences was observed for relative weight of the testicles and lungs of the pups of treated dams compared to the control group.

Please also refer to the field "Attached background material" below.
Dose descriptor:
LOAEL
Generation:
F1
Effect level:
5 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios
other: Decreased body length and tail length in pups during the lactation period.
Dose descriptor:
LOAEL
Generation:
F1
Effect level:
2.1 mg/kg bw/day (nominal)
Based on:
element
Remarks:
vanadium
Sex:
male/female
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios
other: Decreased body length and tail length in pups during the lactation period.
Critical effects observed:
not specified
Reproductive effects observed:
not specified
Conclusions:
In this study, fertility was not affected by treatment of male and female rats with sodium vanadate and there were no signs of maternal toxicity. Data from pups obtained on postnatal days 1, 4 and 21 did not reveal an effect of treatment on number of litters, living and dead offspring per litter and average body weights, on day 1 and 4 except for the average body weight in the high dose group (20 mg/kg bw/d) pups. In addition, some significant diefferences in these parameters could be observed on day 21. An effect of vanadium treatment via their mothers was observed in male and female pups during the lactation period. Body weights, body lenght and tail lenght were significantly decreased, and the relative organ weights (spleen, liver, kidneys) of pups sacrificed on postnatal day 21 were significantly lower.

In conclusion, based on the results of the study an effect on male and female fertility could not be established. The results give some evidence that vanadium treatment of offspring during the ealy life phase until postnatal day 21 via their mothers resulted in effects on pup development already at 5 mg NaVO3/kg bw/d (equivalent to 2.1 mg vanadium/kg). It needs to be noted that the number of animals evaluated is low and that some effects, although statistically signifaicantly, are only slightly affected without a clear dose-response trend, and there are no historical control data available to verify these findings.
Endpoint:
developmental neurotoxicity
Type of information:
experimental study
Adequacy of study:
disregarded due to major methodological deficiencies
Reliability:
other: not rated according to Klimisch et al.
Rationale for reliability incl. deficiencies:
other: see "Remarks"
Remarks:
The references contained in this summary entry represents in vivo experiments with investigations on developmental neurotoxicity with very limited value for risk assessment purposes. The references do not fulfil the criteria for quality, reliability and adequacy of experimental data for the fulfilment of data requirements under REACH and hazard assessment purposes (ECHA guidance R4 in conjunction with regulation (EC) 1907/2006, Annexes VII-X). The information contained therein were included for information purposes only.
Principles of method if other than guideline:
Wang, D.-C. et al. (2015):
Test substance: sodium metavanadate (Sigma-Aldrich, USA)
Dose: 3 mg/kg/day (equivalent to 1.25 mg vanadium/kg/day)
Nursing dams were administered the test substance daily during the lactation period. The male pups were investigated in this study.
No. of animals per sex per dose: 36 male pups
Exposure duration: 21 days (birth until weaning on postnatal day 21)
Exposure frequency: daily
Negative / vehicle control: disitilled water
Positive control: not specified
The weaned male offspring were trained to treadmill running (exercise) and then examined for their motor coordination.
Parameters investigated: motor coordination (extensor thrust response and rotarod test (postnatal days 59 and 60, respectively)), brain-derived neurotrophic factor in plasma and cerebellum (ELISA assay; postnatal day 61)

Mustapha, O. et al. (2014):
Test substance: sodium metavanadate (Sigma-Aldrich, St. Louis, USA)
Dose: 3 mg/kg bw/day
Nursing dams were administered the test substance daily during the lactation period. The pups were investigated following the exposure period.
No. of animals per sex per dose: 12 pups
Exposure duration:
1) 14 days (postnatal day 1 until postnatal day 15; acute vanadium exposure)
2) 21 days (postnatal day 1 until postnatal day 21; chronic vanadium exposure)
Exposure frequency: daily
Negative / vehicle control: sterile water
Positive control: not specified
Parameters investigated: body weights, behavioural tests (open field test (line crossing, centre square entries, centre square duration, rearing, stretch attend postures, grooming, freezing) and negative geotaxis) and immunohistochemical investigation of the brain (GFAP staining (investigation of astrocytic activation) and MBP staining (investigation of myelination))

Todorich, B. et al. (2011):
Test substance: sodium metavanadate (Sigma)
Dose: 3 mg/kg bw/day
No. of animals per sex per dose: 5 pups
Exposure duration: 14 days (starting on postnatal day 1)
Exposure frequency: daily
Negative / vehicle control: saline
Positive control: not specified
Parameters investigated: body weights, motor function (rotarod test; postnatal day 15) and immunohistochemical investigation of the brain

Olopade, J. O. et al. (2011):
Test substance: sodium metavanadate
Dose: 3 mg/kg bw/day
Dams were administered the test substance daily during the lactation period. The pups were investigated following the exposure period.
No. of animals per sex per dose: number of pups not specified
Exposure duration: 14 days (starting on postnatal day 1)
Exposure frequency: daily
Negative / vehicle control: sterile water
Positive control: not specified
Parameters investigated: body weight (postnatal day 7 - 13), behavioural tests (open field test (locomotion, number of rearings, and number of groomings), forelimb support and negative geotaxis; postnatal day 15), brain weight, and immunohistochemical investigation of the brain (GFAP staining)

Azeez, I. A. et al. (2015):
Test substance: sodium metavanadate (pH 7.7)
Dose: 3 mg/kg bw
Test substance exposure of suckling pups via treatment of lactating dams and after weaning.
No. of animals per sex per dose: 8 pups (males and females)
Exposure duration: postnatal days 1 through 21 (treatment of pups via lactating dams) and then postnatal days 22 through 89 (direct treatment of pups)
Exposure frequency:
1) postnatal days 1 through 21: daily
2) postnatal days 22 through 89: 3 times per week
Negative / vehicle control: sterile water
Parameters investigated: body weights (postnatal days 1 - 22: daily; postnatal day 23 - 86: twice weekly), behavioural testing (hanging wire test (postnatal days 60 and 89) and open field test (locomotion, no. of rearings, no of grooms; postnatal day 89)), histopathology (brain only; postnatal day 90) and Western blotting (brain only; postnatal day 90)
Species:
other: Wang, D.-C. et al. (2015) & Todorich, B. et al. (2011): rat (Sprague-Dawley); Mustapha, O. et al. (2014): mouse (CD-1); Olopade, J. O. et al. (2011): rat (strain unknown); Azeez, I. A. et al. (2015): mouse (BALB/c)
Sex:
male/female
Route of administration:
other: Wang, D.-C. et al. (2015), Mustapha, O. et al. (2014), Todorich, B. et al. (2011), Olopade, J. O. et al. (2011) & Azeez, I. A. et al. (2015): intraperitoneal
Remarks on result:
other: see "Remarks"
Remarks:
Wang, D.-C. et al. (2015): the extensor thrust response was not affected by vanadium treatment and the ratios thrust to body weight was influenced by exercise (treadmill running) but not treatment. In the rotarod test at RPMs > 20, latency to fall of the rod and the coordination index was significantly influenced by treatment and exercise showing a positive effect of excercise on the motor coordination in vanadium treated animals. Plasma and brain brain-derived neurotrophic factor levels were statistically significantly influenced by treatment and exercise showing a significant correlation between plasma brain-derived neurotrophic factor level and coordination index indicating higher plasma brain-derived neurotrophic factor levels exhibited better coordination index in the rats. Plasma and cerebellar brain-derived neurotrophic factor levels were decreased significantly in the vanadium treated group. Immunohistochemistry of calbindin-positive neurons illustrated that the no. of Purkinje cells was not influenced by the treatment or exercise. Expression of calbindin was also not infuenced by treatment or exercise. However, the phosphorylation of TrkB receptor was slightly affected by treatment with vanadium and statistically significantly by exercise.
Remarks on result:
other: see "Remarks"
Remarks:
Mustapha, O. et al. (2014): the daily body weights of the acute vanadium exposed mice pups did not differ significantly beween day postnatal day 1 and 15. A significant difference in mean body weight between the vanadium treated animals and controls was only seen when measured between postnatal day 15–21. Behavioural tests showed in most instances a reduction in locomotor activity and a negative effect on geotaxis in the vanadium exposed groups. Furthermore, astrocytic activation and demyelination were observed in vanadium exposed groups compared to the controls.
Remarks on result:
other: see "Remarks"
Remarks:
Todorich, B. et al. (2011): vanadium-treated rat pups showed a slightly decreased (statistically significant) rate of weight gain, and vanadium injections had a substantial impairment in motor function at postnatal day 15 as demonstrated by rotarod testing. Upon histological examination of the brains (corpus callosum) of the postnatal day 15 rat pups, a 2.5-fold decrease in the number of oligodendrocyte progenitors of vanadium-treated rats compared to controls was observed. The number of astrocytes was increased in the vanadium-treated group and had more extensive branching suggestive of activation. The data suggested that vanadium was cytotoxic to oligodendrocytes, but stimulated astrogliosis.
Remarks on result:
other: see "Remarks"
Remarks:
Olopade, J. O. et al. (2011): exposure of rat pups via their mother resulted in statistically significant reduction in body weight gain of pups exposed to vanadium compared to the control. Behavioural tests showed a reduction in locomotor activity, forelimb support, and negative geotaxis in the vanadium exposed group compared to the control. The mean brain weight of the vanadium-only pups at postnatal day 15 was significantly less than that of the controls. Immunohistochemistry revealed a high astrocytic response (glial fibrillary acidic protein (GFAP) staining) in vanadium exposed group.
Remarks on result:
other: see "Remarks"
Remarks:
Azeez, I. A. et al. (2015): the findings showed that vanadium exposure (3 mg/kg bw NaVO3 ip) throughout the first 3 months of life resulted in reduction of body weight gain with conserved muscle strength but decreased locomotor and exploratory activity. Histopathological analysis at the end of the long-term exposure demonstrated myelin damage in a region-dependent pattern that involved fiber tracts as well as gray matter. Axon damage and activation of both microglia and astrocytes was observed. Glial cell activation mostly matched myelin damage but was also observed in fiber bundles, such as the internal capsule, in which myelin was preserved. Furthermore, induction of TNF and IL-1b expression was demonstrated showing that TNF is induced in reactive astrocytes in vanadium toxicity.
Conclusions:
No conclusion can be drawn from the above publications due to lack of quality, reliability and adequacy of the experimental data for the fulfilment of data requirements under REACH.
The references contained in this summary entry represent in vivo experiments with investigations in developemtnal neurotoxicity with very limited value for risk assessment purposes. The references do not fulfil the criteria for quality, reliability and adequacy of experimental data for the fulfilment of data requirements under REACH and hazard assessment purposes (ECHA guidance R4 in conjunction with regulation (EC) 1907/2006, Annexes VII-X). The information contained therein were included for information purposes only.

