Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

No fertility, reproductive, or developmental toxicity data of sufficient quality are available for tungsten dioxide (target substance). However, reproductive toxicity data are available for sodium tungstate (source substance), which will be used for reading across. Due to lower water solubility and lower toxicity for the target substance compared to the source substance, the resulting read across from the source substance to the target substance is appropriate as a conservative estimate of potential toxicity for this endpoint. In addition, read across is appropriate because the classification and labelling is more protective for the source substance than the target substance, the PBT/vPvB profile is the same, and the dose descriptors are, or are expected to be, lower for the source substance. For more details, refer to the attached description of the read-across approach.

Reproductive Toxicity

 

A weight of evidence (WoE) approach was applied to fulfillAnnex X, Section 8.7.3.; test method: EU B.56./OECD TG 443.

 

This approach takes several peer reviewed published rat oral studies on the read-across source substance sodium tungstate to cover the Extended One-Generation Reproductive Toxicity (EOGRT) study extent.

 

In our opinion, when all the 90-day repeated exposure, fertility, reproductive, developmental and immunotoxicity of tungstate oral results are considered collectively, no additional testing should be required as the toxicity tests already conducted offer key information on the intrinsic toxicity of tungstate to parental and F1 groups.

 

In other words, studying the toxicity of a substance in separate studies (see figure in attachment in this section) does not diminishes the scientific reliability compared to a study that uses an extended one-generation approach.

 

When comparing the EOGRT requirements against the available fertility, reproductive and developmental studies on sodium tungstate (see figure in attachment in this section) some of the testing OECD requirements are not completely cover, however the results of the individual fertility, reproductive, developmental, neuro- and immuno-toxicity cover key toxicity endpoints and additional testing is not warranted.

 

1.   Fertility/Reproductive Toxicity ‒ Weight of Evidence

 

The fertility endpoints are covered by Ballester et al (2005 and 2007) studies which exposed unmatched male and female rats to sodium tungstate in drinking water. Reproductive and developmental toxicity are covered by McInturf et al (2007, 2008, and 2011) gavage study together with the US NTP perinatal drinking water study and Osterburg et al (2014) one-generation study in mice. The developmental neuro- and immuno-toxicity endpoints are covered by McInturf et al (2007, 2008, and 2011) gavage study, and the Fastje et al (2012) and Osterburg (2014). The figure in the attachment in this section depicts where each of these studies fall within the EOGRT fertility/reproductive duration and required cohorts. Below each of the studies included in the fertility/reproductive weight of evidence are discussed.

 

a.    Ballester et al (2005 & 2007) 90-day Rat Oral Fertility Studies

 

Two separate drinking water (2 mg/ml, equivalent to 160 and 190 mg/kg/d for females and male rats, respectively) studies on sodium tungstate exposed adult male (Ballester et al, 2005) and female (Ballester et al, 2007) rats for 3 months. The reproductive performance and other reproductive parameters were evaluated and are briefly discussed below.

 

Male rats (15 animals per group) weregiven a solution of 2 mg/mL sodium tungstate in 0.9% NaCl(Ballester et al. 2005). The treatment was carried out for 3 months. The time period of 3 months was chosen to allow tungstate to exert a complete effect on testicular function. At the end of the experiment, all the ratswere alive.

 

To evaluate reproductive performance, after 10 weeks of treatment, individual males were placed in a cage with 1 healthy adult female. The animals were kept together overnight, and the following morning they were separated. Immediately after separation, a vaginal examination and smearswere performed to determine whetherovernight mating hadoccurred. The reproductive performance of the male rats was determined by calculating the percentage offertilemales out of the total number tested.

 

Results showed that tungstate administration to healthy male rats (at an estimated dose of 190 mg/kg/d for 3 months) affected the body-weight gain, howeverdid not: 1)alter the reproductive performance of healthy animals; 2) modify the appearance of number of Leydig cells, or 3) change the serum levels ofluteinizing hormone (LH), follicle-stimulating hormone (FSH) and testosterone.

 

Female rats weregiven a drinking solution of 2 mg/ml sodium tungstate(estimated dose to 160 mg/kg/d)in 0.9% NaCl(Ballester et al 2007). The treatment was carried out for 12 weeks. At the end of the experiment, all the rats in the groups were still alive.

Sexual and reproductive function was assessed in all rats before decapitation. Blood was collected immediately to measure serum parameters.

 

To evaluate reproductive performance, after 10 weeks of treatment, individual females were placed in a cage with one healthy adult male. The animals were kept together overnight, and then separated the following morning. Immediately after each separation, a vaginal examination and smearswere carried out to determine ifmatinghad occurred. Whenvaginal smears werepositive (presence of a vaginal tap and/or spermatozoa). At parturition, litter size was determined and the mother was anesthetized and killed by decapitation. The neonates were killed by CO2inhalation.

 

The reproductive performance of the rats was measured as the proportion ofinseminatedfemales to the total number tested (the percentage of positive vaginalsmears, ie those with presence of spermatozoa, with respect to the total number of vaginalsmearsperformed in one experimental group). This parameter was named as ‘mating index’. The number of vaginalsmearsvaried depending on the time required by animals to show positivemating. Furthermore,thepercentage of parturitionswascalculated with respect to the number of positive smears. This parameter was named ‘fertility’. Finally, the mean litter size was also calculated.

 

Results showed that in healthy ratstungstate treatment caused a decrease in the body weight gainbutdid not modify daily food and water consumption.Tungstate treatment did not modifyalanine aminotransferase (ALT)activity,progesterone, FSHorLH. In addition,tungstate treatmentdid not affect any reproductive parameteror affected theexpression of the estrogen receptor.However, in ovariestungstate treatment had a considerable effect on the expression ofthe progesteronereceptor.In contrast, the uterine expression of the progesterone receptor was not affected by tungstate treatment.

 

b.    Osterburg et al (2014) 1-Gen Mice Study

 

A one-generation oral study in mice included in the immunotoxicity (developmental) study published byOsterburg et al (2014).Parental mice were dosed with0, 2, 62.5, 125, or 200 mgsodiumtungstate /kg bw/d. Both P and F1 micewere maintained on these doses for the course of the study. Thesequantities of tungstate were selected based onprevious workbyMcInturf et al(2011) that used similar doses in rats.

Mice were exposed to tungstate for90-days prior to mating (Weeks 1–12). The next 7 weeks comprisedgestation and weaning (Weeks 13–19). After pups (F1)were weaned, the parents (P) were necropsied, approximately19 weeks after initiation of the study. The F1 generation wasexposed to tungstate for a further 90-days after weaning and thennecropsied. Mice were housed singly during the course of thestudy and pair mated for breeding. After confirmation ofpregnancy, males were removed. During all phases of the one-genstudy mice were kept on the appropriate tungstate dose(Osterburg et al 2014).

Results showed nostatistically significant changes in bodyweight due to any tungstate dose levels. The 200 mg/kg bw/d males in the P generation show a consistenttrend towards decreased weights. This observation,however, was not statistically significant. Additionally,nostatistically significant changes in the number of livebirths, litter size, or sex ratio at any dose of tungstate tested(Osterburg et al. 2014).

c.    McInturft et al (2007, 2008, & 2011) Reproductive Screening Rat Oral Study

 

A study conducted following EPA OPPTS 870.3650 – Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test evaluated the reproductive, systemic and developmental effects of sodium tungstate in rats following 70 days of daily pre-and postnatal exposure via oral gavage to 5, 62.5 and 125 mg/kg/d through mating, gestation and weaning (PND 0 -20). The results of this study were reported in three separate publications and an unpublished summary report (McInturf et al., 2007, 2008 & 2011).

 

It is important to mention that preliminary results were presented on a Scientific Poster at the Society of Toxicology in 2006, showed that initially the study design included a 250 mg/kg bw/d dose group but not the 62.5 mg/kg/d group(Johnson et al 2006). Although for unknown reasons the results of this dose group were never officially published outside of this scientific poster.

 

Briefly, female and male Sprague-Dawley rats were orally dosed with 250 mg/kg bw/d, 125 mg/kg bw/d ay or 5 mg/kg bw/d ay of sodium tungstate or dH2O (n=40/sex/group) for 70 consecutive days. The rats were mated after 14 days and dosing continued through pregnancy up to post-natal day (PND) 21. The gestational effects of oral sodium tungstate as well as early growth and development of the offspring were measured.

 

Sodium tungstate exposure (250 mg/kg bw/d) significantly decreased body-weight gain in the P0males and gestational weight gain (about 20% decrease) as well as increasing gestational length (1.2 days) in the dams. At 250 mg/kg bw/d the litter size and the average weight per pup decreased, however the effect was not significant. No clinical signs or effects on pup viability were observed(Johnson et al 2006).

 

Gestation lengths (22.08 ± 0.089) in days for the 125-mg/kg/d group were significantly different (n > 37) from controls (21.548 ± 0.097) without affecting average gestational weight in adults and offspring, and average litter sizes. However, this effect is not considered to be toxicologically significant as the gestation length in the 125-mg/kg/d dose group did not have effects on average gestational weight gain across treatments, and in the pups, there were no differences in average number of pups born (McInturf et al., 2007, 2008 & 2011).

