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No neurotoxicity data of sufficient quality are available for tungsten carbide (target substance). However, neurotoxicity data are available for sodium tungstate (source substance), which will be used for reading across. The neurotoxicity of sodium tungstate is reported three publications by McInturf et al. (2007, 2008, 2011). McInturf et al. (2007) represents a study report, and the 2008 publication is based on exactly these data. According to the results presented, the 2011 publication uses the same data already published in 2008, but with the extension of one additional dose group and more reproductive parameters. The 2008 paper is not referenced in the 2011 one.

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 latency when 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 water maze 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 because 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 is 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 because 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/pre-pulse 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 pre-pulse inhibition responses. No histopathology effects were noted that indicate effects in the brain.

The following information is considered for hazard / risk assessment:

All in all, though being of some academic and methodological value, the regulatory value of the results presented is strongly limited by both shortcomings in the study design selected and the interpretation of the results in regulatory context. These points will be further specified below (and in Annex 2 attached to this summary).

1.      Study Design

The authors reported that the “study followed methodologies defined in the USEPA Guideline OPPTS 870.3650 Combined Repeated Dose Toxicity Study with the

Reproduction/Developmental Toxicity Study”. However, the dose selection was not appropriately conducted. The guidelines recommend to at least three dose levels and a concurrent control should be used. The dose levels should be spaced to produce a gradation of toxic effects. The highest dose level should be chosen with the aim to induce some maternal toxicity (e.g., clinical signs, decreased body weight gain (not more than 10%) and/or evidence of dose-limiting toxicity in a target organ). The lowest dose level should aim to not produce any evidence of either maternal or developmental toxicity including neurotoxicity. A descending sequence of dose levels should be selected with a view to demonstrating any dose-related response and a No-Observed-Adverse Effect Level (NOAEL), or doses near the limit of detection that would allow the determination of a benchmark dose. Two-to four-fold intervals are frequently optimal for setting the descending dose levels, and the addition of a fourth dose group is often preferable to using very large intervals (e.g., more than a factor of 10) between dosages.”

In the McInturf et al (2008) publication, only two doses were reported, 5 and 125 mg/kg (resulting in a stagger of 25), while in the 2011 one, a third intermediate group appears with 62.5 mg/kg, but no behavioral results are reported for this one, which would have been important for setting NOAEL values (see below). Therefore, the problem with subtle measures as behavior is, due to their variability, they may easily produce both false positive as well as false negative results. Consequently, especially for these parameters it is highly important to show a dose-response relationship using small staggers between the groups. This, however, was not done in the sodium tungstate studies.

As for the methods applied, they in part exceed guideline requirements, which add methodological value to the studies, but due to the above-mentioned shortcomings of dose selection, regulatory use of these data is most limited.

2.      Interpretation of Results

In their 2008 paper, the authors summarize their results as follows:

Neurobehavioral test

Sodium tungstate (mg/kg/day)

 

5

125

Righting reflex (pups)

No effect

No effect

Separation distress (pups)

No effect

Increased counts

Maternal retrieval (dams)

No effect

No effect

Watermaze (dams)

No effect

No effect

Accoustic startle PPI (dams)

No effect

No effect

Spontaneaous locomotor behavior (dams)

Increased exploration

Increased stereotypy

 

2.1.Behavioral Data in Dams

The only significant effects they found in dams was one on spontaneous locomotor behavior, while maternal pup retrieval, acoustic startle pre-pulse inhibition as well as learning and memory in Morris water maze were unaffected.

 Spontaneous locomotor behavior was tested in an open filed situation. This test was established by Hall (1934) to investigate the complex interaction of exploration on one hand, and emotionality on the other hand, on rodent behavior. The full complexity of these interactions is best described by Denenberg (1969). Based on this complexity, this test can be influenced in several ways (Walsh & Cummins, 1976), and is, therefore, prone to yield contradicting results. This is exactly true for the present paper, when the authors found increased exploration in the (very) low dose, but increased stereotypy in the high one. To prevent such unclear results, it is important to find out dose response relationships and threshold levels for a given effect to occur. Here again it is the main shortcoming of the paper that they chose testing just two doses with a stagger as high as 25, which makes a sound interpretation of these data for regulatory purposes impossible. Consequently, the effects the authors describe may need further investigation, but the results from McInturf at al. (2008), as they are reported, by no means can be considered prove of an influence of tungstate on adult behavior.

2.2. Behavioral Data in Offspring

In pups, only two endpoints were investigated and reported, namely tests of the righting reflex and on separation distress calls emitted by the pups when removed from the nest. The test of the righting reflex did only reveal effects of the pup sex on performance of this test, but no effects of substance exposure could be detected.

 As for the separation distress calls, the authors report that “Pups showed dose-related effects in the number of ultrasonic distress vocalizations recorded. Specifically, those in the control and 5 mg/kg/day groups vocalized significantly less than those in the 125 mg/kg/day treatment group during the 60-seconds time-period (19.5±3.2 (control), 23.1± 3.8 (5 mg/kg/day), and 34.4±4.1 (125 mg/kg/day), p < 0.05).” The test of the emission of separation calls by rodent pups is one the tests specifically applied to study anxiety in this model (Olivier et al, 1994). In the context of the study of McInturf et al. (2008), this endpoint appears to be someway random, so their observation may or may not be an indication of an adverse effect of prenatal tungstate treatment. Again, to better interpret these data, dose response relationships would be of crucial importance, but the chosen study design precludes this. Consequently, the effects the authors describe may warrant further investigation, but the results from Mclnturf et al. (2008), as they are reported, by no means can be considered prove of an adverse influence of tungstate on early pup behavior.