Wang, D.-C. et al. (2015):
The publication is not relevant for human health risk assessment. Firstly, in this non-regulatory mechanistic study, the test item is not fully characterized. In addition, the route of exposure (intraperitoneal) is not relevant for human risk assessment. Furthermore, one dose levels was only tested, whereas normally three doses should be tested. Overall, this study was not designed to evaluate a dose-response relationship or a no effect level and the study endpoints were limited to the purpose of the study.

Mustapha, O. et al. (2014):
The publication is not relevant for human health risk assessment. Firstly, in this non-regulatory mechanistic study, the test item is not fully characterized. In addition, the route of exposure (intraperitoneal) is not relevant for human risk assessment. Furthermore, the study was conducted with only one dose level.. The study was not designed to evaluate a dose-response relationsship or a no effect level and the study endpoints were limitted to the purpose of the study. Lastly, the study was conducted with a very limited number of animals.

Todorich, B. et al. (2011):
The publication is not relevant for human health risk assessment. Firstly, in this non-regulatory mechanistic study, the test item is not fully characterized. In addition, the route of exposure (intraperitoneal) is not relevant for human risk assessment. The study was only conducted with one dose level and was not designed to evaluate the dose-response relationship or a no effect level. The endpoints were limitted to the very specific goal of the study.

Olopade, J. O. et al. (2011):
Firstly, in this non-regulatory mechanistic study, the test item is not fully characterized. In addition, the route of exposure (intraperitoneal) is not relevant for human risk assessment. The study, using only one dose level, was not conducted to evaluate a dose-response relationship or a no effect level. Furthermore, pups from only one dam were used per group and endpoints were limitted to the purpose of the study. This study must be regarded as supportive only. It gives some evidence of an effect of vanadium treatment on the developing brain in rat pups when treated early post-natally via their mothers, but only a very small cohort was used .

Azeez, I. A. et al. (2015):
The publication is not relevant for human health risk assessment. Firstly, in this mechanistic study, the test item is not fully characterized. In addition, the route of exposure (intraperitoneal) is not relevant for human risk assessment. This study, using only one dose level, was not designed to show a dose-response relationship or a no effect level and the evaluated endpoints are limited to the study purpose. The number of pups used for the study is relatively low with 8 animals (males and females), but a matched control group was included to evaluate the findings against the vehicle used. The interpretation of the cellular and molecular findings in the brain remain to be further verified.
Endpoint:
developmental neurotoxicity
Type of information:
experimental study
Adequacy of study:
disregarded due to major methodological deficiencies
Study period:
not specified
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
This non-regulatory mechanistic study was conducted with only one effective dose level of 3 mg NaVO3/kg body weight/day (1.25 mg V/kg bw/day) in comparison to a control group and were not designed to evaluate a dose-response relationship or a no effect level. Furthermore, a non-relevant route of exposure (intraperitoneal injection) was used. Lastly, the publication showed deficiencies with respect to reporting of methods and results.
Qualifier:
no guideline followed
Principles of method if other than guideline:
In the current study dams of treated litters (4 litters containing 8 pups (4 male and 4 female pups, if possible)) were intraperitoneally injected with 3 mg sodium metavanadate/kg body weight/day in distilled water (equivalent to 1.25 mg vanadium/kg bw/day) during postnatal day 10 to postnatal day 21. A control group receiving saline only was run concurrently by using the same procedure as for the treatment group. The following parameters were investigated: clinical signs (dams and pups), body weights (dams and pups), and brain weight (pups). Lastly, brain areas were removed for lipid peroxidation assay by the thiobarbituric acid reaction, the other four were transcardially perfused-fixed and their brains were removed and cut with a cryostat. Brain sections were processed for: NADPHd histochemistry and anti-HSP70, anti-GFAP and anti-S100 immunohistochemistry. The relative optical density of the NADPHd stained layers and of S100 (+) astrocytes and the GFAP (+) astrocyte surface area in cerebellum and hippocampus were measured.
GLP compliance:
not specified
Remarks:
not specified in the publication
Limit test:
no
Justification for study design:
not applicable
Specific details on test material used for the study:
not specified
Species:
rat
Strain:
Wistar
Details on species / strain selection:
not specified
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS - OFFSPRING
- Age at study initiation: 1 day after birth
- Housing: offspring, together with their mother, were housed in plastic breeding cages
- Diet (ad libitum)
- Water (ad libitum)

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Relative humidity: 40 – 60 %
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
intraperitoneal
Vehicle:
other: distilled water
Details on exposure:
Newborn rats were grouped into litters of eight animals (four males and four females, if possible) to ensure good nutrition. Eight litters were randomly assigned to one of the following animal groups:
- Vanadium-treated group: consisted of offspring of 4 dams. The dams were were treated with the test item in distilled water from the 10th to the 21st postnatal day.
- Control group: consisted of offspring of 4 dams. The dams were treated with an equal volume (400 to 500 µL) of saline solution during the same period as the treatment group.
Details on mating procedure:
not specified
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
postnatal day 10 to postnatal day 21 (test item treatment of dams)
Frequency of treatment:
daily (test item treatment of dams)
Details on study schedule:
not specified
Dose / conc.:
3 mg/kg bw/day (nominal)
Remarks:
equivalent to 1.25 mg vanadium/kg bw/day
No. of animals per sex per dose:
4 litters containing 8 pups (four males and four females/litter, if possible)(total number of pups/dose: 32)
Control animals:
yes
Details on study design:
- Dose selection rationale: the dose of vanadium was dictated by previous results, which indicated that the administration of similar amounts of NaVO3 resulted in a delay in eye opening, decreased muscular strength and locomotion and decreased myelin staining in neonate rats (Soazo and Garcia, 2007)*.

*Reference:
- Soazo M, Garcia GB. Vanadium exposure through lactation produces behavioural alterations and CNS myelin deficit in neonatal rats. Neurotoxicol Teratol 2007;29(4): 503–10.
Positive control:
not specified
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: not specified

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: at the end of test item administration

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data


Oestrous cyclicity (parental animals):
not specified
Sperm parameters (parental animals):
not specified
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in offspring:
- clinical signs and mortality were recorded.
- control and test item-exposed pups' body weight was recorded daily.
Postmortem examinations (parental animals):
not specified
Postmortem examinations (offspring):
- Biochemical determinations:
Sixteen animals of each group (4 pups/litter, two males and two females) were sacrificed and selected brain areas (hypothalamus, prefrontal cortex, striatum, hippocampus, cerebellum andmidbrain) were removed according to Heffner et al. (1980)*. The tissues were weighed and homogenized with cold distilled water. The homogenates were sonicated and then centrifuged. An aliquot of the supernatant was used for protein determination by the Bradford method (Bradford, 1976)* using bovine serum albumin as a standard. Lipid peroxidation was determined by the thiobarbituric acid reaction as follows: two volumes of TCA/TBA/HCl (15%:0.375%:0.25 N) for each volume of the homogenate were added and the mixture was incubated. After centrifugation, absorbance was determined at 535 nm in a Beckman DU 640 spectrophotometer (Hall and Andrus, 2000)*. Malondialdehyde concentrationwas obtained using themolar extinction coefficient.

- Histological studies:
Four pups of each litter were anesthetized and transcardially perfused-fixed with 4% paraformaldehyde in a buffer phosphate 0.1 M (pH 7.4) solution. A wash with 0.9% w/v NaCl with 0.4 M NaNO2 and heparin was passed through their circulatory system prior to fixation. Brains were kept in the same fixative solution for 2 – 4 hours and immersed in 20% sucrose overnight or until they fell down. 40 μm-thick longitudinal brain sections were cut with a cryostat and kept at −20 °C cryoprotected with 30% sucrose in PBS.

- NADPHd histochemistry:
Since the histochemical detection of NADPHd-producing neurons is indicative of nitric oxide synthase activity in fixed tissues (Hope et al., 1991; Matsumoto et al., 1993)*, brain free floating sections were stained with the Nitro Blue Tetrazolium modified method of Vincent and Kimura (1992)*. Two animals per litter were used. Control and treated brain sections were incubated in a 0.1 M phosphate buffer (pH 7.4) solution containing: 1 mg/mL β-NADPH, 0.1 mg/ml NBT and 0.3% Triton X-100. To stop the reaction, sections were transferred to phosphate buffer and rinsed with distilled water. Then, they were mounted on gelatinized standard glass slides, cover slipped and examined using a light microscope.