 

No marked effects on pup survival, M: F ratio, litter size, or clinical signs were observed in the F1 litters. No significant treatment-related effects were reported on the gestational weight gain in the dams, number of pups born, or physical birth defects. Based on the lack of toxicologically significant effects directly attributable to Na2WO4, the NOAEL for reproductive toxicity was 125 mg Na2WO4/kg/d (McInturf et al., 2008 & 2011).

 

d.    US NTP Rat Perinatal Oral Study

 

The study design included drinking water doses of0, 125, 250, 500, 1000, or 2000 mg/L (an estimated oral dose between 10-180 mg/kg bw/d for rats).The in-life study phase has been completed but no study report has been issued. Currently, preliminary results contained in graphs and tables are available on theUS NTP website. Furthermore, at the2012 Annual Meeting of theSociety of Toxicology, a Scientific Posterwas presented detailing the following preliminary results showed:

·      No treatment related effects on the percentage of dams delivering, litter size, or litter weights.

·      Reduced body weights greater than 10% relative to control were observed in 2000 mg/L dams, PND 4 pups, PND 21 pups, and adult rats.

 

e.    McCain 90-day Repeated Exposure Oral Study

 

No effects on the male or female reproductive organs were observed following 90-d repeat exposure to sodium tungstate via the oral route in rats (Biotechnics, 2009).

 

Fertility/Reproductive WoE Conclusions:

 

The overall fertility reproductive parameters measured were unchanged in mice and rats after sodium tungstate exposure.No marked effects on pup survival, M: F ratio, litter size, or clinical signs were observed in the F1 rat litters. No significant treatment-related effects were reported on the gestational weight gain in the dams, number of pups born, or physical birth defects. Based on the lack of toxicologically significant effects directly attributable to sodium tungstate, the NOAEL for reproductive toxicity was 125 mg Na2WO4/kg bw/d (McInturf et al, 2008 & 2011). Based on the current WoE, it is not expected that sodium tungstate is a fertility/reproductive toxicant.

Link to relevant study records

Referenceopen allclose all

Endpoint:
fertility, other
Remarks:
based on test type (migrated information)
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Study conducted only on female rats. Reproductive toxicity indicators included estrogen, progesterone and prolactin receptors, luteinizing hormone, progesterone, and follicle-stimulating hormone serum concentrations.
Justification for type of information:
REPORTING FORMAT FOR THE CATEGORY APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH: The hypothesis is that properties are likely to be similar or follow a similar pattern because of the presence of a common metal ion, in this case tungstate.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES):
Source: Sodium tungstate
Target: Tungsten Dioxide
3. CATEGORY APPROACH JUSTIFICATION: See Annex 1 in CSR
4. DATA MATRIX: See Annex 1 in CSR
Reason / purpose:
read-across: supporting information
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
GLP compliance:
not specified
Limit test:
yes
Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
Adult female Wistar rats (200 g) were kept under a constant 12-hour light-dark cycle and were allowed to eat and drink ad libitum.
Route of administration:
oral: drinking water
Vehicle:
other: 0.9% NaCl
Details on exposure:
The treatment was carried out for 12-weeks. During this period, glycemia and body weight were measured regularly.
Details on mating procedure:
To evaluate reproductive performance, after 10 weeks of treatment, individual females were placed in a cage with one healthy adult male (body weight: 250 g). The animals were kept together overnight, and then separated the following morning. Immediately after each separation, a vaginal examination and scrape were carried out to determine if sexual intercourse had occurred. When intercourse was positive (presence of a vaginal tap and/or spermatozoa), the night/day routine was discontinued and the female was housed individually during the estimated period of gestation. When females showed no sexual contact with the male during 9 consecutive days, they were not used for further mating and were labelled ‘unable’, whereas those that showed sexual activity were labelled ‘able’.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
12-weeks
Frequency of treatment:
Daily via drinking water
Dose / conc.:
2 000 mg/L drinking water
Remarks:

nominal in water
No. of animals per sex per dose:
24 female rats per group.
Control animals:
yes, concurrent vehicle
Details on study design:
All the animals were anesthetized with diethyl ether and sacrificed by decapitation.
Parental animals: Observations and examinations:
- Histological analyses of ovaries
- Wester blot analyses for estrogen receptor, progesterone receptor, insulin receptor and FSH receptor.
Litter observations:
At parturition, litter size was determined
Postmortem examinations (parental animals):
Blood was collected immediately to measure serum parameters. All the animals were then anesthetized with diethyl ether and killed by decapitation. Ovaries horns were prepared in two ways. First, part of the tissues were immediately fixed in 3% formaldehyde in a buffer solution containing 54 mM NaH2PO4 and 28 mM Na2HPO4 (pH 7.4) at 48C (buffered formaldehyde). This material can be stored for up to 5 weeks. These tissues were used for optical microscope histology. The rest of the tissues were immediately frozen in liquid N2 and stored at 2908C until Western blot and semi-quantitative RT–PCR analyses were performed.
Statistics:
All values are means + SEM. Statistical comparisons of the means were performed by analysis of variance, followed by the Student– Neumann–Keuls test. This test makes multiple pairwise comparisons, where the cut-off point to which comparisons are made changes between them. Thus, this methodology shows greater sensitivity than other similar multiple pairwise comparison tests, such as the Student’s t-test. A P-value of (0.05 was considered to be statistically significant.
Reproductive indices:
The reproductive performance of the rats was measured as the proportion of ‘able’ females to the total number tested (the percentage of positive vaginal scrapes, i.e. those with presence of spermatozoa, with respect to the total number of vaginal scrapes performed in one experimental group). This parameter was named as ‘mating index’. The number of vaginal scrapes varied depending on the time required by animals to show positive intercourse. Furthermore, we also calculated the percentage of parturitions with respect to the number of positive scrapes. This parameter was named ‘fertility’. These definitions do not necessarily coincide with the common definitions for mating index, fertility and prolificacy published elsewhere.
Offspring viability indices:
The mean litter size was also calculated and was defined as ‘prolificacy’. These definitions do not necessarily coincide with the common definitions for mating index, fertility and prolificacy published elsewhere.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Description (incidence):
At the end of the experiment, all the rats in the tugstate treated healthy (TH) rats were alive.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Tungstate treatment also caused a decrease in the body weight gain of healthy rats
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Tungstate treatment did not modify daily food intake in rats.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Tungstate treatment did not modify daily water consumption in healthy rats
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Tungstate treatment did not modify serum levels of glucose, ALT activity, insulin, progesterone, FSH and LH in healthy rats
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Histological analyses of ovaries did not show evident differences between groups. Thus, active ovaries showing evolutive follicles, corpora lutea and follicular glands were detected in all groups. Ovaries were generally covered by a significant amount of adipose tissue.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
- Tungstate did not significantly affect LH receptor mRNA content in healthy rats
- Tungstate slightly decreases western blot band intensity of GLUT 3 hexose expression in ovaries of healthy rats.
- No differences in the expression of the estrogen receptor in tungstate treated healthy rats.
- Tungstate does not affect the amount of ovarian prolactin receptor mRNA.
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Description (incidence and severity):
Tungstate treatment did not modify Leydig cell function in healthy rats
Reproductive performance:
no effects observed
Description (incidence and severity):
Tungstate administration did not alter the reproductive performance of healthy female rats.
- All the rats in the conrol and tretated tungstate group survived.
- Tungstate exposed rats have lower body weight gain than control animals.
- Tungsten treatment had no effect on serum glucose, insulin, alanine aminotransferase, luteinizing hormone, progesterone, and follicle-stimulating hormone concentrations.
- Tungstate treatment did not affect any reproductive parameter in healthy rats.
- Tungsten treatment does not affect the expression of estrogen, progesterone and prolactin receptors as determined by western blotting.
Key result
Dose descriptor:
NOAEL
Effect level:
> 2 000 mg/L drinking water
Based on:
test mat.
Sex:
female
Basis for effect level:
reproductive performance
Clinical signs:
not examined
Mortality / viability:
not examined
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Tungsten treatment did not alter prolifacy (litter size)
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Reproductive effects observed:
no
Conclusions:
Oral administration (2 mg/ml in drinking water) of sodium tungstate to adult female for 12 weeks did not show hypoglycemia, did not alter the reproductive performance of healthy females or any alteration in estrogen, progesterone and prolactin receptors. In adddition, tungsten treatment had no effect on luteinizing hormone, progesterone, and follicle-stimulating hormone serum concentrations. Tungstate orally did not alter the reproductive performance of healthy females.
Executive summary:

No fertility, reproductive, or developmental toxicity data of sufficient quality are available for tungsten dioxide (target substance). However, reproductive toxicity data are available for sodium tungstate (source substance), which will be used for reading across. Due to lower water solubility and lower toxicity for the target substance compared to the source substance, the resulting read across from the source substance to the target substance is appropriate as a conservative estimate of potential toxicity for this endpoint. In addition, read across is appropriate because the classification and labelling is more protective for the source substance than the target substance, the PBT/vPvB profile is the same, and the dose descriptors are, or are expected to be, lower for the source substance. For more details, refer to the read-across category approach included in the Category section of this IUCLID submission and/or as an Annex in the CSR.