2.3.Gestation Length

McInturf et al., (2008) reported an increased gestation length in the high dose group (22.08 vs. 21.55 days in the control group). In McInturf et al., (2011) publication in addition to the results already published in 2008, the authors refer to one more group, namely one treated with 62.5 mg/kg/day. For this group, mainly parameters on dams were reported, and no other behavior-related ones were provided. Interestingly, in this 62.5 mg/kg/day dose group no such prolonged gestation length was reported. However, no other effects on dam or early pup development were reported even for the high dose group (e.g. “sodium tungstate treatments did not affect the average gestational weight gain in adults and offspring"). It is difficult to judge the adversity of this increased gestation length in the absence of any effects on offspring development.

3.      Conclusions

The sodium tungstate neurodevelopmental study described by Mclnturf et al. (2007, 2008, 2009) suffer from severe shortcomings in the study design, which limit their usability for regulatory purposes. The potential adverse results from this sodium tungstate study is reported in three separate documents and include activity and behavioral data in adults, increased number of distress calls in pups when separated from the nest, and increase in gestation length. A critical review of all these effects by no means can be considered prove of an adverse influence of tungstate on adult behavior, early pup behavior or an indicator of developmental toxicity and/or neurotoxicity in rats. Based on this, none of the data from these publications warrant any classification for tungstate as a reproductive toxicant per CLP. Overall, there is enough information to assess the developmental neurotoxicity of tungstate (the tungsten ion bioavailable at physiological conditions), and based on the weight of evidence, the inclusion of cohorts 2A and 2B in a potential EOGRT study is not justified.

References:

Denenberg VH. Open-field Behavior in the Rat: What Does It Mean? Annals of the New York Academy of Sciences 159 (1969) 852–859.

Hall CS, Emotional behavior in the rat. I. Defecation and urination as measures of individual differences in emotionality. Journal of Comparative Psychology 18 (1934) 385–403.

McInturf SM, Bekkedal MYV, Olabisi A, Arfsten D, Wilfong E, Casavant R, Jederberg W, Gunasekar PG, Chapman GD. Neurobehavioral effects of sodium tungstate exposure on rats and their progeny, Naval Health Research Center Detachment, Environmental Health Effects Laboratory, June 30, 2007.

McInturf SM, Bekkedal MYV, Wilfong E, Arfsten D, Gunasekar PG, Chapman GD, Neurobehavioral effects of sodium tungstate exposure on rats and their progeny, Neurotoxicology and Teratology 30 (2008) 455–461.

McInturf SM, Bekkedal MYV, Wilfong E, Arfsten D, Chapman GD, Gunasekar PG. The potential reproductive, neurobehavioral and systemic effects soluble sodium tungstate exposure in Sprague-Dawley rats. Toxicology and Applied Pharmacology 254 (2010) 133-137.

OECD Guidelines for the Testing of Chemicals, Section 4, Test No. 426: Developmental Neurotoxicity Study, October 15, 2007. http://www.oecdilibrary.org/docserver/download/9742601e.pdf?expires=1480249396&id=id&accname=guest&checksum=B96BE9C3F3DE225AF49E92DE76A8D60F

Olivier B, Molewijk E, van Oorshot R, van der Poel G, Zethof T, van der Heyden J, Mos J New animal models of anxiety; European Neuropsychopharmacology 4 (1994) 93-102.

United Nations, Globally Harmonized System of Classification and Labeling of Chemicals (GHS), 2011https://www.unece.org/fileadmin/DAM/trans/danger/publi/ghs/ghs_rev04/English/ST-SG-AC10-30-Rev4e.pdf

United States Environmental Protection Agency, Prevention, Pesticides and Toxic Substances (7101), EPA 712–C–00–368, July 2000. Health Effects Test Guidelines, OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Testhttps://www.regulations.gov/document?D=EPA-HQ-OPPT-2009-0156-0016

United States Environmental Protection Agency, Prevention, Pesticides and Toxic Substances (7101), EPA 712–C–96–239, June 1998. Health Effects Test Guidelines, OPPTS 870.3650, Developmental Neurotoxicity Study https://www.regulations.gov/document?D=EPA-HQ-OPPT-2009-0156-0042

 Walsh, RN., Cummins RK, The open-field test: A critical review. Psychological Bulletin 83 (1976) 482-504.


Key value for chemical safety assessment

Effect on neurotoxicity: via oral route

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
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 data of sufficient quality are available for tungsten carbide (target substance). However, neurotoxicity data are available for sodium tungstate (source substance), which will be used for reading across. The sodium tungstate neurodevelopmental study described by Mclnturf et al. (2007, 2008, 2011) suffer from severe shortcomings in the study design, which limit their usability for regulatory purposes (see Annex 2 attached to this endpoint summary). The potential adverse results from this sodium tungstate study is reported in three separate documents and include activity and behavioral data in adults, increased number of distress calls in pups when separated from the nest and increase in gestation length. A critical review of all these effects by no means can be considered prove of an adverse influence of tungstate on adult behavior, early pup behavior or an indicator of developmental toxicity and/or neurotoxicity in rats. Based on this, none of the data from these publications warrant any classification for tungsten carbide as a developmental neurotoxicant per CLP.

In addition, there is reliable evidence that bioaccessibility of metal ion in simulated gastric fluid correlates well within 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 carbide bioaccessibility (0.035 +/- 0.00071%) 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 carbide as a neurodevelopmental toxicant.