- Immunohistochemical procedures:
Brain floating sections from two exposed and control pups per litter were processed. The same protocol was used for each step of immunohistochemical staining according to Sternberger's peroxidase–antiperoxidase (PAP) technique (Sternberger et al., 1970)* using polyclonal anti-GFAP antibody (developed in rabbit) 1:8000 and anti-S100 1:8000 and monoclonal anti-HSP70 antibody (developed in mouse) 1:2000 as primary antibodies. Development of peroxidase activity was performed with DAB/nickel ammonium sulfate in acetate buffer (0.1 M, pH 6) at room temperature for GFAP and S100 and with DAB in phosphate buffer (0.1 M, pH 7.4) at room temperature
for HSP70. Two to three sections of each animal were mounted on gelatinized standard glass slides for light microscopy study.

- Computerized image analysis:
The relative optical density of the NADPHd stained cerebellar molecular and granular layers and of S100 immunoreactive (IR) astrocytes and the GFAP-(IR) astrocyte surface area in cerebellum and hippocampus were measured using a NIH imaging analysis system. Images were obtained through an Olympus D-560 zoom digital camera attached to an Olympus BX40 microscope.
The mean gray value of the Nitro Blue Tetrazolium stained cerebellar molecular and granular layers was measured using a rectangular area of interest (AOI). This AOI was located 4–5 times in each layer and the mean gray values were taken. Relative optical density was obtained after a transformation of mean gray values into relative optical density using the formula: Relative optical density = log (256/mean gray). The relative optical density value was chosen to evaluate the intensity of Nitro Blue Tetrazolium staining. Relative optical density was calculated and averaged within the experimental group in each animal per layer.
In order to evaluate astrogliosis in hippocampus and cerebellum, images from anatomically matched regions of each experimental group were used. After image segmentation, the cross sectional area of all GFAP-IR astrocytes was measured. The average for each experimental group represents the mean surface area of GFAP-IR cells present per individual. In S100 immunocytochemistry, relative optical density of all S100-IR astrocytes was measured after image segmentation.

*Reference:
- Heffner TG, Hartman JA, Seiden LS. A rapid method for the regional dissection of the rat brain. Pharmacol Biochem Behav 1980;13(3): 453 – 6.
- Bradford M. A rapid and sensitive method for the quantification of microgram quantities of protein utilising the principle of protein–dye binding. Anal Biochem 1976;72: 248 – 54.
- Hall E, Andrus K. Measurement of oxygen radicals and lipid peroxidation in neural tissues. In: Gerfen C, Holmes A, Rogawsky M, Sibley D, Skolnick P, Wray S, editors.
Current protocols in neuroscience. John Wiley and Sons; 2000.
- Vincent SR, Kimura H. Histochemical mapping of nitric oxide synthase in the rat brain. Neuroscience 1992;46:755–84.
- Sternberger LA, Hardy PH, Cuculis JJ, Mayer HG. The unlabeled antibody enzyme method of immunohistochemistry. Preparation and properties of soluble antigen–antibody
complex (horseradish peroxidase–antihorseradish peroxidase) and its use in identification of spirochetes. J Histochem Cytochem 1970;28:315–33.
- Hope BT, Michael GJ, Knigge KM, Vincent SR. Neuronal NADPH-diaphorase is a nitric oxide synthase. Proc Natl Acad Sci U S A 1991;88:2811–4.
- Matsumoto T, Nakane M, Pollock JS, Kuk JE, Forstermann U. A correlation between soluble brain nitric oxide synthase and NADPH-diaphorase activity is only seen after
exposure of the tissue to fixative. Neurosci Lett 1993;155:61–4.
Statistics:
All data are presented as litters' means ± SD. Litters' means were tested by the Student's t test. Data of each litter were previously evaluated by ANOVA looking for interaction between sexes. In all cases, the significance level was considered to be p < 0.05.
Reproductive indices:
not specified
Offspring viability indices:
not specified
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not specified
Mortality:
not specified
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- At the end of test item administration, body weight gain in treated dams (−6.25 ± 2.87 g) was significantly lower (p = 0.04) compared to control dams (2.00 ± 5.75 g)
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:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
not specified
CLINICAL SIGNS:
- no sings of illness were observed in the dams.
Remarks on result:
not determinable because of methodological limitations
Critical effects observed:
not specified
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not specified
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
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:
no effects observed
Gross pathological findings:
not specified
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
HSP70 IMMUNOHISTOCHEMISTRY:
In the cerebellum, HSP70 immunoreactivity was detected in molecular and granular layers, and Purkinje cells also became immunostained after test item exposition. In the hippocampus, HSP70 immunoreactivity was diffusely distributed in all layers of control and test item-exposed pups.

GFAP IMMUNOHISTOCHEMISTRY:
GFAP immunostaining specifically labels the intermediate filaments of the astroglial cytoskeleton, allowing the study of astrocyte morphology. GFAP immunostaining shows the cell body of astrocytes as well as their numerous processes. GFAP-IR astrocytes in cerebellum, mainly those in the white matter, were markedly larger in the test item-exposed animals. Computerized image analysis data confirmed these observations: average GFAP-IR astrocytic surface area was 36.30 ± 3.0 μm² in test item-exposed pups while it was 30.50 ± 2.8 μm² for control pups, p = 0.03. In the hippocampus, GFAP-IR cells appeared more densely grouped in the hilus and in the subiculum in both experimental groups. In the hippocampal hilus of test item-exposed pups, astrocytes presented enlarged thick processes and increased soma size, the average GFAP-IR astrocytic surface area was 33.79 ± 2.60 μm² in test item-exposed pups, while in the control group it was 25.53 ± 1.39 μm², p = 0.010.
Other effects:
not specified
Behaviour (functional findings):
not specified
Developmental immunotoxicity:
not specified
CLINICAL SIGNS:
- signs of overall toxicity such as pallor, asphyxia, lethargy or diarrhea were not observed in the pups.

BODY WEIGHT AND WEIGHT CHANGES:
- at the end of test item administration, there were no significant differences in body weight (p = 0.06) between control (46.30 ± 9.12 g) and test item-exposed pups (39.23 ± 3.84 g) of both sexes.

ORGAN WEIGHT
- at the end of test item administration, there were no significant differences in brain weight (p = 0.10) between control (1.28 ± 0.03 g) and test item-exposed pups (1.21 ± 0.05 g) of both sexes.

NADPHd HISTOCHEMISTRY:
Histochemical detection of NADPHd activity was assumed as a measure of nitric oxide synthase activity in brain sections. Computerized image analysis of Nitro Blue Tetrazolium staining could be interpreted as a specific augmentation of nitric oxide synthase expression in neurons. However, in Cer (p = 0.08), cortex (p = 0.16) and striatum (p = 0.23) of test item-exposed pups, Nitro Blue Tetrazolium staining was not statistically different to control ones.

GFAP IMMUNOHISTOCHEMISTRY:
GFAP immunostaining specifically labels the intermediate filaments of the astroglial cytoskeleton, allowing the study of astrocyte morphology. GFAP immunostaining shows the cell body of astrocytes as well as their numerous processes. No differences between test item-exposed and control pups in GFAP-IR astrocytic surface area in hippocampal subiculum (p = 0.09) were detected.

S100 IMMUNOHISTOCHEMISTRY:
S100 proteins appeared to be confined to the cytosol of both normal and reactive astrocytes. S100 immunostaining labeled the astrocyte cell body and some cytoplasmic projections. S100 immunostaining was observed in the astroglial cells of every analyzed brain region in control and treated groups although, the immunostaining intensity was similar both in control and test item-exposed pups in cerebellum (p = 0.30) and hippocampus [hilus (p = 0.69) and subiculum (p = 0.28)]

BIOCHEMICAL DETERMINATIONS - MDA detection:
No differences in the final concentrations of thiobarbituric acid-reactive material were observed in any brain area homogenates of test item-exposed pups when compared to the corresponding control homogenate. There were no differences between sexes.
Please also refer to the field "Any other information on results incl. tables" below.
Remarks on result:
not determinable because of methodological limitations
Critical effects observed:
not specified
Reproductive effects observed:
not specified

Table 1: Concentrations of MDA/mg protein in various brain areas.

Brain regions

Control

NaVO3-exposed

p

Hypothalamus

1.18 ± 0.39

1.49 ± 0.78

0.52

Prefrontal cortex

2.06 ± 0.84

1.85 ± 0.62

0.72

Striatum

1.50 ± 0.33

1.43 ± 0.49

0.85

Hippocampus

1.91 ± 0.89

1.66 ± 0.76

0.64

Midbrain

1.18 ± 0.19

1.14 ± 0.17

0.30

Cerebellum

1.67 ± 0.26

1.70 ± 0.47

0.89

Results are expressed as nmol MDA/mg protein. Each value is the mean ± SD of 4 litters.

Conclusions:
Cuesta et al. (2013) administered sodium metavanadate (3 mg/kg bw/day; equivalent to 1.25 mg vanadium/kg bw/day) via intraperitoneal injection to dams that were nursing pups during postnatal day 10 to postnatal day 21 (4 litters containing 8 pups; 4 male and 4 female pups, if possible). There were no clinical signs observed in the dams, but body weight was significantly decreased in the test item treated dams compared to the control animals.

No mortality or clinical sigs were observed in the offspring. Furthermore, no significant differences were observed in body or brain weights between the treatment and control group. In addition, MDA levels, S100 immunostaining and NADPHd activity did not show differences between experimental and control groups, but astrogliosis and HSP70 activitation were detected in cerebellum, while only the former was detected in the hippocampal hilus of vanadium-exposed pups.