Endpoint:
fertility, other
Remarks:
based on test type (migrated information)
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Study conducted only on male rats. Reproductive toxicity indicators included Leydig cell density and morphology, serum testosterone, luteinizing hormone, follicle-stimulating hormone, and percent of able males.
Justification for type of information:
REPORTING FORMAT FOR THE CATEGORY APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH: The hypothesis is that properties are likely to be similar or follow a similar pattern because of the presence of a common metal ion, in this case tungstate.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES):
Source: Sodium tungstate
Target: Tungsten Dioxide
3. CATEGORY APPROACH JUSTIFICATION: See Annex 1 in CSR
4. DATA MATRIX: See Annex 1 in CSR
Reason / purpose:
read-across: supporting information
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
GLP compliance:
not specified
Limit test:
yes
Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
Adult male Wistar rats (200 g) were kept under a constant 12-hour light-dark cycle and were allowed to eat and drink ad libitum.
Route of administration:
oral: drinking water
Vehicle:
other: 0.9% NaCl
Details on exposure:
The treatment was carried out for 3 months. During this period, glycemia and body weight were measured regularly. The time period of 3 months was chosen to allow tungstate to exert a complete effect on testicular function.
Details on mating procedure:
To evaluate reproductive performance, after 10 weeks of treatment, individual males were placed in a cage with 1 healthy adult female (body weight: 250 g). The animals were kept together overnight, and the following morning they were separated. Immediately after separation, a vaginal examination and scrape were performed to determine whether sexual intercourse had occurred. When intercourse was positive (presence of a vaginal tap with or without spermatozoa), the night routine was discontinued. When the male showed no sexual contact with the female during 9 consecutive days, this individual was not used for further mating and was termed ‘‘unable,’’ while those that showed sexual activity, as determined above, were termed ‘‘able.’’
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
3 months (time period was chosen to allow tungstate to exert complete effect on testicular function)
Frequency of treatment:
Daily via drinking water
Dose / conc.:
2 000 mg/L drinking water
Remarks:
Nominal in water
No. of animals per sex per dose:
12 male rats
Control animals:
yes, concurrent vehicle
Details on study design:
All the animals were anesthetized with diethyl ether and sacrificed by decapitation.
Parental animals: Observations and examinations:
Gycemia and body weights measured regularly
Sperm parameters (parental animals):
Time period was chosen to allow tungstate to exert complete effect on testicular function
Postmortem examinations (parental animals):
Blood was collected immediately to measure serum parameters. Testes were prepared in 3 ways: 1) Tissues were immediately fixed in 3% formaldehyde in a buffered solution containing 54 mM NaH2PO4 and 28 mM Na2HPO4 (pH 7.4) at 48C (buffered formaldehyde). This material can be stored for up to 5 weeks. These tissues were used for optical microscope histology and immunohistochemistry; 2) Tissues were immediately fixed in 2.5% glutaraldehyde in ice-cold 0.1 M sodium cacodylate buffer (pH 7.4). These tissues were used for transmission electronic microscope histology; and 3) Tissues were immediately frozen in liquid N2 and store at 290C until Western blot analyses were performed.
Reproductive indices:
The reproductive performance of the male rats was determined by calculating the percentage of ‘‘able’’ males out of the total number tested.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Description (incidence):
All the rats in the tungstate healthy rat group were alive
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight decease in treated animals
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Body weight decease in treated animals
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Tungstate administration to healthy rats did not modifiy the apperance or number of Leydig cells
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
To check whether tungstate has a direct effect on Leydig cells, the effects on the mLTC-1 cell line were evaluated, which is derived from Leydig cells. For this purpose, the effect of tungstate on the phosphorylation state of MAP-kinase and GSK-3, 2 were evaluated. Incubation with 1 mM tungstate induced a clear increase in the phosphorylation signal of MAP-kinase of these cells, which reached a maximum after 10 minutes of incubation. No effect of 1 mM tungstate was observed on the levels of total MAP-kinase. Incubation with 1 mM tungstate caused a significant increase in the GSK-3 phosphorylation state. No effects of tungstate were observed on the total amount of GSK-3.
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Tungstate treatment did not modify Leydig cell function in healthy rats
Reproductive performance:
no effects observed
Description (incidence and severity):
Tungstate administration did not alter the reproductive performance of healthy animals
- All the rats in the conrol and tretated tungstate group survived.
- Tungstate exposed rats have lower body weight gain than control animals.
- Tungstate administration to healthy rats did not modify the appearance or number of Leydig cells
- Administration of tungstate to healthy rats did not modify the expression of insulin receptor.
- To check whether tungstate has a direct effect on Leydig cells, the effects on the mLTC-1 cell line were evaluated, which is derived from Leydig cells. For this purpose, the effect of tungstate on the phosphorylation state of MAP-kinase and GSK-3, 2 were evaluated. Incubation with 1 mM tungstate induced a clear increase in the phosphorylation signal of MAP-kinase of these cells, which reached a maximum after 10 minutes of incubation. No effect of 1 mM tungstate was observed on the levels of total MAP-kinase. Incubation with 1 mM tungstate caused a significant increase in the GSK-3 phosphorylation state. No effects of tungstate were observed on the total amount of GSK-3.
- Tungsten treatment had no effect on serum glucose, insulin, luteinizing hormone, and follicle-stimulating hormone concentrations.
.
Key result
Dose descriptor:
NOAEL
Effect level:
> 2 000 mg/L drinking water
Based on:
test mat.
Sex:
male
Basis for effect level:
reproductive performance
Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
not examined
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Reproductive effects observed:
no
Conclusions:
Oral administration (2 mg/ml in drinking water) of sodium tungstate to adult male rats for 3 months did not show hypoglycemia, did not alter the reproductive performance of healthy males or any alteration in Leydig cell function, as shown by serum testosterone levels.. Tungstate orally exposed rats did not alter the reproductive performance of healthy males or any alteration in Leydig cell function, as shown by serum testosterone levels.
Executive summary:

No fertility, reproductive, or developmental toxicity data of sufficient quality are available for tungsten dioxide (target substance). However, reproductive toxicity data are available for sodium tungstate (source substance), which will be used for reading across. Due to lower water solubility and lower toxicity for the target substance compared to the source substance, the resulting read across from the source substance to the target substance is appropriate as a conservative estimate of potential toxicity for this endpoint. In addition, read across is appropriate because the classification and labelling is more protective for the source substance than the target substance, the PBT/vPvB profile is the same, and the dose descriptors are, or are expected to be, lower for the source substance. For more details, refer to the read-across category approach included in the Category section of this IUCLID submission and/or as an Annex in the CSR.

Endpoint:
one-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Well documented scientfically sound study similar to OECD guidelines with sufficient information provided on materials and methods to evaluate results. However as this study is used in the context of a read across, Klimisch 2 is assigned.
Justification for type of information:
REPORTING FORMAT FOR THE CATEGORY APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH: The hypothesis is that properties are likely to be similar or follow a similar pattern because of the presence of a common metal ion, in this case tungstate.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES):
Source: Sodium tungstate
Target: Tungsten Dioxide
3. CATEGORY APPROACH JUSTIFICATION: See Annex 1 in CSR
4. DATA MATRIX: See Annex 1 in CSR
Reason / purpose:
read-across: supporting information
Qualifier:
equivalent or similar to
Guideline:
other: EPA OPPTS 870.3650 "Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Study
Deviations:
yes
Remarks:
The protocol was extended beyond the minimum 54 days of treatment to accomodate evaluation of the development of the offspring through postnatal day 20 as well as their dams following the last dose on day 70.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories (Wilmington, MA)
- Age at study initiation: 8 weeks
- Housing: The adults (e.g., P1) were singly housed (except during mating)
- Acclimation period: 14 days
Route of administration:
oral: gavage
Vehicle:
other: deionized water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The powder readily dissolved in a deionized water (diH2O) vehicle for the concentrations used in this study at 5, 62.5 and 125 mg/mL. The solution was concentrated to administer a volume of 1 mL/kg body weight not to exceed 2 mL. Fresh solution was made daily and administered via oral gavage


VEHICLE
- Justification for use and choice of vehicle (if other than water): no data
- Concentration in vehicle: no data
- Amount of vehicle (if gavage): no data
- Lot/batch no. (if required): no data
- Purity: no data
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: not specified
- Length of cohabitation: 14 days
- Verification of same strain and source of both sexes: not specified
- Proof of pregnancy: vaginal plug referred to as day 1 of pregnancy
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
70 days
Frequency of treatment:
Daily pre- and postnatal exposure
Details on study schedule:
Day 13: 24 hr usine/feces
Day 14: Mating begins
Day 28: Mating ends
Day 70: Last dosing
Dose / conc.:
5 mg/kg bw/day (actual dose received)
Dose / conc.:
62.5 mg/kg bw/day (actual dose received)
Dose / conc.:
125 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
40 rats per sex per treatment group
Control animals:
yes, concurrent vehicle
Details on study design:
Dosing continued for 70 days and encompassed mating following 14 days of treatment, that continued through the 14 day mating period, the 22 day gestational period, and through to postnatal day (PND)20. In order to ensure a total of 70 day exposure, adults were dosed for any additional days necessary following weaning. Prenatal pup exposure was from sodium tungstate crossing through the placenta and postnatal exposure was indirect via dams' milk. Offspring (F1 generation) were monitored until PND70. On PND1 (day of parturition=PND 0), the number of pups in each litter was recorded. Litters were culled on PND4 to eight pups, and litters were weighed. Selection of pups was pseudo-random to maintain a 4 male/4 female ratio whenever possible, and all pups remained with their biological mothers.
Parental animals: Observations and examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Throughout the exposure period, dams and the males used for mating were observed for clinical signs of toxicity.

