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Neurotoxicity

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Description of key information

No neurotoxicity data of sufficient quality are available for tungsten metal (target substance). However, neurotoxicity 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.

A study conducted following EPA OPPTS 870.3650 evaluated the reproductive and developmental (teratogenic) 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/ day through mating, gestation and weaning (Postnatal day, PND 0–20). In this study, a range of neurobehavioral capacities in sodium tungstate exposed dams and their offspring were assessed. The tests evaluated reflexive responding, emotionality and spatial learning and memory in the low and high dose groups, but not in the mid dose group.

The following neurobehavioral test batteries were performed on pups and adult females after exposure to sodium tungstate. The righting reflex and separation distress were done on PD4 and PND7, respectively. The adult females were tested for maternal retrieval latencywhen pups were age PND2, and spontaneous locomotor activity (SLA) on post-dosing day 7,acoustic Startle/Pre Pulse Inhibition (AS/PPI) on post-dosing day 8, and watermaze navigation on post-dosing days 15–18 (McInturf et al., 2007, 2008 & 2011).

Results from one of the two tests in the pups, separation distress, suggest neurobehavioral perturbations as a result of exposure to sodium tungstate (McInturf et al., 2007, 2008 and 2011). The high dose group was reported to have a greater number of ultrasonic distress vocalizations when separated from the dam and littermates. However,in the absence of single animal data from the study and historical control data, this effect cannot be evaluated. The other pup assessment, righting reflex latency, showed sex differences where males demonstrated faster righting than females, however, the effects were not dose-dependent.In the absence of single animal and historical control data the relevance of this finding cannot be evaluated. In addition, in Table 2 of the publication (McInturf et al., 2008) state that no effects were observed in the pups for this endpoint.The authors of the study determined that the collection of results are insufficient to delineate a clear dose response in either the pups, and the pattern of behavioral perturbations do not provide a clear indication of areas of the brain that may be more susceptible to neurotoxic effects as a result of exposure to sodium tungstate.Thus the study does not provide clear evidence of developmental neurotoxicity.

McInturf et al (2008) indicated that only two neurobehavioral tests were used in the pups, and they measured very early, reflexive behavioral responses. In addition, no effects of sodium tungstate exposure at either dose were found in the dams for latency of maternal retrieval, or water maze navigation latency or distance traveled, and acoustic startle/prepulse inhibition. Exposure effects in the dams were detected for some measures of spontaneous locomotor activity. However, the altered stereotypical behavior was not apparent in the measures of gross motor movements in the open field, or in the reflexive acoustic startle or prepulse inhibition responses. No histopathology effects were noted that indicate effects in the brain.

The one-generation study on sodium tungstate study results does not provide a clear indication for neurotoxic effects, the collection of results is insufficient to delineate a clear dose response in either the pups or dams, and the pattern of behavioral perturbations do not provide a clear indication of areas of the brain that may be more susceptible to neurotoxic effects as a result of exposure to tungstate.

Key value for chemical safety assessment

Effect on neurotoxicity: via oral route

Link to relevant study records
Reference
Endpoint:
neurotoxicity: sub-chronic oral
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 metal
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:
according to
Guideline:
other: EPA OPPTS 870.3650 "Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Study
GLP compliance:
not specified
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
- 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).
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:
Day 13: 24 hr usine/feces
Day 14: Mating begins
Day 28: Mating ends
Day 70: Last dosing
Observations and clinical examinations performed and frequency:
Adults (10 of each sex) from each dose group were necropsied on day 70, and pups (one male/one female) from each litter were necropsied at PND20 and PND70. Weight of pregnant dams and gestation length were recorded as was the litter size. Litters were culled on PND4 to maintain a 4 male/4 female ratio whenever possible, and all pups remained with their biological mothers. Animals were observed for clinical signs of toxicity throughout the exposure period.
Neurobehavioural examinations performed and frequency:
The following neurobehavioral test batteries were performed on pups and adult females after exposure to soidum tugstate. The righting reflex and separation distress were done on PD4 and PND7, respectively. The adult females were tested for maternal retrieval latency when pups were age PND2 , and spontaneous locomotor activity (SLA) on post-dosing day 7.
Sacrifice and (histo)pathology:
At the end of experimentation, rats were perfusion-fixed with 4% paraformaldehyde. 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
Other examinations:
Inductively coupled plasma mass spectrometry (ICP-MS) was used to measure tungsten concentrations in brain tissue of adults and pups from the control and highest dose groups only and in dam mammary secretion in all control, low and high dose P1 groups
Statistics:
All statistical testing was performed using ANOVA with repeated measures factors. Tukey's HSD analysis was used to evaluate pair-wise comparisons
Clinical signs:
no effects observed
Description (incidence and severity):
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 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
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Distress vocalizations were elevated in F1offspring from the high dose group, whereas righting reflex showed unexpected sex differences where males demonstrated faster righting than females; however, the effects were not dose-dependent. Locomotor activity was affected in both low and high-dose groups of F1 females. Low-dose group showed increased distance traveled, more time in ambulatory movements and less time in stereotypic behavior than controls or high dose animals. The high-dose group had more time in stereotypical movements than controls, and less time resting than controls and the lowest exposure group. Maternal retrieval was not affected by NaW exposure
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Pathological examination revealed that no treatment related deaths or histopathological changes in any organs except P0 male hearts at 125 mg/kg bw/d. Two animals of five displayed myocarditis with cardiomyocyte degeneration and necrosis.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not specified
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Migrated information from 'Further observations for developmental neurotoxicity study'