This non-regulatory mechanistic study was conducted with only one effective dose level of 3 mg NaVO3/kg body weight/day (1.25 mg V/kg bw/day) in comparison to a control group and were not designed to evaluate a dose-response relationship or a no effect level. Furthermore, a non-relevant route of exposure (intraperitoneal injection) was used. Lastly, the publication showed deficiencies with respect to reporting of methods and results.
Endpoint:
developmental neurotoxicity
Remarks:
mechanistic study
Type of information:
experimental study
Adequacy of study:
disregarded due to major methodological deficiencies
Study period:
not specified
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
Study conducted with a non-relevant route of exposure (intraperitoneal route); only one dose level used, no NOEL determined
Qualifier:
no guideline followed
Principles of method if other than guideline:
The study was performed to assess the vanadium-induced developmental neurotoxicity in sucklings of Wistar rats. Dams of treated litters (4 litters containing 8 pups (4 male and 4 female pups, if possible)) were intraperitoneally injected with 3 mg sodium metavanadate/kg body weight/day in distilled water (equivalent to 1.25 mg vanadium/kg bw/day) during postnatal day 10 to postnatal day 21. A control group receiving saline only was run concurrently by using the same procedure as for the treatment group. The following parameters were investigated: clinical signs (dams and pups), body weights (dams and pups), eye opening (pups), brain weight (pups) and behavioural tests (surface righting reflex, negative geotaxis, forelimb support and open field (number of crosses, rearing, grooming and defecation); pups). Lastly, brain sections were prepared from the brain of the pups and they were myelin histochemically as well as anti-myelin basic protein immunohistochemcally investigated.
GLP compliance:
not specified
Remarks:
not specified in the publication
Limit test:
no
Justification for study design:
not applicable
Specific details on test material used for the study:
not specified
Species:
rat
Strain:
Wistar
Details on species / strain selection:
not specified
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS - OFFSPRING
- Age at study initiation: 1 day after birth
- Housing: offspring, together with their mother, were housed in plastic breeding cages
- Diet (ad libitum)
- Water (ad libitum)

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C,
- Relative humidity: 40 – 60 %
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
intraperitoneal
Vehicle:
other: distilled water
Details on exposure:
Newborn rats were grouped into litters of eight animals (four males and four females, if possible) to ensure good nutrition. Eight litters were randomly assigned to one of the following animal
groups:
- Vanadium-treated group: consisted of offspring of 4 dams. The dams were were treated with the test item in distilled water from the 10th to the 21st postnatal day.
- Control group: consisted of offspring of 4 dams. The dams were treated with an equal volume of saline solution during the same period as the treatment group.
Details on mating procedure:
not specified
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
postnatal day 10 to postnatal day 21 (test item treatment of dams)
Frequency of treatment:
daily (test item treatment of dams)
Details on study schedule:
not specified
Dose / conc.:
3 mg/kg bw/day (nominal)
Remarks:
equivalent to 1.25 mg vanadium/kg bw/day
No. of animals per sex per dose:
4 litters containing 8 pups (four males and four females/litter, if possible)(total number of pups/dose: 32)
Control animals:
yes
Details on study design:
- Dose selection rationale: the dose of vanadium was dictated by previous results (Garcia et al., 2004)*, which indicated that the administration of similar amounts of sodium metavanadate resulted in changes in the locomotor activity, specific myelin staining and lipid peroxidation in some brain areas in adult rats.


*Reference:
- G.B. Garcia, A. Quiroga, N. Strütz, A. Martinez, M.E. Biancardi, Morphological alterations of central nervous system (CNS) myelin in vanadium (V) exposed adult rats, Drug Chem. Toxicol. 27 (2004) 1–13.
Positive control:
not specified
Parental animals: Observations and examinations:
NOTE: dams only were observed and examined during the study

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at the end of administration
- Cage side observations checked: clinical signs

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE: No data
Oestrous cyclicity (parental animals):
not specified
Sperm parameters (parental animals):
not specified
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in offspring:
- clinical signs and mortality were recorded.
- pups' body weight were recorded daily from postnatal day 6 until postnatal day 20.
- eye opening (open eyes noted) was observed from postnatal day 6 until postnatal day 20

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY:
The behavioural tests were carried out in a dimly lit room which was maintained at about 25 °C. They were performed on all pups (control and treated) every 48 hours, always at the same time of the day (between 10.00 AM and 12.00 PM) with the exception of the open field test which was performed only on the postnatal day 21. Each neonate was subjected to the following tests:

a) surface righting reflex:
This test was conducted from postnatal day 8 to postnatal day 18. Pups were directly placed on their back on a flat table top and released. Time was recorded until all four paws were placed on to the table surface.

b) negative geotaxis:
This test was performed from postnatal day 8 to postnatal day 18. Pups were placed in the middle of a sandpaper covered 30° inclined surface plane, in a head down position and the latency to turn 180° to a head-up position was recorded.

c) forelimb support:
The pups' forepaws were placed on a horizontally suspended wire (1 mm in diameter), 47 cm above a soft foam rubber landing area. Each pup was timed from the moment it was placed on the wire until it was unable to remain on the wire. This test was conducted from postnatal day 11 until postnatal day 20.

d) open field:
The apparatus was made of wood and consisted of a 42.5×42.5 cm floor with 40.0 cm high sides. The floor was divided into squares with black ink. For the observations each pup was placed in the center of the floor and the following parameters were measured over a period of 10 minutes: locomotion (the number of times each pup crossed from one square to another entering at least its two front paws), rearing (the number of times the animal stood on hind legs), grooming (sets of heterogeneous constituents comprising face washing, body licking, paw licking, head and body shaking, scratching and genital licking) and defecation (number of faecal pellets). The open field board was washed with a 5% alcohol/water solution before placing the subsequent animals on it in order to obviate possible biasing effects due to odour clues left by previous rats.

All data were analyzed considering the litter as the smallest unit.
Postmortem examinations (parental animals):
not specified
Postmortem examinations (offspring):
HISTOPATHOLOGY / ORGAN WEIGTHS
Ten 22-day-old rats from each group were anaesthetized with sodium thiopental and transcardially perfused with paraformaldehyde (w/v), in phosphate buffer, pH 7.4. Prior to fixation a saline solution, 0.9% w/v NaCl with 0.4M NaNO2 and heparin was passed through their circulatory system. Brains were dissected and kept in the same fixative solution for 2–4 hours and then they were longitudinally cut. One hemisphere was used for the histochemical staining and the other one for immunohistochemistry.

a) histochemical staining:
Hemispheres were postfixed in the same fixative solution plus 20% sucrose at 4 °C overnight. They were longitudinally freeze-sectioned on a cryostat at 30 μm thickness and the sections were kept in phosphate buffer saline at 4 °C until the staining procedure was started. The myelin staining was performed according to the Schmued's histochemical staining technique (Schmued, 1990)*, using a 0.2% solution of gold chloride (AuCl), modified for free floating sections.

b) myelin basic protein immunohistochemical staining:
Hemispheres were immersed in phosphate buffer saline plus 20% sucrose overnight or until they fell down. 40 μm-thick brain longitudinal sections were cut and kept at −20 °C cryoprotected with 30% sucrose in phosphate buffer saline. Immunohistochemistry was performed by the Sternberger's peroxidase–anti peroxidase (PAP) technique (Sternberger et al., 1970)*. Free-floating sections from exposed and control animals were simultaneously processed: they were washed in phosphate buffer saline, placed in methanol containing 0.3% H2O2 for 30 minutes and incubated in 3% normal goat serum (NGS) in phosphate buffer saline with 0.3% Triton X-100 (PBSX) for 30 minutes. Tissue sections were then incubated with polyclonal anti-MBP antiserum 1:1000 (primary antibody) for 72 hours at −4 °C in PBSX with 1% NGS. Development of peroxidase activity was performed with diaminobenzidine hydrochloride in phosphate buffer. After the enzymatic incubation step, sections were washed in phosphate buffer three times and once in distilled water, mounted on gelatin-coated standard glass slides, dehydrated and coverslipped for light microscopy observation.

Computerized image analysis:
The relative optical density (ROD) of the AuCl or a-MBP stained myelin tracts in the cerebellum and in the corpus callosum were measured using a NIH imaging analysis system. Images were obtained through an Olympus D-560 zoom digital camera attached to an Olympus BX40 microscope.
The mean gray value of the stained myelin tracts were measured using a rectangular area of interest (AOI). This AOI was located n times in stained myelin and the n mean gray values were taken. Relative optical density was obtained after a transformation of mean gray values into relative optical density using the formula: relative optical density = log (256/mean gray). The relative optical density value was chosen to evaluate the intensity of AuCl and a-MBP staining. Relative optical density was calculated and averaged within the experimental group in each animal.