BODY WEIGHT: Yes
- Time schedule for examinations: Pregnant dams were weighed on gestation days (GD) 1, 10, 15 and 20, and gestational weight gain and gestation length were recorded.

Various organ tissues (heart, spleen, kidney, liver, lungs, brain, testes, ovaries, thymus, bone, gastrointestinal tract, etc,) were carefully removed and postfixed in 4% paraformaldehyde no longer than 24 h. After dehydration, tissues embedded in paraffin and 40 μm coronal sections were made on glass slides for histological evaluation using hematoxylin and eosin.
Litter observations:
PND 0-4: SIze of Litter (#males/#females), weight of litter, and gross abnormalities
PND 4: Liiter culled to 8 pups (4 males and 4 females)
PND 5: Individual pups weight starting on PND 5 and weekly therefe
Postmortem examinations (parental animals):
Tungsten concentrations in brain tissue were measured in adults and pups for the control and highest dose groups only. Ten male and female adults from each dose group were necropsied on the day of the last dose were necropsied on PND20 and PND70. For all necropsies, animals were anesthetized via CO2 overdose followed by exsanguination via the vena cava. Concentrations of the tungstate anion were determined using inductively coupled plasma mass spectrometry (ICP-MS).

Various organ tissues (heart, spleen, kidney, liver, lungs, brain, testes, ovaries, thymus, bone, gastrointestinal tract, etc,) were carefully removed and postfixed in 4% paraformaldehyde no longer than 24 h. After dehydration, tissues embedded in paraffin and 40 μm coronal sections were made on glass slides for histological evaluation using hematoxylin and eosin (H&E) stain.
Postmortem examinations (offspring):
Tungsten concentrations in brain tissue were measured in pups for the control and highest dose groups only. Ten male and female adults from each dose group were necropsied on the day of the last dose were necropsied on PND20 and PND70. For all necropsies, animals were anesthetized via CO2 overdose followed by exsanguination via the vena cava. Concentrations of the tungstate anion were determined using inductively coupled plasma mass spectrometry (ICP-MS).
Statistics:
All statistical testing was performed using ANOVA with repeated measures factors. Tukey'svHSD analysis was used to evaluate pair-wise comparisons.
Clinical signs:
no effects observed
Description (incidence and severity):
Animals were observed for clinical signs of toxicity throughout the exposure period, although none were noted. Additionally, no deaths were recorded for the adult females or males at the doses tested.
Mortality:
no mortality observed
Description (incidence):
No deaths were recorded for the adult females or males at the doses tested
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Sodium tungstate treatments did not have an effect on average gestational weight gain in adults
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Sodium tungstate treatments did not have an effect on average gestational weight gain in adults
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):
effects observed, treatment-related
Description (incidence and severity):
1) A significant effect of tungstate exposure on spontaneous locomotor activity was detected especially with low dose treated dams. Compared to control and the high dose treated animals, the low dose treated dams spent more time on ambulatory time and distance traveled and less time in stereotypies. On the contrary, in high dose treated dams less time resting and more time in stereotypic movements than the controls or low dose group were markedly observed. Maternal retrieval test revealed that sodium tungstate exposure to dams had no effect on latency in both treatment groups. In the dams, opposing exposure effects were observed in the low and high dose groups related to locomotor activity and exploration.

2) On PND 20, the mean tungstate ion concentrations in males and females of the 125 mg/kg/day treatment group were 0.008 ± 0.006 ppm and 0.002±0.002 ppm, respectively. No tungstate ion was detected in the males or females of the control group. Furthermore, no tungstate ion was detected in the brains of males or females in the control or high dose group on PND 70.

3) Tungstate ion concentrations in dam milk secretions measured PND10–14 were significantly greater in the 125 mg/kg/day treatment group (0.45 ± 0.007 ppm) than in the low dose group (0.021 ± 0.001 ppm) or controls (0.005±0.0 ppm). Concentrations in the low dose group did not significantly differ from controls.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
The heart showed histological lesions, histiocytic inflammation from minimal to mild with cardiomyocyte degeneration and necrosis in several P0 animals of 125 mg NW dose group
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathological examination showed no severe chronic injury in the organs tested. However, the heart showed histological lesions, histiocytic inflammation from minimal to mild with cardiomyocyte degeneration and necrosis in several P0 animals of 125 mg NaW dose group.
Histopathological findings: neoplastic:
not specified
Other effects:
effects observed, treatment-related
Description (incidence and severity):
- The study results indicate that a significant difference was found for one of the treatment groups on measures of reproductive toxicity. In the dams, particularly 125 mg dose group animals showed longer gestational period when compared to control, 5 and 62.5 mg groups, but did not have an effect on average gestational weight gain in adults and offspring.
- Tungstate ion concentrations in the male and female adult and pup brains after sodium tungstate exposure were significantly greater in the high dose (125 mg) treated rats than control. Similarly, in dam milk secretions tungstate ion concentrations was significantly greater in the 125 mg treatment group than in the low dose group or controls. Further, we also observed increased concentration of tungstate ion distribution in other major organs like heart, spleen, kidney, thymus, testes, lungs, liver, femur bone and gastrointestinal regions in both male and female treated adult and pups.
Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed
Description (incidence and severity):
Daily administration of sodium tungstate produced no overt evidence of toxicity and had no apparent effect on mating success
- Tungstate ion concentrations in the male and female adult rats after sodium tungstate exposure were significantly greater in the high dose (125 mg) treated rats than control
- Further, we also observed increased concentration of tungstate ion distribution in other major organs like heart, spleen, kidney, thymus, testes, lungs, liver, femur bone and gastrointestinal regions in both male and female treated adult rats.
- Histopathological examination showed no severe chronic injury in the organs tested.
Key result
Dose descriptor:
NOAEL
Effect level:
125 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
gross pathology
histopathology: non-neoplastic
reproductive performance
Key result
Dose descriptor:
LOEL
Effect level:
125 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
NOAEL
Effect level:
125 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Behaviour
Key result
Dose descriptor:
LOAEL
Effect level:
125 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Critical effects observed:
yes
Lowest effective dose / conc.:
125 mg/kg bw/day (actual dose received)
System:
cardiovascular
Organ:
heart
Treatment related:
yes
Dose response relationship:
no
Relevant for humans:
not specified
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Sodium tungstate treatments did not have an effect on average gestational weight gain in adults and offspring
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 examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
Daily administration of sodium tungstate produced no overt evidence of toxicity on offspring physical development.
Histopathological findings:
no effects observed
Description (incidence and severity):
Daily administration of sodium tungstate produced no overt evidence of toxicity on offspring physical development and no significant test article-related lesions
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Tungstate ion concentrations in the pup brains after sodium tungstate exposure were significantly greater in the high dose (125 mg) treated rats than control. Similarly, in dam milk secretions tungstate ion concentrations was significantly greater in the 125 mg treatment group than in the low dose group or controls. Further, we also observed increased concentration of tungstate ion distribution in other major organs like heart, spleen, kidney, thymus, testes, lungs, liver, femur bone and gastrointestinal regions in pups.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
For righting reflex, a significant sex effect was found on male where males were faster than females. However, there was no dose related effect on this activity (Fig. 3). Further, on ultrasonic distress vocalization test, pups showed dose-related effects during 60 s time period. The high dose treated pups exhibited more vocalization than control and 5 mg groups
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 125 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
clinical signs
mortality
body weight and weight gain
gross pathology
histopathology: non-neoplastic
Key result
Generation:
F1
Based on:
test mat.
Sex:
male/female
Basis for effect level:
developmental neurotoxicity
Remarks on result:
not determinable because of methodological limitations
Key result
Reproductive effects observed:
no
Conclusions:
Daily administration of sodium tungstate produced no overt evidence of toxicity and had no apparent effect on mating success, and did not cause a change in gestation length and weight gain in adult females. No significant test article related histopathological findings were observed in the F1 treatment groups. The 2011 publication mentions the investigation of neurobehavioral findings only in the control, low and high dose groups but not from the mid dose (only body weight gain, tungsten organ distribution data and histopathology) for the mid dose group animals are reported. The neurobehavioral results in the 2008 publication resemble the results reported in the 2011 publication. However, the 2011 paper does not quote the 2008 publication of the same group. This causes some doubt on the study design.

Executive summary:

No fertility, reproductive, or developmental toxicity data of sufficient quality are available for tungsten dioxide (target substance). However, reproductive toxicity data are available for sodium tungstate (source substance), which will be used for reading across. Due to lower water solubility and lower toxicity for the target substance compared to the source substance, the resulting read across from the source substance to the target substance is appropriate as a conservative estimate of potential toxicity for this endpoint. In addition, read across is appropriate because the classification and labelling is more protective for the source substance than the target substance, the PBT/vPvB profile is the same, and the dose descriptors are, or are expected to be, lower for the source substance. For more details, refer to the read-across category approach included in the Category section of this IUCLID submission and/or as an Annex in the CSR.