Developmental landmarks (offspring):Distress vocalizations were elevated in the highest dose group. There was no treatment related effect on righting reflex latencies, however, the males had significantly shorter latencies than the females. (migrated information)

Details on results (for developmental neurotoxicity):Dams:
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.

Pups:
- 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.
- 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 low dose groups. This result indicates that both 5 and 125 mg exposed male (not female) pups motor reflex were affected due to sodium tungstate. However, only high dose exposed males were ffected by emotionality.
(migrated information)
Details on results:
- Tungstate exposure caused spontaneous locomotor activity 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.
- 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.
- 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.
- Maternal retrieval test revealed that sodium tungstate exposure to dams had no effect on latency in both treatment groups.
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:
behaviour (functional findings)
Key result
Based on:
test mat.
Sex:
male/female
Basis for effect level:
behaviour (functional findings)
Remarks on result:
not determinable because of methodological limitations

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.

Conclusions:
The tests were selected to provide a screening-level assessment of a range of neurobehavioral capacities in sodium tungstate exposed dams and their offspring. The tests evaluated reflexive responding, emotionality and spatial learning and memory. Exposure-related effects were observed both in the dams and developing pups, and for low and high dose exposures. Overall, the results of this study suggest pre- and postnatal oral exposure to sodium tungstate may produce slight neurobehavioral effects in offspring related to motor activity and emotionality.

While the results of this study provide evidence for neurological effects in F1 offspring, the collection of results are insufficient to delineate a clear dose response in either the pups or dams, and the pattern of behavioral perturbations do not provide a clear indication of areas of the brain that may be more affected.

In the pups, only two tests were used, and they measured very early, reflexive behavioral responses. A more thorough assessment covering a longer period of development would help delineate the reversibility of these deficits, and the impact they may have on subsequent neurobehavioral integrity susceptible to neurotoxic effects as a result of exposure to sodium tungstate.
Executive summary:

No neurotoxicity data of sufficient quality are available for tungsten metal (target substance). However, neurotoxicity 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 conclusion
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 neurotoxicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Species:
rat
Quality of whole database:
Well documented scientfically sound study 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 neurotoxicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

An inhalation study reported that sodium tungstate is not appreciably transported via the olfactory pathway to the brain following a single 90-min exposure in rats, although this pathway is known to transport a number of other metals (Radcliffe et al, 2009). Sodium tungstate exposure was reported in one study to produced oxidative stress in brains from rats exposed. However, the study did not elucidate and correlate these oxidative changes with behavioral and functional alterations (Sachdeva et al, 2015).

Justification for classification or non-classification

No neurotoxicity studies are available for tungsten metal. However, data were available on sodium tungstate, which were used for read-across.

The one-generation study on sodium tungstate study results does not provide a clear indication for neurotoxic effects, the collection of results is insufficient to delineate a clear dose response in either the pups or dams, and the pattern of behavioral perturbations do not provide a clear indication of areas of the brain that may be more susceptible to neurotoxic effects as a result of exposure to tungstate.

In addition, there is reliable evidence that bioaccessibility of metal ion in simulated gastric fluid correlates well with in vivo systemic bioavailability and/or toxicity (European Commission, 2015), and represents a worst-case fasting exposure scenario for a conservative bioaccessibility assessment (Hillwalker and Anderson, 2014). A low tungsten metal bioaccessibility (0.23 +/- 0.013%) in simulated gastric fluids (pH= 1.5) (see Section 5.1.3) (IITRI, 2010a) suggests a low oral bioavailability, and based on this no classification is warranted for tungsten metal as a neurodevelopmental toxicant.