*Reference:
- L.C. Schmued, A rapid sensitive histochemical staining for myelin in frozen brain sections, J. Histochem. Cytochem. 38 (5) (1990) 717 – 720.
- L.A. Sternberger, P.H. Hardy, J.J. Cuculis, H.G. Mayer, The unlabeled antibody enzyme method of immunohistochemistry. Preparation and properties of soluble antigen–antibody complex (horseradish peroxidase–antihorseradish peroxidase) and its use in identification of spirochetes, J. Histochem. Cytochem. 28 (1970) 315–333.
Statistics:
All data are presented as litter means ± SEM. Data were statistically evaluated by ANOVA. The Student's t-test was used as a post hoc test when no interaction between sexes was observed. Some data were tested by the Mann–Whitney test when the difference between SDs was significant. Optical density data of Vanadium-exposed and control groups were statistically evaluated by an unpaired t-test. The acceptable significance level was set at p < 0.05.
Reproductive indices:
not specified
Offspring viability indices:
not specified
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At the end of vanadium administration, treated dams showed lethargy and piloerection without any other sign of illness.
Dermal irritation (if dermal study):
not specified
Mortality:
not specified
Body weight and weight changes:
no effects observed
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:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
not specified
CLINICAL SIGNS AND BODY WEIGHT / WEIGHT CHANGES:
Although the difference was not statistically significant, body weight gain was decreased (-4 %) in vanadium-treated dams (249 ± 23 g) compared to control (259 ± 19 g). Control dams and their litters displayed none of the signs observed in vanadium-exposed animals.
Remarks on result:
not determinable because of methodological limitations
Critical effects observed:
not specified
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not specified
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
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:
no effects observed
Gross pathological findings:
not specified
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
Gold chloride histochemical and MBP immunohistochemical stainings showed a decreased myelin fiber density in different brain areas in vanadium-exposed pups. Gold chloride staining showed a decreased myelin staining in cerebellum and corpus callosum in vanadium-exposed pups' brains. Computerized image analysis data confirmed these observations: relative optical density of the gold chloride stained myelin in cerebellum on vanadium-exposed pups, expressed as mean ± SEM, was 0.33 ± 0.005, while it was 0.41 ± 0.006 for controls (diffferences between groups was statistically significant ; p < 0.0001). In the corpus callosum relative optical density was 0.17 ± 0.007 in vanadium-exposed pups, while in the control group it was 0.25 ± 0.008 (diffferences between groups was statistically significant ; p < 0.0001).
Anti-MBP immunostaining also showed a statistically significant (p < 0.0001) decreased myelin staining in vanadium-exposed pups' cerebellum and cerebrum. Relative optical density of the a-MBP immunostained myelin in cerebellum on vanadium-exposed pups was 0.072 ± 0.007, while it was 0.098 ± 0.002 for controls. In corpus callosum relative optical density was 0.016 ± 0.001 in vanadium-exposed pups and 0.045 ± 0.002 in the control group. Staining differences between sexes were not detected either with gold chloride histochemistry or with anti-MBP immunohistochemistry.

Please also refer to the field "Attached background material" below.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
EYE OPENING.
A statistically significant (p<0.01) exposure-related effect was observed when achieving eye opening. The eye opening mean day in vanadium-exposed pups was 16.3 ± 0.2, while it was 15.3 ± 0.3 for control ones. No differences were observed between sexes.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
a) Forelimb support:
A statistically significant (p < 0.05) decreased forelimb support latency was observed in vanadium-exposed pups with respect to controls on postnatal day 20.

b) Open field
Vanadium-exposed pups manifested a statstically significant (p < 0.01) decrease of locomotor activity – expressed as number of crosses – with respect to control pups.

Please also refer to the field "Attached background material" below.
Developmental immunotoxicity:
not specified
CLINICAL SIGNS
Signs of vanadium toxicosis (pallor, asphyxia, lethargy, diarrhoea) were not observed.

MORTALITY / VIABILITY
Before and during dams' treatment, newborn mortality was not observed.

BODY WEIGHT AND WEIGHT CHANGES
Body weight gain was not different between control and vanadium-exposed pups.

ORGAN WEIGHTS
There were no significant differences in brain weight between control and vanadium-exposed pups.

DEVELOPMENTAL NEUROTOXICITY
a) Surface righting reflex:
Both vanadium-exposed and control pups show a progressive improvement in righting response performance from postnatal day 8 to postnatal day 18. Although it was not statistically significant, progressive decrease in righting time was less marked in vanadium-exposed pups.

b) Negative geotaxis:
Vanadium-exposed and control pups showed a similar response in this test from postnatal day 8 to postnatal day 18. The latency time to turn 180 ° to a head-up position was similar in both groups.

c) Open field
There were no statistically significant differences between vanadium-exposed and control groups in the number of rearing, grooming and faecal pellets.

Please also refer to the field "Attached background material" below.
Remarks on result:
not determinable because of methodological limitations
Critical effects observed:
not specified
Reproductive effects observed:
not specified
Conclusions:
Soazo & Gracia (2007) administered sodium metavanadate (3 mg/kg bw/day) via intraperitoneal injection to dams that were nursing pups during postnatal day 10 to postnatal day 21 (4 litters containing 8 pups; 4 male and 4 female pups, if possible). At the end of the vanadium administration, dams showed lethary and piloerection without any other signs of illness. Furthermore, body weight gain was decreased (-4 %, not statistically significant) in vanadium-treated dams compared to control.

Investigation of pups revealed no signs of pallor, asphyxia, lethargy or diarrhoea as well as no mortality. Furthermore, body weight gain and brain weight were not different between control and vanadium-exposed pups. In addition, no test item-related effect was observed for surface righting reflex, negative geotaxis, rearing, grooming and faecel pelltes. However, forelimb support latency and locomotor activity (number of crosses) in the open field were statistically signficant decreased in vanadium-exposed pups with respect to controls. A statstically significant delay in eye opning was observed as well in vanadium-exposed pups compared to control pups. Lastly, histochemical and immunohistochemical investigations showed a decreased myelin fiber density in different brain areas in vanadium treated pups.

Study was conducted using a non-relevant route of exposure (intraperitoneal route) and the study only utilised one dose level. A NOEL could not be determined from the results of the study. Thus, the study is not appropriate for using in a regulatory context.
Effect on fertility: via oral route
Quality of whole database:
Several studies with oral exposure are available, but the reliability is low and the results are not considered relevant for human exposure.
Effect on fertility: via inhalation route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
4 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
One reliable study (RL1) with inhalation exposure of rat and mice with V2O5, and several studies with RL3 and apprpriate routes of exposure are available. Additional studies with exposure by a non-relevant route (ip) were disregarded.
4 mg/m3 V2O5 corresponds to 2.25 mg V/m3
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Read-across:The read-across approach based on dissolved vanadium is based on the assumption that once inorganic vanadium compounds dissolve or become bioavailable, this will be in tetra- or pentavalent vanadium forms.In bioaccessibility tests of tetra- and pentavalent vanadium substances, tetra- and pentavalent forms dissolved completely within 2h in various media selected to simulate relevant human-chemical interactions (i.e. PBSmimickingthe ionic strength of blood, artificial lung, lysosomal, and gastric fluid as well as artificial sweat).Pentavalent vanadium substances are released and retained as pentavalent forms in physiological media, with the exception of artificial lysosomal fluid in which tetravalent V dominates after 2h and is the only form present after 24h.Thus, read-across of reproductive toxicity data from soluble pentavalent vanadium substances is justified.

The registrant is aware that the National Toxicology Programme (NTP) in the US nominated tetra- and pentavalent vanadium forms (sodium metavanadate, NaVO3, CAS # 13718-26-8; and vanadium oxide sulphate, VOSO4, CAS # 27774-13-6), i.e. species present in drinking water and dietary supplements in 2007 (http://ntp.niehs.nih.gov/). A comprehensive characterisation via the oral route of exposure of

(i) chronic toxicity,

(ii) carcinogenicity, and 

(iii) multi-generation reproductive toxicity

is planned.

 

The NTP testing program began with sub-chronic drinking water studies on VOSO4& NaVO3as follows:

- Genetic toxicology studies, i.e. the Salmonella gene mutation assays, with NaVO3 and VOSO4 - negative

- 14 days with Harlan Sprague-Dawley rats and B6C3F1/N mice (Dose: R&M: 0, 125, 250, 500, 1000, 2000 mg/L) - already completed

- 90-d oral toxicity studies (dosed feed: NaVO3; dosed water: VOSO4) with Harlan Sprague-Dawley rats and B6C3F1/N (dose: rats and mice: 0, 31.3, 62.5, 125, 250, or 500 ppm - ongoing

- Organ systems toxicity, i.e. 28-d immunotoxicity study of NaVO3 (dosed-water) with female B6C3F1/N mice (dose: 0, 31.3, 62.5, 125, 250, or 500 ppm) - ongoing

- Perinatal dose-range finding study: gestation day 6 (GD 6) until postnatal day 42 (PND 42) with Harlan Sprague-Dawley rats - ongoing

It can reasonably be anticipated that these studies will be of high quality and relevance, and thus will serve as a more robust basis than the current data base with all its shortcomings. In addition, repeated-dose inhalation toxicity studies (14, 28, and 90 days) with various vanadium substances are planned within the Vanadium Safety Readiness Safety Program. These studies will address issues for which to date equivocal or no data at all exist. Further information on these studies can be found in section 7.5. Only upon availability of the results from these studies, it will be possible to render a more meaningful decision on whether or not testing for reproductive toxicity is required. Therefore for the time being this data requirement should be waived in consideration of animal welfare.

EBRC (2007) HERAG fact sheet - Assessment of occupational dermal exposure and dermal absorption for metals and inorganic metal compounds, EBRC Consulting GmbH, Hannover, Germany, August 2007, 49 pages.

Effects on developmental toxicity

Description of key information

An extensive literature/data search and evaluation programme on animal and human data relating to possible adverse effects of vanadium substances on developmental toxicity has recently been conducted. As an outcome, some data are available for very soluble tetra- and pentavalent substances (VOSO4 and Na3VO4) via the oral route.