Endpoint:
one-generation reproductive toxicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
REPORTING FORMAT FOR THE CATEGORY APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH: The hypothesis is that properties are likely to be similar or follow a similar pattern because of the presence of a common metal ion, in this case tungstate.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES):
Source: Sodium tungstate
Target: Tungsten Dioxide
3. CATEGORY APPROACH JUSTIFICATION: See Annex 1 in CSR
4. DATA MATRIX: See Annex 1 in CSR
Reason / purpose:
read-across: supporting information
Qualifier:
according to
Guideline:
other: USEPA Guideline OPPTS 870.3650
Principles of method if other than guideline:
This study examined the consequences of tungstate exposure on the immune response of mice after activation of the immune system with either Staphylococcal enterotoxin B (SEB) or in a delayed-type hypersensitivity (DTH) Type IV model. The study here investigated the effects of tungstate in a 28-day and a one-gen exposure to determine acute as well as longer-term exposure toxicities.. Experiments were then conducted as described in Current Protocols in Immunology (Luo & Dorf, 2001)
GLP compliance:
not specified
Limit test:
no
Justification for study design:
Support for read-across from water-soluble tungsten compounds to ammonium metatungstate (AMT) is based on consideration of structural similarities, physicochemical properties, release of ionic tungsten species, and/or toxicity data for AMT, ammonium paratungstate (APT), sodium metatungstate, and sodium tungstate. This approach is applicable for all systemic effect endpoints for all routes of exposure (more imformation is available in Annex 1 of the CSR attached in Section 13)
Species:
mouse
Strain:
C57BL
Details on species / strain selection:
c57BL6
Sex:
male/female
Details on test animals and environmental conditions:
C57BL6 (28-day study ¼ 8–12-week-old, 19–22 g; one-gen study=8–12-week-old, 15–18 g) mice were obtained from Charles River Laboratories (Wilmington, MA). Animals were housed in an Association for Assessment and Accreditation of Laboratory
Animal Care (AAALAC) approved facility under animal protocols approved by the Wright-Patterson Air Force Base and/or University of Cincinnati Institutional Animal Care and Use Committee (IACUC). Mice were allowed to acclimatize to the animal facility for 7 days before commencement of the experimental phase. During the course of the experiment, animals had a 12 h day/night cycle in a temperature-controlled room (22 C). All animals had ad libitum access to filtered water and a low molybdenum diet; CA.170481 AIN-76 A Purified Diet
Route of administration:
oral: drinking water
Vehicle:
water
Details on exposure:
Individual mice were identified by ear punch. Body weights were recorded weekly and quantities of tungstate adjusted appropriately in water bottles Water consumption was determined using graduated water bottles. Measurements were made daily of water consumed by mice. The tungstate in the water bottles was administered to calculate approximate ingested doses of tungstate based on an estimated water consumption of 4.5 ml/mouse/day. In the one-gen study parental mice were dosed with 0, 2, 62.5, 125, or 200 mg/kg/day of tungstate.
Details on mating procedure:
Mice were housed singly during the course of the study and pair mated for breeding. After confirmation of pregnancy, males were removed
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
Both P and F1 mice were maintained on these doses for the course of the study. Water bottles were changed 2–3 times weekly, always using water from the source and a 1 M sodium tungstate stock supply. In the one-gen exposure, mice were exposed to tungstate for 90 days prior to mating (Weeks 1–12). The next 7 weeks comprised gestation and weaning (Weeks 13–19). After pups (F1) were weaned, the parents (P) were necropsied as described, &19 weeks after initiation of the study. The F1 generation was exposed to tungstate for a further 90 days after weaning and then necropsied.
Dose / conc.:
0.2 mg/kg bw/day (nominal)
Dose / conc.:
62.5 mg/kg bw/day (nominal)
Dose / conc.:
125 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
Control animals:
yes, concurrent vehicle
Parental animals: Observations and examinations:
After euthanasia, splenocytes and blood were collected and stained with lymphocyte and/or myeloid immunophenotyping panels and analyzed by flow cytometry. I
Statistics:
All statistical tests were performed with Systat (v11 or v13, Systat Software Inc., Chicago, IL). Statistical significance was assumedat p<0.05. Comparisons between treatments (tungstate dose, immune challenge) were performed as two-way analysis of variance (ANOVA). Differences in weights were determined by repeated measures analysis of variance (RM-ANOVA). When assumptions of normality and/or equal variances were violated, non-parametric Kruskal-Wallis tests were performed. Specific post-hoc tests were performed and multiple test corrections performed when appropriate.
Reproductive indices:
Litter size, and sex ratio
Offspring viability indices:
Live births
Clinical signs:
not specified
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No statistically significant changes in body weight due to any tungstate dose levels in one-gen study. The 200 mg/kg/day males in the P generation show a consistent trend towards decreased weights. This observation, however, was not statistically significant.
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
There were no significant changes in water consumption due to the quantity of tungstate present in the water.
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
There were no significant changes in any of the parameters measured in response to tungstate, with the exception of the percent monocytes (%MO). There were fewer lymphocytes in the F1 generation compared to the P generation (p<0.023), but this was not dose-related. Additionally, the red blood cell distribution width (RDW) was higher in the P generation vs the F1 pups (p<0.004). The percentage of monocytes was dose-dependently lower at higher concentrations when compared to control (p<0.003). Other parameters suggest a dose-dependent trend (e.g. hematocrit); however, these trends were not statistically significant.
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
effects observed, treatment-related
Description (incidence and severity):
- The one-gen exposures resulted in reduced quantities of CD71þ TH cells in the parental mice, 6.21 0.39% for the controls and 2.28+/-0.41% for the 200 mg/kg/day dose group. The F1 controls in the SEB-treated groups were 7.20+/-0.76% and 2.85+/-0.53% for the 200 mg/kg/day tungstate groups (p<0.001). No statistically significant differences were noted in the overall quantity of CD3+ CD4+ TH cells. Additionally, there were no significant effects of generation on the suppression of CD71+TH cells in SEB-treated mice exposed to tungstate.
- The cytotoxic CD3+ CD8þ CD71+ cells in the parental mice were 7.98+/-0.49% in the 0 mg/kg/day SEB-challenged groups and 1.58+/-0.23% in the corresponding 200 mg/kg/day group. The F1 mice were 6.33+/-0.49% for the controls and 2.52+/-0.25% in the 200 mg/kg/day tungstate group (p<0.001).
- Interferon gamma in the P mice of the one-gen exposure had a reduction from the control value of 7.11 1.7 to 3.74 2.9 pg/ml (p<0.001). Although not statistically significant, the F1 mice had an overall reduced IFNg response, especially at the 62.5 mg/kg/day dose, compared to their parents
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed
Description (incidence and severity):
No observed statistically significant changes in the number of live births, litter size, or sex ratio at any dose of tungstate tested.
Key result
Dose descriptor:
NOEL
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Key result
Dose descriptor:
NOEL
Effect level:
125 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Immunological: reduction in the quantity of CD71+ helper and cytotoxic T-cells present in the high-dose tungstate exposure groups (200 mg/kg/day) when challenged with SEB
Key result
Dose descriptor:
LOEL
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: immunological: reduction in the quantity of CD71+ helper and cytotoxic T-cells present in the high-dose tungstate exposure groups (200 mg/kg/day) when challenged (co-exposure) with SEB, and this efect cannot be attributed to tungstate only.
Clinical signs:
not specified
Mortality / viability:
no mortality observed
Description (incidence and severity):
No observed statistically significant changes in the number of live births
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No statistically significant changes in body weight due to any tungstate dose levels in one-gen study. The 200 mg/kg/day males in the P generation show a consistent trend towards decreased weights. This observation, however, was not statistically significant.
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
There were no significant changes in any of the parameters measured in response to tungstate, with the exception of the percent monocytes (%MO). There were fewer lymphocytes in the F1 generation compared to the P generation (p<0.023), but this was not dose-related. Additionally, the red blood cell distribution width (RDW) was higher in the P generation vs the F1 pups (p<0.004). The percentage of monocytes was dose-dependently lower at higher concentrations when compared to control (p<0.003). Other parameters suggest a dose-dependent trend (e.g. hematocrit); however, these trends were not statistically significant.
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Histopathological findings:
not specified
Other effects:
no effects observed
Description (incidence and severity):
Not observed statistically significant changes in the number of llitter size, or sex ratio at any dose of tungstate tested.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
effects observed, treatment-related
Description (incidence and severity):
- The one-gen exposures resulted in reduced quantities of CD71+ TH cells in the parental mice, 6.21 0.39% for the controls and 2.28+/-0.41% for the 200 mg/kg/day dose group. The F1 controls in the SEB-treated groups were 7.20+/-0.76% and 2.85+/-0.53% for the 200 mg/kg/day tungstate groups (p<0.001). No statistically significant differences were noted in the overall quantity of CD3+ CD4+ TH cells. Additionally, there were no significant effects of generation on the suppression of CD71+TH cells in SEB-treated mice exposed to tungstate.
- The cytotoxic CD3+ CD8+ CD71+ cells in the parental mice were 7.98+/-0.49% in the 0 mg/kg/day SEB-challenged groups and 1.58+/-0.23% in the corresponding 200 mg/kg/day group. The F1 mice were 6.33+/-0.49% for the controls and 2.52+/-0.25% in the 200 mg/kg/day tungstate group (p<0.001).
- Interferon gamma in the P mice of the one-gen exposure had a reduction from the control value of 7.11 1.7 to 3.74 2.9 pg/ml (p<0.001). Although not statistically significant, the F1 mice had an overall reduced IFNg response, especially at the 62.5 mg/kg/day dose, compared to their parents
Key result
Dose descriptor:
NOEL
Generation:
F1
Effect level:
125 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: reduction in the quantity of CD71+ helper and cytotoxic T-cells present in the high-dose tungstate exposure groups (200 mg/kg/day) when challenged (co-exposure) with SEB, and this efect cannot be attributed to tungstate only.
Key result
Dose descriptor:
LOEL
Generation:
F1
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
developmental immunotoxicity
Key result
Reproductive effects observed:
no
Conclusions:
No observed statistically significant changes in the number of live births, litter size, or sex ratio at any dose of tungstate tested.
Executive summary:

No fertility, reproductive, or developmental toxicity data of sufficient quality are available for tungsten dioxide (target substance). However, reproductive toxicity data are available for sodium tungstate (source substance), which will be used for reading across. Due to lower water solubility and lower toxicity for the target substance compared to the source substance, the resulting read across from the source substance to the target substance is appropriate as a conservative estimate of potential toxicity for this endpoint. In addition, read across is appropriate because the classification and labelling is more protective for the source substance than the target substance, the PBT/vPvB profile is the same, and the dose descriptors are, or are expected to be, lower for the source substance. For more details, refer to the read-across category approach included in the Category section of this IUCLID submission and/or as an Annex in the CSR.

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Justification for type of information:
REPORTING FORMAT FOR THE CATEGORY APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH: The hypothesis is that properties are likely to be similar or follow a similar pattern because of the presence of a common metal ion, in this case tungstate.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES):
Source: Sodium tungstate
Target: Tungsten Dioxide
3. CATEGORY APPROACH JUSTIFICATION: See Annex 1 in CSR
4. DATA MATRIX: See Annex 1 in CSR
Reason / purpose:
reference to same study
Reason / purpose:
read-across: supporting information
Qualifier:
equivalent or similar to
Guideline:
other: EPA Health Effects Test Guidelines OPPTS 870.3650 (which is similar to OECD TG 422)
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
- Sodium tungstate dihydrate was selected because it is the naturally occurring form of tungsten and the most water soluble
- Sodium tungstate dihydrate (purity ≥ 99%)
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Route of administration:
oral: drinking water
Vehicle:
water
Remarks:
Deionized drinking water
Details on exposure:
Drinking water was selected as the most likely route of exposure for the general population
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
Gestation Day (GD) 6 to Postnatal Day (PND) 21 (rats)
Frequency of treatment:
Daily
Dose / conc.:
0 mg/L drinking water
Dose / conc.:
125 mg/L drinking water
Dose / conc.:
250 mg/L drinking water
Dose / conc.:
500 mg/L drinking water
Dose / conc.:
1 000 mg/L drinking water
Dose / conc.:
2 000 mg/L drinking water
No. of animals per sex per dose:
The perinatal phase started with 8 dams/group. On PND 4, unacceptable litters (<8 pups or <2 pups of a sex) were culled and a maximum of 7 litters were standardized to 8 pups (4/sex; 2 or 3/sex was acceptable
Control animals:
yes, concurrent vehicle
Details on study design:
NTP conducted 13-week studies of sodium tungstate dihydrate in rats and mice; the rat study included perinatal exposure to evaluate potential effects resulting from in utero and lactational exposure
Positive control:
Not applicable
Parental animals: Observations and examinations:
- Survival, body weights, water consumption, clinical observations
- Hematology; clinical chemistry and urinalysis in rats
- Gross pathology, histopathology, and organ weights
- Tungsten concentrations in blood and urine
- Genotoxicity (micronucleus and Comet assay)
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One 2000 mg/L moribund dam and litter were terminated on postnatal day 6.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Reduced body weights greater than 10% and 18% relative to control were observed in 1000 and 2000 mg/L dams, respectively.
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Water consumption decreased on the 500 (11% reduction), 1000 (18% reduction) and 2000 (27% reduction) mg/L exposed groups.
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
Gross pathological findings:
not specified
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed
Description (incidence and severity):
There were no treatment related effects on the percentage of dams delivering, litter size, or litter weights.
Key result
Dose descriptor:
LOAEL
Remarks:
10% body weight reduction
Effect level:
ca. 1 000 mg/L drinking water
Based on:
test mat.
Sex:
female
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 500 mg/L drinking water
Based on:
test mat.
Sex:
female
Basis for effect level:
body weight and weight gain
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
1 000 mg/L drinking water
Food consumption and compound intake (if feeding study):
not specified
Ophthalmological findings:
not specified
Reproductive performance:
not examined
Clinical signs:
not specified
Dermal irritation (if dermal study):
not specified
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Reduced body weights greater than 10% relative to control were observed in 2000 mg/L PND 4 pups and PND 21 pups.
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 examined
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Histopathological findings:
not specified
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
LOAEL
Generation:
F1
Effect level:
ca. 2 000 mg/L drinking water
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
ca. 1 000 mg/L drinking water
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Conclusions:
Perinatal studies concluded that were no treatment related effects on the percentage of dams delivering, litter size, or litter weights. There is a reduced body weights greater than 10% relative to control were observed in 2000 mg/L dams, PND 4 pups, PND 21 pups, and adult rats.
Executive summary:

No fertility, reproductive, or developmental toxicity data of sufficient quality are available for tungsten dioxide (target substance). However, reproductive toxicity data are available for sodium tungstate (source substance), which will be used for reading across. Due to lower water solubility and lower toxicity for the target substance compared to the source substance, the resulting read across from the source substance to the target substance is appropriate as a conservative estimate of potential toxicity for this endpoint. In addition, read across is appropriate because the classification and labelling is more protective for the source substance than the target substance, the PBT/vPvB profile is the same, and the dose descriptors are, or are expected to be, lower for the source substance. For more details, refer to the read-across category approach included in the Category section of this IUCLID submission and/or as an Annex in the CSR.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
125 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Well documented scientfically sound study similar to OECD guidelines with sufficient information provided on materials and methods to evaluate results. However as this study is used in the context of a read across, Klimisch 2 is assigned.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Description of key information

No developmental toxicity data of sufficient quality are available for tungsten dioxide (target substance). However, developmental (teratogenic) toxicity data are available for sodium tungstate (source substance), which will be used for reading across. Due to lower water solubility and lower toxicity for the target substance compared to the source substance, the resulting read across from the source substance to the target substance is appropriate as a conservative estimate of potential toxicity for this endpoint. In addition, read across is appropriate because the classification and labelling is more protective for the source substance than the target substance, the PBT/vPvB profile is the same, and the dose descriptors are, or are expected to be, lower for the source substance. For more details, refer to the attached description of the read-across approach.

The US Navy has sponsored a study conducted followingUS EPA Guideline OPPTS 870.3650 (equivalent to OECD TG 422) – Combined Repeated Dose Toxicity Study with the Reproduction-Developmental Toxicity Study. The study evaluated the reproductive, systemic and developmental effects of sodium tungstate in rats following 70 days of daily pre-and postnatal exposure via oral gavage to 5, 62.5 and 125 mg/kg/d through mating, gestation and weaning (PND 0–20). Preliminary results presented on a Scientific Poster at the Society of Toxicology in 2006, showed that initially the study design included a 250 mg/kg bw/d dose group (Johnson, EW Boeckman, HJ Arfsten et al. 2006). However, for unknown the results of this dose group were never peer reviewed and published.

The objective of the study was to evaluate the reproductive, systemic and developmental effects of sodium tungstate in rats following 70 days of daily pre-and postnatal exposure via oral gavage to 5, 62.5 and 125 mg/kg/d through mating, gestation and weaning (PND 0 -20). The results of this study were reported in two separate peer review publications (McInturf et al 2008 & 2011) and on an unpublished summary report (McInturf et al., 2007).Unfortunately, a review of the laboratory final study report has not been conducted as is not publicly available and all the information described here are coming from the fore mentioned documents.

Daily administration of sodium tungstate produced no overt evidence of toxicity and had no apparent effect on mating success or offspring physical development. At the 125 mg/kg bw/d no effects on pup survival, M/F ratio, liter size, clinical signs, or physical birth defects were observed in the F1 litters. At 250 mg/kg bw/d the litter size and the average weight per pup decreased, however the effect was not significant. No clinical signs or effects on pup viability were observed (Johnson, EW Boeckman, HJ Arfsten et al. 2006), but as it was mentioned previously the results of the 250 mg/kg/d dose group were never peer reviewed and published.