Studies via the oral or inhalation route are not available for divanadium pentaoxide, but for other soluble vanadium substances. One study (k_Sanchez 1991) with administration of different dose levels of sodium orthovanadate of 0, 7.5, 15, 30 and 60 mg/kg body weight/day per gavage to pregnant mice on gestation days 6-15 was identified. In this study, maternal toxicity was observed at the highest doses levels, as evidenced by a significant number of deaths (60 and 30 mg/kg body weight/day) and reduced weight gain and food consumption a 30 and 15 mg/kg body weight/day. Embryo-lethality and teratogenicity were not observed at maternally toxic doses and below, but fetal toxicity was evidenced by a significant delay in the ossification process of some skeletal districts at 30 mg/kg body weight/day. The NOAEL for maternal toxicity was 7.5 mg/kg body weight/day and 15 mg/kg body weight/day represented a NOAEL for developmental toxicity in mice under the conditions of this study. However, it needs to be considered that fetal toxicity was observed at maternal toxic dose levels and can thus be regarded as secondary. Thus, the dose level of 30 mg/kg bw/d represents the LOEL for developmental effects and 15 mg/kg bw/d the NOEL for developmental effects at maternally toxic dose levels (reduced body weight gain and food consumption). These values correspond to 8.3 mg V/kg bw/d (LOEL) and 4.2 mg V/kg bw/d (NOEL).

One supportive developmental toxicity study (s_Paternain_1990) with administration of different dose levels of vanadyl sulphate to Swiss mice was identified. Vanadyl sulphate was evaluated for its embryotoxic, fetotoxic and teratogenic potential in Swiss mice. The compound was administered by gavage to pregnant mice at doses of 0, 37.5, 75 or 150 mg/kg body weight/day on days 6 -15 of pregnancy. Maternal toxicity was observed in all vanadium-treated animals, evident by reduced weight gain, reduced body weight on gestation day 18 (corrected for gravid uterine weight) and decreased absolute liver and kidney weights at 75 and 150 mg/kg body weight/day. The number of total implants, live and dead foetuses, late resorptions, the sex ratio and the post-implantation losses were not significantly different between the vanadium-treated mice and the control. However, there was a significant increase in the number of early resorptions per litter at all dose levels. Fetotoxicity was evident by lower fetal weights and lengths and the presence of developmental variations. Malformation incidence also increased by administration of vanadium. Thus, the NOEL for maternal toxicity, embryo-/fetotoxicity and teratogenicity for vanadyl sulphate pentahydrate under these test conditions can be expected below 37.5 mg/kg body weight/day (11.72 mg V/kg bw/d) in Swiss mice.

In a study with intragastric administration of sodium metavanadate (Paternain et al. 1987), an oral NOAEL of 20 mg/kg bw/d was determined at the high dose level corresponding to 8.4 mg V/kg bw/d. There is no clear evidence of direct developmental effects in foetuses of dams exposed during gestational day 6-14 to different dose levels. However, no information on maternal toxicity was reported in the study. Additional supportive studies with administration of V2O5 by non-relevant routes of exposure (ip or iv) (Altamirano-Lozano, M. et al. 1993, Altamirano et al. 1991, Wide, 1984), or other vanadium compounds, e.g. ammonium metavandate (Morgan and El-Tawil, 2003, Carlton et al 1982) or sodium metavandate (Domingo et al. 1986, Gomez et al., 1992) could be found, but were not further considered.

From the study results it can be concluded that foetotoxic effects occurred in absence of unspecific parental toxicity, and therefore there is evidence that vanadium compounds induce developmental toxicity.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
no data available
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
only summary results but no details or individual data presented
Qualifier:
no guideline followed
Principles of method if other than guideline:
Evaluation of the effects of vanadate (V5+) when administered once daily by gavage as sodium orthovanadate to mice throughout organogenesis.
GLP compliance:
no
Limit test:
no
Species:
mouse
Strain:
Swiss
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Animals were obtained from Letica (Barcelona, Spain).
- Weight at study initiation: 26-29g
- Housing: mice were housed in solid-bottom plastic cages with stainless steel wire lids.
- Diet: ad libitum, standard laboratory chow
- Water: ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-23
- Humidity (%): 45 +/- 5
- Photoperiod: 12 hours dark/light cycle
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- The test substance was dissolved in deionised water.

Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No details are given.
Details on mating procedure:
After acclimatisation, females were mated overnight with adult males of the same strain. The morning on which a copulation plug was detected was considered as day 0 of gestation. At this time, animals were randomly assigned to either the control or the vanadate-treated group.
Duration of treatment / exposure:
10 days
Frequency of treatment:
once daily on days 6-15 of pregnancy
Duration of test:
till day 18 of pregnancy
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
7.5 mg/kg bw/day (nominal)
Dose / conc.:
15 mg/kg bw/day (nominal)
Dose / conc.:
30 mg/kg bw/day (nominal)
Dose / conc.:
60 mg/kg bw/day (nominal)
No. of animals per sex per dose:
14-20 dams per dose group
Control animals:
yes
Details on study design:
- Dose selection rationale: The choice of the dosage levels was based on data from a previous study on the developmental toxicity of vanadyl sulfate in mice (Paternein, J.L.; et al. 1990).
Maternal examinations:
CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: general appearance of pregnant mice were monitored daily.

BODY WEIGHT: Yes
- Time schedule for examinations: body weight of pregnant mice were monitored daily.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Food consumption of pregnant mice were monitored daily.

WATER CONSUMPTION: No data

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 18 with an overdose of ether.
- All dams were evaluated for body weight, liver and kidney weights.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: No data
- Number of implantations: Yes
- Number of resorptions: Yes
- Number of dead and live fetuses: Yes
Fetal examinations:
- External examinations: all live fetuses were dissected from the uterus and evaluated for body weight, sex and external abnormalities.
- Soft tissue examinations: 1/3 of the fetuses from each litter were placed in Bouin's fluid to be examined for soft tissue abnormalities.
- Skeletal examinations: 2/3 of the fetuses from each litter were cleared and stained with alizarin red S before examination for skeletal malformations and variations.
- Head examinations: No data
Statistics:
Homogeneity of variance was analysed by Barlett's test. If variances were homogenous, a one-way analysis of variance (ANOVA) was used to test all dose groups simultaneously. The Kruskal-Wallis test was used when variances were not homogenous. Differences between control and orthovanadate-treated groups were analysed by Student's t-test. Incidence data were analysed using the chi-square test. The level of significance for all analyses was p<0.05.
Indices:
no details given
Historical control data:
no data
Clinical signs:
not specified
Dermal irritation (if dermal study):
not specified
Mortality:
mortality observed, treatment-related
Description (incidence):
- In the high dose group, 17 dams (from 19 pregnant females dosed) died during the treatment period. Consequently, the two remaining dams were not included in the teratological evaluation of orthovanadate.
- At 30 mg/kg/day, 4 dams (from 18 pregnant females does) were found dead during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- Maternal weight gain was significantly reduced below control values in the 30 mg/kg/day dose group on gestational days 6-15.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- Food consumption exhibited a significant decrease in the 15 and 30 mg/kg/day dose groups on gestational days 0-18.
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
Description (incidence and severity):
- There was an increase in relative kidney weight at 30 mg/kg/day, which were statistically significant versus controls.
Gross pathological findings:
not specified
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Details on results:
Maternal toxic effects:yes

Details on maternal toxic effects:
Doses of 30 and 60 mg/kg/day of sodium orthovanadate resulted in maternal toxicity.
- Exposure to 7.5 or 15 mg/kg/day of the test substance did not result in any maternal death.
- At scheduled termination on gestation day 18, there were no significant decreases in body weight, gravid uterine weight, corrected body weight and change in corrected body weight.
- There was a decrease in absolute and relative liver weight in the 15 mg/kg/days dose group and an increase in relative kidney weight at 30 mg/kg/day, which were statistically significant versus controls. However, the decreases in liver weight were not dose-related and therefore they were not attributed to treatment.
Number of abortions:
not specified
Pre- and post-implantation loss:
not specified
Total litter losses by resorption:
not specified
Early or late resorptions:
not specified
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not specified
Changes in number of pregnant:
not specified
Other effects:
no effects observed
Details on maternal toxic effects:
- Evaluation of gestational parameters for the mice indicated no treatment-related effects on number of total implantations per litter, number of live and dead/resorbed fetuses per litter, sex ratio, fetal body weights and the number of stunted fetuses.
Dose descriptor:
NOAEL
Effect level:
7.5 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
mortality
Abnormalities:
not specified
Fetal body weight changes:
not specified
Reduction in number of live offspring:
not specified
Changes in sex ratio:
not specified
Changes in litter size and weights:
not specified
Changes in postnatal survival:
not specified
External malformations:
no effects observed
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
- Treatment-related changes were found during the examination of the incidence and type of skeletal anormalies: significant decreases in the number of ossified sacrococcygeal vertrebrae, as well as the number of ossified forelimb and hindlimb proximal phalanges were sen at 30 mg/kg bw/d.
Visceral malformations:
no effects observed
Other effects:
not specified
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes. Remark: skeletal anormalities

Details on embryotoxic / teratogenic effects:
- Sodium orthovanadate did not induce significant incidence of gross and visceral malformations or variations in mouse fetuses.
- Treatment-related changes were found during the examination of the incidence and type of skeletal anormalies at 30 mg/kg bw/d.
- No significant increases in the number of fetuses with reduced ossification of occipital and parietal bones or sternebrae were observed.
Dose descriptor:
NOAEL
Effect level:
15 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
skeletal malformations
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
At 30 and 60 mg sodium orthovandate/kg body weight, deaths occurred among the dams (4/18 and 17/19). Body weight gain was significantly reduced (approximately 20%) at 15 mg/kg b.w. No differences were reported in final body weight, gravid uterine weight, or corrected body weight.
NOAEL for maternal toxicity: 7.5. mg/kg. b.w./day
NOAEL for foetotoxicity 15 mg/kg b.w./day
Executive summary:

Sodium orthovanadate in deionised water was administered once daily by gavage on gestational days 6 -15 to mice at doses of 0, 7.5, 15, 30 and 60 mg/kg body weight/day. Dams were killed on day 18 of pregnancy, and fetuses were examined for external, visceral and skeletal defects. Maternal toxicity was observed at the highest doses levels, as evidenced by a significant number of deaths (60 and 30 mg/kg body weight/day) and reduced weight gain and food consumption (30 and 15 mg/kg body weight/day). Embryolethality and teratogenicity were not observed at maternally toxic doses and below, but fetal toxicity was evidenced by a significant delay in the ossification process of some skeletal districts at 30 mg/kg body weight/day. The NOAEL for maternal toxicity was 7.5 mg/kg body weight/day and 15 mg/kg body weight/day represented a NOAEL for developmental toxicity in mice under the conditions of this study.