The US EPA Guideline OPPTS 870.3650 calls for a gross necropsy of the offspring which includes visceral malformations (but not skeletal ones), and on this subject, “all pups were inspected for physical birth defects, including missing digits, however, none were found”. Based on the lack of F1 significant effects, the NOAEL for developmental toxicity is 125 mg/kg/d. (McInturf et al., 2008 & 2011).

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
REPORTING FORMAT FOR THE CATEGORY APPROACH
1. HYPOTHESIS FOR THE ANALOGUE APPROACH: The hypothesis is that properties are likely to be similar or follow a similar pattern because of the presence of a common metal ion, in this case tungstate.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES):
Source: Sodium tungstate
Target: Tungsten Dioxide
3. CATEGORY APPROACH JUSTIFICATION: See Annex 1 in CSR
4. DATA MATRIX: See Annex 1 in CSR
Reason / purpose:
read-across: supporting information
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
other: EPA OPPTS 870.3650 "Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Study
Deviations:
yes
Remarks:
The protocol was extended beyond the minimum 54 days of treatment to accomodate evaluation of the development of the offspring through postnatal day 20 as well as their dams following the last dose on day 70.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories (Wilmington, MA)
- Age at study initiation: 8 weeks
- Housing: The adults (e.g., P1) were singly housed (except during mating)
- Acclimation period: 14 days
Route of administration:
oral: gavage
Vehicle:
other: deionized water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The powder readily dissolved in a deionized water (diH2O) vehicle for the concentrations used in this study at 5 mg/mL and 125 mg/mL. The solution was concentrated to administer a volume of 1 mL/kg body weight not to exceed 2 mL. Fresh solution was made daily and administered via oral gavage

VEHICLE
- Justification for use and choice of vehicle (if other than water): no data
- Concentration in vehicle: no data
- Amount of vehicle (if gavage): no data
- Lot/batch no. (if required): no data
- Purity: no data
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
Concentrations of the tungstate anion were determined using inductively coupled plasma mass spectrometry (ICP-MS).
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: not specified
- Length of cohabitation: 14 days
- Verification of same strain and source of both sexes: not specified
- Proof of pregnancy: vaginal plug referred to as day 1 of pregnancy
Duration of treatment / exposure:
70 days
Frequency of treatment:
daily pre- and postnatal exposure
Dose / conc.:
5 mg/kg bw/day (actual dose received)
Dose / conc.:
125 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
40 rats per sex per treatment group
Control animals:
yes, concurrent vehicle
Details on study design:
In order to ensure a total of 70 days exposure, adults were dosed for any additional days necessary following weaning. Prenatal pup exposure was from sodium tungstate crossing through the placenta and postnatal exposure was indirect via dams' milk.
Maternal examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Throughout the exposure period, dams and the males used for mating were observed for clinical signs of toxicity.

BODY WEIGHT: Yes
- Time schedule for examinations: Pregnant dams were weighed on gestation days (GD) 1, 10, 15 and 20, and gestational weight gain and gestation length were recorded.

OTHER: N/A



Ovaries and uterine content:
no data
Fetal examinations:
- External examinations: No data
- Soft tissue examinations: Yes: control and high dose pups
- Skeletal examinations: No data
- Head examinations: Yes
Statistics:
All measures were analyzed using ANOVA. Repeated measures ANOVA were used where appropriate. All animals in the pre-weanling litter were tested for righting reflex and separation distress so the litter was used as the unit of analysis for the ANOVA. The fiducial limit was pb 0.05 and post-hoc comparisons were performed using Tukey's HSD analysis for pair-wise comparisons as appropriate.
Clinical signs:
no effects observed
Description (incidence and severity):
Animals were observed for clinical signs of toxicity throughout the exposure period, although none were noted.
Mortality:
no mortality observed
Description (incidence):
No deaths were recorded for the adult females or males at the doses tested
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Sodium tungstate treatments did not have an effect on average gestational weight gain in adults.
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):
effects observed, treatment-related
Description (incidence and severity):
A significant effect of tungstate exposure on spontaneous locomotor activity was detected especially with low dose treated dams. Compared to control and the high dose treated animals, the low dose treated dams spent more time on ambulatory time and distance traveled and less time in stereotypies. On the contrary, in high dose treated dams less time resting and more time in stereotypic movements than the controls or low dose group were markedly observed.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
effects observed, treatment-related
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathological examination showed no severe chronic injury in the organs tested. However, the heart showed histological lesions, histiocytic inflammation from minimal to mild with cardiomyocyte degeneration and necrosis in several P0 animals of 125 mg dose group
Histopathological findings: neoplastic:
not specified
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Tungstate ion concentrations in the male and female adult and pup brains after sodium tungstate exposure were significantly greater in the high dose (125 mg) treated rats than control (data not shown). Similarly, in dam milk secretions tungstate ion concentrations was significantly greater in the 125 mg treatment group than in the low dose group or controls. Increased concentration of tungstate ion distribution in other major organs like heart, spleen, kidney, thymus, testes, lungs, liver, femur
bone and gastrointestinal regions in both male and female treated adult.
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:
not specified
Changes in pregnancy duration:
effects observed, treatment-related
Description (incidence and severity):
The study results indicate that a significant difference was found for one of the treatment groups on measures of reproductive toxicity. In the dams, particularly 125 mg dose group animals showed longer gestational period when compared to control, 5 and 62.5 mg groups.

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): effects observed, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): The study results indicate that a significant difference was found for one of the treatment groups on measures of reproductive toxicity. In the dams, particularly 125 mg dose group animals
Changes in number of pregnant:
not specified
Other effects:
effects observed, treatment-related
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS):
Astatistically significant difference was found for one of the treatment groups on measures of reproductive toxicity. In the dams, average gestation duration was longer for the 125 mg/kg/day dose group than for the control and 5 mg/kg/day groups. Average gestational weight gain did not differ across treatments.

Key result
Dose descriptor:
NOAEL
Effect level:
> 125 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
clinical signs
mortality
Key result
Dose descriptor:
LOEL
Remarks:
125 mg dose group animals showed longer gestational period when compared to control, 5 and 62.5 mg groups
Effect level:
125 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
effects on pregnancy duration
Dose descriptor:
LOAEL
Effect level:
125 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
histopathology: non-neoplastic
Key result
Abnormalities:
effects observed, treatment-related
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): Sodium tungstate treatments did not have an effect on average gestational weight gain in offspring
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:
no effects observed
Visceral malformations:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
- Increased concentration of tungstate ion distribution in other major organs like heart, spleen, kidney, thymus, testes, lungs, liver, femur bone and gastrointestinal regions in both male and female treated pups
- On PND 20, the mean tungstate ion concentrations in males and females of the 125 mg/kg/day treatment group were 0.008 ± 0.006 ppm and 0.002±0.002 ppm, respectively. No tungstate ion was detected in the males or females of the control group. Furthermore, no tungstate ion was detected in the brains of males or females in the control or high dose group on PND 70.
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There were no differences in average number of pups born.
All pups were inspected for physical birth defects, including missing digits, however, none were found.
The high dose group demonstrated a greater number of ultrasonic distress vocalizations when separated from the dam and littermates.

Key result
Dose descriptor:
NOAEL
Effect level:
> 125 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
changes in litter size and weights
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Tungstate ion concentrations in the male and female adult and pup brains after sodium tungstate exposure were significantly greater in the high dose (125 mg) treated rats than control. Similarly, in dam milk secretions tungstate ion concentrations was significantly greater in the 125 mg treatment group than in the low dose group or controls. Further, an increased concentration of tungstate ion distribution was observed in other major organs like heart, spleen, kidney, thymus, testes, lungs, liver, femur bone and gastrointestinal regions in both male and female treated adult and pups.

Conclusions:
This study evaluated the reproductive, systemic and developmental effects of sodium tungstate in rats following 70 days of daily pre-and postnatal exposurevia oral gavage to 5, 62.5 and 125 mg/kg/ day through mating, gestation and weaning (PND 0–20). Daily administration of sodium tungstate produced no overt evidence of toxicity and had no apparent effect on mating success or offspring physical development.
Executive summary:

No fertility, reproductive, or developmental toxicity data of sufficient quality are available for tungstic acid (target substance). However, developmental toxicity data are available for sodium tungstate (source substance), which will be used for reading across. Due to lower water solubility and lower toxicity for the target substance compared to the source substance, the resulting read across from the source substance to the target substance is appropriate as a conservative estimate of potential toxicity for this endpoint. In addition, read across is appropriate because the classification and labelling is more protective for the source substance than the target substance, the PBT/vPvB profile is the same, and the dose descriptors are, or are expected to be, lower for the source substance. For more details, refer to the read-across category approach included in the Category section of this IUCLID submission and/or as an Annex in the CSR.