Although this publication shows some deficiencies with respect to reporting of methods and results it was conducted in a suficient number of female mice and can be regarded as relevant and approriate.

Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
no data available
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well reported study. The relevance of the study is limited since all developmental effects were associated by pronounced maternal toxicity. Purity of the test substance not stated.
Guideline:
other: no information available if a guideline was followed
GLP compliance:
no
Limit test:
no
Species:
mouse
Strain:
Swiss
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: adult mice were obtained from Panlab (Barcelone, Spain).
- Weight at study initiation: 25-30 g
- Diet: ad libitum, commercial chow
- Water: ad libitum
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-23
- Humidity (%): 45 +/- 5
- Photoperiod: 12 hours dark/light cycle
NO further details are given.
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: no data


Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No details are reported.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/2; for mating, one male and two females were placed into a breeding cage.
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
No further details are given.
Duration of treatment / exposure:
on gestational days 6-15
Frequency of treatment:
once daily
Duration of test:
till day 18 of gestation
Remarks:
Doses / Concentrations:
0 mg/kg body weight/day
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
37.5 mg/kg body weight/day
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
75 mg/kg body weight/day
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
150 mg/kg body weight/day
Basis:
nominal conc.
No. of animals per sex per dose:
Three groups of 22 sperm-positive females were randomly assigned to each group.
Control animals:
yes
Details on study design:
- Dose selection rationale: the doses are approximately 1/12, 1/6 and 1/3 of the acute oral LD50 of VOSO4 * 5H2O for adult mice (LLobet, J.M.; Domingo, J.L., 1984).
Maternal examinations:
CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: mice were observed daily for mortality and morbidity throughout the study.

BODY WEIGHT: Yes
- Time schedule for examinations: body weights were computed for the pre-treatment, treatment and post-treatment periods from daily records of these parameters.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: food consumption was computed for the pre-treatment, treatment and post-treatment periods from daily records of these parameters.

WATER CONSUMPTION: No data

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 18 by over-exposure to ether.
- Organs examined: the uteri were excised and weighed.
- Dams were examined for visceral gross pathology.

OTHER: Yes
- Maternal liver, kidney, spleen and placenta were analysed for the concentration of vanadium. Vanadium concentrations were determined by atomic absorption spectrophotometry.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: No data
- Number of implantations: Yes
- Number of resorptions: Yes
- Other: post-implantation losses were calculated for each litter as follows: (No. of implants - No. of live fetuses)/No. of implants x 100. The number of stunted fetuses (under 2/3 average body weight) was also recorded.
Fetal examinations:
The fetuses were taken by caesarean section.
- External examinations: fetuses were removed from their placentas and examined externally. Sex, weight and length were recorded.
- Soft tissue examinations: after the external observation was completed, approximately 1/3 of the remaining fetuses from each group were fixed in Bouin's solution and examined for soft tissue abnormalities.
- Skeletal examinations: remaining fetuses were fixed with Alizarin red solution and examined for skeletal abnormalities.
- Head examinations: No / No data
- Other: 3 fetuses from each dam were used for whole body analyses of vanadium.
Statistics:
Test groups were compared to the control group at a level of significance of P<0.05. Continuous data (e.g. maternal body weight, body weight gain, food consumption etc.) were analysed using one-way analysis of variance with significant F values analysed further using Student's t-test or the Mann-Whitney U test. Statistical comparison of the vanadium analyses were made by Student's t-test (one tailed), with a Bonferroni correction for multiple comparisons between treatment groups. A probability of P<0.05 was used as the criterion of statistical significance. Incidence data were compared using chi-square contingency tables (2x4). Each test group was compared to the control group when chi-square was significant.
Indices:
no details given
Historical control data:
no data
Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: body weight gain, organ weights, food consumption

Details on maternal toxic effects:
- No deaths or treatment-related clinical signs of toxicity were observed in the study. 2 animals died in the low-dose group, but these deaths appeared to be related of dosing accidents.
- There were no treatment-related gross necropsy findings.
- Mean body weight gain during the pre-treatment interval, days 0-6 of gestation, was comparable between treated and control groups. However, there were significant decreases in body weight gain during the exposure period in treated animals. The reduction in maternal weight gain during this period was dose-related and was observed at all dose levels. The body weight gain during the post-exposure interval indicated that the treated animals gained less weight than the controls, but these differences were not statistically significant.
- No significant differences occurred between vanadium-treated groups and the control group in food consumption during pregnancy. Although an apparent decrease in the mean food consumption was observed for the 75 and 150 mg/kg body weight/day groups from day 0 to day 18 of gestation, this decrease was not statistically significant.
- Maternal organ weights at termination, including absolute and corrected body weight, absolute liver weight, absolute kidney weight and placenta weight, were affected by treatment. Absolute maternal liver weight (g), but not relative liver weight (% body weight), was significantly decreased at 75 and 150 mg/kg body weight/day, whereas absolute maternal kidney weight was significantly decreased at 150 mg/kg/day.
- Gravid uterine weights were significantly lower at all dose levels relative to controls.
- There was no effect of vanadium on the number of total implantations, live and dead fetuses, late resorptions per litter, or on the percentage post-implantation losses or the sex ratio. The number of early resorptions per litter exhibited a dose-related increase for all vanadium-treated groups.
- Vanadium could not be detected in control mice. In contrast, it was detected in both maternal and fetal tissues of the treated animals. All these values were significant. However, a significant dose-related increase of vanadium concentrations was observed only in the dam kidney of treated animals.
Dose descriptor:
LOAEL
Remarks:
(lowest dose level)
Effect level:
37.5 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes. Remark: external defects, skeletal variations

Details on embryotoxic / teratogenic effects:
- Mean fetal body weights and mean fetal body lengths were significantly lower in the treated groups when compared to the control group.
- There was a significant incidence of stunted fetuses at the 75 mg/kg/day group (P<0.05) and at the 150 mg/kg/day group (P<0.001) relative to controls.
- Cleft palate and micrognathia were the most significant external malformations observed in the 150 mg/kg/day group.
- The total number of external defects was statistically significant for all vanadium-treated groups relative to controls.
- Hydrocephaly in fetuses from dams given 75 or 150 mg/kg/day of test substance was the only incidental abnormality observed after visceral examination.
- Increased incidence of skeletal variations was seen in all vanadium-treated groups. Decreased ossification of the supraoccipital bone and decreased ossification of the carpus and tarsus were the most common anormalies found. The increase in the total number of skeletal defects was significantly different from the controls in all the treated groups. In addition, this increase was dose-dependent.
Dose descriptor:
LOAEL
Remarks:
(lowest dose level)
Effect level:
37.5 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: embryotoxicity
Dose descriptor:
LOAEL
Remarks:
(lowest dose level)
Effect level:
37.5 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: fetotoxicity
Dose descriptor:
LOAEL
Remarks:
(lowest dose level)
Effect level:
37.5 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: teratogenicity
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
The present study involved oral administration of vanadyl sulphate pentahydrate to mice during organogenesis. Maternal toxicity in terms of decreased weight gain and reduction in body weight appeared to be produced in pregnant mice treated with vanadyl sulphate pentahydrate at doses of 37.5, 75 or 150 mg/kg/day. Embryo-/fetotoxicity was incidenced by an increased number of early resorptions and by lower fetal weights and lengths, as well as by increased incidence of poorly ossified skeletal elements at all dose levels. A significantly increased number of major malformations and minor anormalies indicated teratogenicity.
The NOEL for maternal toxicity, embryotoxicity, fetotoxicity and teratogenicity was <37.5 mg/kg/day of vanadyl sulphate pentahydrate, as signs of maternal and developmental toxicity were observed at this dose.
Executive summary:

Vanadium, as vanadyl sulphate pentahydrate, was evaluated for its embryotoxic, fetotoxic and teratogenic potential in Swiss mice. The compound was administered by gavage to pregnant mice at doses of 0, 37.5, 75 or 150 mg/kg body weight/day on days 6 -15 of pregnancy. On gestation day 18, all live fetuses were examined for external, visceral and skeletal malformations and variations.

Maternal toxicity was observed in all vanadium-treated animals, evidend by reduced weight gain, reduced body weight on gestation day 18 (corrected for gravid uterine weight) and decreased absolute liver and kidney weights at 75 and 150 mg/kg body weight/day. The number of total implants, live and dead fetuses, late resorptions, sex ratio and post-implantation losses were not significantly different between the vanadium-treated mice and the controls. However, there was a significant increase in the number of early resorptions per litter at all dose levels. Fetotoxicity was evidend by lower fetal weights and lengths and the presence of developmental variations. Malformation incidence also increased by administration of vanadium. Thus, the NOEL for maternal toxicity, embryo-/fetotoxicity and teratogenicity for vanadyl sulphate pentahydrate under these test conditions can be expected below 37.5 mg/kg body weight/day in Swiss mice.