Endpoint:
developmental toxicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
REPORTING FORMAT FOR THE CATEGORY APPROACH
1. HYPOTHESIS FOR THE ANALOGUE APPROACH: The hypothesis is that properties are likely to be similar or follow a similar pattern because of the presence of a common metal ion, in this case tungstate.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES):
Source: Sodium tungstate
Target: Tungsten Dioxide
3. CATEGORY APPROACH JUSTIFICATION: See Annex 1 in CSR
4. DATA MATRIX: See Annex 1 in CSR
Reason / purpose:
read-across: supporting information
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
other: EPA OPPTS 870.3650 "Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Study
Deviations:
yes
Remarks:
The protocol was extended beyond the minimum 54 days of treatment to accomodate evaluation of the development of the offspring through postnatal day 20 as well as their dams following the last dose on day 70.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories (Wilmington, MA)
- Age at study initiation: 8 weeks
- Housing: The adults (e.g., P1) were singly housed (except during mating)
- Acclimation period: 14 days
Route of administration:
oral: gavage
Vehicle:
other: deionized water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The powder readily dissolved in a deionized water (diH2O) vehicle for the concentrations used in this study at 5 mg/mL and 125 mg/mL. The solution was concentrated to administer a volume of 1 mL/kg body weight not to exceed 2 mL. Fresh solution was made daily and administered via oral gavage

VEHICLE
- Justification for use and choice of vehicle (if other than water): no data
- Concentration in vehicle: no data
- Amount of vehicle (if gavage): no data
- Lot/batch no. (if required): no data
- Purity: no data
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
Concentrations of the tungstate anion were determined using inductively coupled plasma mass spectrometry (ICP-MS).
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: not specified
- Length of cohabitation: 14 days
- Verification of same strain and source of both sexes: not specified
- Proof of pregnancy: vaginal plug referred to as day 1 of pregnancy
Duration of treatment / exposure:
70 days
Frequency of treatment:
daily pre- and postnatal exposure
Dose / conc.:
5 mg/kg bw/day (actual dose received)
Dose / conc.:
62.5 mg/kg bw/day (actual dose received)
Dose / conc.:
125 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
40 rats per sex per treatment group
Control animals:
yes, concurrent vehicle
Details on study design:
In order to ensure a total of 70 days exposure, adults were dosed for any additional days necessary following weaning. Prenatal pup exposure was from sodium tungstate crossing through the placenta and postnatal exposure was indirect via dams' milk.
Maternal examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Throughout the exposure period, dams and the males used for mating were observed for clinical signs of toxicity.

BODY WEIGHT: Yes
- Time schedule for examinations: Pregnant dams were weighed on gestation days (GD) 1, 10, 15 and 20, and gestational weight gain and gestation length were recorded.

OTHER: N/A



Ovaries and uterine content:
no data
Fetal examinations:
- External examinations: No data
- Soft tissue examinations: Yes: control and high dose pups
- Skeletal examinations: No data
- Head examinations: Yes
Statistics:
All measures were analyzed using ANOVA. Repeated measures ANOVA were used where appropriate. All animals in the pre-weanling litter were tested for righting reflex and separation distress so the litter was used as the unit of analysis for the ANOVA. The fiducial limit was pb 0.05 and post-hoc comparisons were performed using Tukey's HSD analysis for pair-wise comparisons as appropriate.
Clinical signs:
no effects observed
Description (incidence and severity):
Animals were observed for clinical signs of toxicity throughout the exposure period, although none were noted.
Mortality:
no mortality observed
Description (incidence):
No deaths were recorded for the adult females or males at the doses tested
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Sodium tungstate treatments did not have an effect on average gestational weight gain in adults.
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):
effects observed, treatment-related
Description (incidence and severity):
A significant effect of tungstate exposure on spontaneous locomotor activity was detected especially with low dose treated dams. Compared to control and the high dose treated animals, the low dose treated dams spent more time on ambulatory time and distance traveled and less time in stereotypies. On the contrary, in high dose treated dams less time resting and more time in stereotypic movements than the controls or low dose group were markedly observed.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
effects observed, treatment-related
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathological examination showed no severe chronic injury in the organs tested. However, the heart showed histological lesions, histiocytic inflammation from minimal to mild with cardiomyocyte degeneration and necrosis in several P0 animals of 125 mg dose group
Histopathological findings: neoplastic:
not specified
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Tungstate ion concentrations in the male and female adult and pup brains after sodium tungstate exposure were significantly greater in the high dose (125 mg) treated rats than control (data not shown). Similarly, in dam milk secretions tungstate ion concentrations was significantly greater in the 125 mg treatment group than in the low dose group or controls. Increased concentration of tungstate ion distribution in other major organs like heart, spleen, kidney, thymus, testes, lungs, liver, femur
bone and gastrointestinal regions in both male and female treated adult.
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:
not specified
Changes in pregnancy duration:
effects observed, treatment-related
Description (incidence and severity):
The study results indicate that a significant difference was found for one of the treatment groups on measures of reproductive toxicity. In the dams, particularly 125 mg dose group animals showed longer gestational period when compared to control, 5 and 62.5 mg groups.

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): effects observed, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): The study results indicate that a significant difference was found for one of the treatment groups on measures of reproductive toxicity. In the dams, particularly 125 mg dose group animals
Changes in number of pregnant:
not specified
Other effects:
effects observed, treatment-related
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS):
Astatistically significant difference was found for one of the treatment groups on measures of reproductive toxicity. In the dams, average gestation duration was longer for the 125 mg/kg/day dose group than for the control and 5 mg/kg/day groups. Average gestational weight gain did not differ across treatments.

Key result
Dose descriptor:
NOAEL
Effect level:
> 125 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
clinical signs
mortality
Key result
Dose descriptor:
LOEL
Remarks:
125 mg dose group animals showed longer gestational period when compared to control, 5 and 62.5 mg groups
Effect level:
125 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
effects on pregnancy duration
Dose descriptor:
LOAEL
Effect level:
125 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
histopathology: non-neoplastic
Key result
Abnormalities:
effects observed, treatment-related
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): Sodium tungstate treatments did not have an effect on average gestational weight gain in offspring
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:
no effects observed
Visceral malformations:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
- Increased concentration of tungstate ion distribution in other major organs like heart, spleen, kidney, thymus, testes, lungs, liver, femur bone and gastrointestinal regions in both male and female treated pups
- On PND 20, the mean tungstate ion concentrations in males and females of the 125 mg/kg/day treatment group were 0.008 ± 0.006 ppm and 0.002±0.002 ppm, respectively. No tungstate ion was detected in the males or females of the control group. Furthermore, no tungstate ion was detected in the brains of males or females in the control or high dose group on PND 70.
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There were no differences in average number of pups born.
All pups were inspected for physical birth defects, including missing digits, however, none were found.
The high dose group demonstrated a greater number of ultrasonic distress vocalizations when separated from the dam and littermates.

Key result
Dose descriptor:
NOAEL
Effect level:
> 125 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
changes in litter size and weights
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Tungstate ion concentrations in the male and female adult and pup brains after sodium tungstate exposure were significantly greater in the high dose (125 mg) treated rats than control. Similarly, in dam milk secretions tungstate ion concentrations was significantly greater in the 125 mg treatment group than in the low dose group or controls. Further, an increased concentration of tungstate ion distribution was observed in other major organs like heart, spleen, kidney, thymus, testes, lungs, liver, femur bone and gastrointestinal regions in both male and female treated adult and pups.

Conclusions:
This study evaluated the reproductive, systemic and developmental effects of sodium tungstate in rats following 70 days of daily pre-and postnatal exposurevia oral gavage to 5, 62.5 and 125 mg/kg/ day through mating, gestation and weaning (PND 0–20). Daily administration of sodium tungstate produced no overt evidence of toxicity and had no apparent effect on mating success or offspring physical development.
Executive summary:

No fertility, reproductive, or developmental toxicity data of sufficient quality are available for tungsten dioxide (target substance). However, developmental toxicity data are available for sodium tungstate (source substance), which will be used for reading across. Due to lower water solubility and lower toxicity for the target substance compared to the source substance, the resulting read across from the source substance to the target substance is appropriate as a conservative estimate of potential toxicity for this endpoint. In addition, read across is appropriate because the classification and labelling is more protective for the source substance than the target substance, the PBT/vPvB profile is the same, and the dose descriptors are, or are expected to be, lower for the source substance. For more details, refer to the read-across category approach included in the Category section of this IUCLID submission and/or as an Annex in the CSR.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
125 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Toxicity to reproduction: other studies

Additional information

No fertility, reproductive, or developmental toxicity data of sufficient quality are available for tungsten dioxide (target substance). However, reproductive toxicity data are available for sodium tungstate (source substance), which will be used for reading across. Due to lower water solubility and lower toxicity for the target substance compared to the source substance, the resulting read across from the source substance to the target substance is appropriate as a conservative estimate of potential toxicity for this endpoint. In addition, read across is appropriate because the classification and labelling is more protective for the source substance than the target substance, the PBT/vPvB profile is the same, and the dose descriptors are, or are expected to be, lower for the source substance. For more details, refer to the read-across category approach included in the Category section of this IUCLID submission and/or as an Annex in the CSR.

Justification for classification or non-classification

No reproductive/developmental studies are available for tungsten dioxide. However, data were available on sodium tungstate, which were used for read-across. Based on the weight-of-evidence from fertility and one-generation reproductive/developmental studies in which no significant effects were observed on reproductive parameters, a lack of significant developmental effects was reported, as well as a lack of reproductive organ effects following 90-d of oral exposure to sodium tungstate in a repeat dose study, it is not expected that tungsten dioxide is a reproductive/developmental (teratogenic) toxicant. Therefore, based on the lack of reproductive and/or developmental effects observed, no classification is warranted.