Endpoint:
developmental toxicity
Adequacy of study:
supporting study
Study period:
no data available
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: No data regarding maternal toxicity was reported
Remarks:
Material and methods described only briefely and only summary results but no details or individual data presented
Qualifier:
no guideline followed
Principles of method if other than guideline:
In order to obtain an overall understanding of vanadium toxicity the present study was undertaken to further examine the adverse effects of sodium metavanadate in rats when given throughout organogenesis.
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Rats were obtained from Interfauna (Barcelona, Spain).
- Age at study initiation: adult rats
- Weight at study initiation: not less than 250g
- Housing: The animals were kept individually in Makrolon cages.
- Diet: ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-23
- Humidity (%): 40-60 :
- Photoperiod: 12 hours dark/light cycle

No further details are given.
Route of administration:
other: intragastrically
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The sodium metavanadate solutions were prepared to give any dose in a volume of 1 mL/250 g body weight.

DIET PREPARATION
not applicable
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
no data
Details on mating procedure:
The females were caged with males overnight and examined the following morning for the copulating plug or for spermatozoa by vaginal lavage. The day of vaginal plugs or spermatozoa detection was defined as day 1 of pregnancy.
Duration of treatment / exposure:
from gestation day 6 to 14
Frequency of treatment:
daily
Duration of test:
8 days
Remarks:
Doses / Concentrations:
5.0 mg test item/kg
Basis:
nominal in water
Remarks:
Doses / Concentrations:
10 mg tets item/kg
Basis:
nominal in water
Remarks:
Doses / Concentrations:
20 mg test item/kg
Basis:
nominal in water
No. of animals per sex per dose:
Groups of 20 pregnant rats.
Control animals:
other: yes; distilled water
Details on study design:
No further details on study design.
Maternal examinations:
na data
Ovaries and uterine content:
Cesarean sections were performed on the 20th day of gestation and the following examination made and compared with the control values:
- Gravid uterus weight: No data
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of resorptions: Yes
- Number of live and dead fetuses: Yes
- Other: placental weights
Fetal examinations:
- External examinations: Yes: average fetus body weight, fetal body length and fetal tail length. All fetuses were examined for abnormalities and sexed.
- Soft tissue examinations: Yes: One half of the fetuses from each litter were fixed in Bouin's solution and subsequently examined for visceral abnormalities by razor-blade sectioning.
- Skeletal examinations: Yes: The remaining fetuses from each litter were stained with alizarin red S and examined for skeletal anormalies.
- Head examinations: No data
Statistics:
The magnitude of the differences between the groups was calculated either by Student's t-test or Mann-Whitney U test. The litter was the treatment unit on which statistical analyses were based.
Indices:
The incidence of abnormalities in fetuses from treated dams compared to controls was determined.
Historical control data:
no data
Details on maternal toxic effects:
Maternal toxic effects:no data

Details on maternal toxic effects:
The number of apparently non-pregnant animals increased in the group receiving the highest dose of NaVO3. The number of litters decreased when 20 mg/kg/day NaVO3 were administered. The administration of vanadium had no significant adverse effects on the number of corpora lutea or the number of implantations.
The doses of 10 and 20 mg/kg/day NaVO3 caused an increase of the number of resorptions and number of dead fetuses, although no significant effect on the resorption rate could be demonstrated.
The average weights of placentas were similar.
Dose descriptor:
NOAEL
Effect level:
5 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
The incidence of abnormalities in fetuses from treated dams was remarably higher than the incidence in the control group, especially in the group given 20 mg/kg/day NaVO3. The differences between the number of male and female fetuses were not significant.
Slight differences in body weight, body length and tail length were observed between fetuses from treated dams and controls.
Visceral and skeletal examinations of fetuses did not reveal significant abnormalities in any group. However, the incidence of haemorrhage in facial area (18.4%) in fetuses from dams given 20 mg/kg/day NaVO3 was significantly higher than the incidence in the controls (0.0%). Moreover, in some fetuses of the same group, additional abnormalities were observed (hydrocephaly, 1.0%). In the 5 and 10 mg/kg/day groups, a greater number of abnormal fetuses than in the control group were also observed.
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
In this non-guideline study on developmental toxicity, no significant maternal toxicity was determined. The number of apparently non-pregnant females increased in the high dose group with the number of litters beeing 14, 14, 12, 8 in the control and treated groups, respectively. There were no effects on nos. of corpora lutea and implantations. An increased number of abnormal foetuses was observed from 5 mg/kg bw/d onwards without dose-response relation. At 10 and 20 mg/kg/d, an increase of the number of resorptions (not dose-related) and number of dead foetuses was observed although no significant effect on the resorption rate could be demonstrated. No skeletal abnormalities were determined, and the incidence of visceral abnormalities (hydrocephaly, haemorrhages) in foetuses from treated dams at 20 mg/kg/d was remarkably higher compared to the control group. With respect to haemrorhages there were also higher incidences in the low dose group than in the control group.

Overall, there is no clear evidence of direct developmental toxicity following exposure to sodium metavanadate under the condition of this study.
Possibly no observed effect level for embryonic developent: 10 mg NaVO /kg bw.

In conclusion, the study gives no clear evidence of direct developmental toxicity (WHO 2001)
Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
15 mg/kg bw/day
Species:
mouse
Quality of whole database:
Data of the developmental toxicity are available for very soluble tetra- and pentavalent substances via the oral route.
15 mg/kg bw/d sodium orthovanadate corresponds to 4.2 mg V/kg bw/d.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Read-across: The read-across approach based on dissolved vanadium is based on the assumption that once inorganic vanadium compounds dissolve or become bioavailable, this will be in tetra- or pentavalent vanadium forms.In bioaccessibility tests of tetra- and pentavalent vanadium substances, tetra- and pentavalent forms dissolved completely within 2h in various media selected to simulate relevant human-chemical interactions (i.e. PBSmimickingthe ionic strength of blood, artificial lung, lysosomal, and gastric fluid as well as artificial sweat).Pentavalent vanadium substances are released and retained as pentavalent forms in physiological media, with the exception of artificial lysosomal fluid in which tetravalent V dominates after 2h and is the only form present after 24h.Thus, read-across of developmental toxicity data from soluble tetra- and pentavalent vanadium substances is justified.

The registrant is aware that the National Toxicology Programme (NTP) in the US nominated tetra- and pentavalent vanadium forms(sodium metavanadate, NaVO3, CAS # 13718-26-8; and vanadium oxide sulphate, VOSO4, CAS # 27774-13-6), i.e. species present in drinking water and dietary supplements in 2007 (http://ntp.niehs.nih.gov/). A comprehensive characterisation via the oral route of exposure of

(i) chronic toxicity,

(ii) carcinogenicity, and 

(iii) multi-generation reproductive toxicity

is planned.

 

The NTP testing program began with sub-chronic drinking water studies on VOSO4& NaVO3as follows:

- Genetic toxicology studies, i.e. the Salmonella gene mutation assays, with NaVO3 and VOSO4 - negative

- 14 days with Harlan Sprague-Dawley rats and B6C3F1/N mice (Dose: R&M: 0, 125, 250, 500, 1000, 2000 mg/L) - already completed

- 90-d oral toxicity studies (dosed feed: NaVO3; dosed water: VOSO4) with Harlan Sprague-Dawley rats and B6C3F1/N (dose: rats and mice: 0, 31.3, 62.5, 125, 250, or 500 ppm - ongoing

- Organ systems toxicity, i.e. 28-d immunotoxicity study of NaVO3 (dosed-water) with female B6C3F1/N mice (dose: 0, 31.3, 62.5, 125, 250, or 500 ppm) - ongoing

- Perinatal dose-range finding study: gestation day 6 (GD 6) until postnatal day 42 (PND 42) with Harlan Sprague-Dawley rats - ongoing

It can reasonably be anticipated that these studies will be of high quality and relevance, and thus will serve as a more robust basis than the current data base with all its shortcomings. In addition, repeated-dose inhalation toxicity studies (14, 28, and 90 days) with various vanadium substances are planned within the Vanadium Safety Readiness Safety Program. These studies will address issues for which to date equivocal or no data at all exist. Further information on these studies can be found in section 7.5. Only upon availability of the results from these studies, it will be possible to render a more meaningful decision on whether or not testing for reproductive toxicity is required. Therefore for the time being this data requirement should be waived in consideration of animal welfare.

Justification for selection of Effect on developmental toxicity: via oral route:

In the absence of other reliable information, the lowest NOEL for developmental effects at a maternal toxic dose level was selected.

Justification for selection of Effect on developmental toxicity: via dermal route:

Data of developmental toxicity via the dermal route are not available for any vanadium substance. Following the HERAG guidance for metals and metal salts (see section 7.1.2 of the technical dossier: dermal absorption), negligible percutaneous uptake based on minimal penetration, i.e. a dermal absorption rate in the range of maximally 0.1 - 1.0 %, can be anticipated. Dermal absorption in this order of magnitude is not considered to be “significant”. Thus, regarding developmental toxicity of vanadium substances, the dermal exposure route is not expected to be the most relevant.

References:

EBRC (2007) HERAG fact sheet - Assessment of occupational dermal exposure and dermal absorption for metals and inorganic metal compounds, EBRC Consulting GmbH, Hannover, Germany, August 2007, 49 pages.

Justification for classification or non-classification

An extensive literature/data search and evaluation programme on animal and human data relating to possible adverse effects of relevant vanadium substances on fertility and developmental toxicity has been conducted.

Divanadium pentaoxide is classified as reproductive toxicant category 2 according to commission Regulation (EC) No 1272/2008.The placing of a substance in Category 2 is done on the basis of evidence in animal studies, which is not sufficiently convincing to place the substance in Category 1A or 1B.

Additional information