Tungsten

Endpoint summary

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 (target substance). However, reproductive toxicity data are available for sodium tungstate (source substance), which are used for read-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 in the Category section of this IUCLID submission or Annex 3 in the CSR.

There are no reproductive and/or pre- or post- natal developmental toxicity studies on sodium tungstate conducted under OECD guidelines. However, a tripartite study on sodium tungstate conducted under the International Conference of Harmonisation (ICH) Harmonised Tripartite Guideline on Detection of Toxicity to Reproduction for Medicinal Products & Toxicity to Male Fertility S5 (R2) [finalised (Step 4) November 2005] and following Good Laboratory Principles (GLP) can be adapted to fulfill this endpoint. ICH has produced a comprehensive set of safety Guidelines for human pharmaceuticals to uncover potential risks like carcinogenicity, genotoxicity and reproductive toxicity.

This tripartite study together with the Osterburg et al (2014) fulfill the EOGRT Cohorts as follows:

 

REACH Requested Test (OECD TG 443)	Studies Requesting Adaptation
Cohort 1A	Segment I - ICH Testing Guideline  (4.1.1. Fertility Study)
Cohort 2A Cohort 2B	Segment III - ICH Testing Guideline (4.1.2. Pre- and postnatal study)   McInturf et al (2007, 2008 & 2011)
Cohort 3	Osterburg et al (2014)

 

1. Segment I (Cohort 1A) - Fertility and Early Embryonic Development (ICH 4.1.1 Fertility Study)

A test adaptation is requested as no rat OECD TG 421 or 422 study is available on sodium tungstate. Instead, we are submitting a rat Fertility and Early Embryonic Development (ICH Segment III) on sodium tungstate study conducted according to the ICH Harmonised Tripartite Guideline on Detection of Toxicity to Reproduction for Medicinal Products & Toxicity to Male Fertility S5 (R2) [finalised (Step 4) November 2005] and following Good Laboratory Principles (GLP).

Doses were selected based on a dose-range finding study that exposed females rats to 0, 50, 100 or 150 mg sodium tungstate/kg/day by oral gavage. The groups exposed from Day 6 to Day 17 of gestation. Exposure to 50 or 100 mg/kg/day was well tolerated with no significant maternal or embryo-foetal toxicity. However, administration of 250 mg/kg/day sodium tungstate elicited marked maternal toxicity. Consequently, a dose level below 250 mg/kg/day is considered a suitable high dose level for the subsequent pivotal embryo-foetal development study.

 

The effects of sodium tungstate on the fertility and early embryonic development of the rat, were evaluated when administered orally, by gavage, to males for at least 14 days before and during pairing and until the day before necropsy, and to females for at least 14 days before and during pairing and then to Day 6 of gestation.  Females were killed on Day 13 of gestation. Four groups of 20 male and 20 female Crl:CD(SD) rats were dosed with 0 (purified water), 40, 80 or 160 mg/kg/day sodium tungstate daily, by oral gavage, at a constant dose volume of 5 mL/kg body weight. Clinical observations, body weight and food intake were recorded at regular intervals.  Oestrous cycles were monitored daily from 10 days before pairing and until mating was confirmed.  The females were killed on Day 13 of gestation and a full uterine examination was conducted.  Males were killed approximately two weeks after completion of the mating period and a necropsy was performed. Testes and ovaries were weighed, then these and the epididymides, prostate and seminal vesicles were retained. There was no mortality or clinical signs associated with sodium tungstate.  In males given 160 mg/kg/day, mean body weight gain was lower than Controls throughout the study, with some sporadic individual body weight losses and consequently, mean absolute body weight at the end of the study was statistically lower than Controls (-8.2 %).  In females given 160 mg/kg/day, mean body weight gains were also lower throughout the dosing period, but these changes were less apparent than effects in males and after the cessation of dosing, compensatory body weight gains resulted in mean absolute body weight being comparable with Controls on Day 13 of gestation.  Body weight gains in males and females given 40 or 80 mg/kg/day were similar to Controls and there was no adverse effect of sodium tungstate on food intake at any dose level in either sex. There was no effect of sodium tungstate on the number of oestrous cycles recorded during the pre-pairing period, or on the average cycle length.  All paired females mated and there was no effect of sodium tungstate on the time taken to mate or on the fertility of either the males or females; only one Control female was not pregnant. Pregnancy data were similar in all groups. There were no macroscopic abnormalities in females which were considered to be test item related, however, six out of 20 males given 160 mg/kg/day had kidney abnormalities (generally large and abnormally coloured).  There was no effect of sodium tungstate on mean ovary or testis weights. Administration of sodium tungstate to the Crl:CD(SD) rat for 14 days before pairing until Day 6 of gestation in females, or necropsy in males (five weeks), was generally well tolerated, with only non-adverse body weight changes seen in both sexes. On this basis, the No Observed Adverse Effect Level (NOAEL) for female fertility and early embryonic development, or male fertility, was considered to be 160 mg/kg/day.

Conclusion: There is enough toxicological information from the rat Segment I study conducted following ICH guidelines to assess the fertility of sodium tungstate and based on this evidence, the addition of Cohort 1A in an EOGRT study is not justified.

2. Segment III, ICH 4.1.2 Pre and Postnatal Study (Cohort 2A and 2B)

A test adaptation is requested as no rat OECD TG 443 study is available on sodium tungstate. Instead, we are submitting a Pre- and Post- Natal Development Study (ICH 4.1.2) on sodium tungstate rat study conducted according to the ICH Harmonised Tripartite Guideline on Detection of Toxicity to Reproduction for Medicinal Products & Toxicity to Male Fertility S5 (R2) [finalised (Step 4) November 2005] and following GLP.

OECD TG 443 developmental neurotoxicity (DNT) in addition to brain histopathology include auditory startle, functional observation, motor activity endpoints. ICH Segment III includes P1 generation learning and memory, auditory function and motor activity, and in F1 offspring first day of eye and ear open occurrence, static righting reflex, and startle response.

The effects of the sodium tungstate on embryonic, foetal and post-natal development of the rat were assessed via oral gavage. Four groups of 22 time-mated female Crl: CD(SD) rats were dosed at dose levels of 0 (vehicle - purified water), 40, 80 or 160 mg/kg/day sodium tungstate once daily, from Day 6 of gestation to Day 20 of lactation, inclusive, at a dose volume of 5 mL/kg body weight.  

Parental (P generation) females were observed daily from the start of dosing and body weights and food intake were recorded at regular intervals. The females were allowed to litter and rear their offspring to Day 21 of lactation. Pups not selected for the filial (F1) generation were killed on Day 21 of age together with all P generation dams and a macroscopic necropsy was conducted.  For the F1 generation, 20 males and 20 females per group were selected at weaning and allowed to mature, undosed [see McInturf et al (2007, 2008 & 2011) below for study design that exposed for 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)]. The effects on growth, development, behaviour and reproductive performance were assessed.

 

There was no mortality or clinical signs associated with sodium tungstate. Maternal body weight gains at 160 mg/kg/day were lower than Controls during gestation; however, although body weights were lower at the start of lactation, these females gained more weight than Controls over the whole lactation period. Body weight gains for females given 40 or 80 mg/kg/day were similar to Controls and there was no effect of Sodium Tungstate on food intake at any dose level. There were 21, 19, 21 and 20 females that littered given 0, 40, 80 or 160 mg/kg/day, respectively. There were no effects of sodium tungstate on pregnancy parameters or litter survival. Pup weights were similar to Control values and there was no effect of maternal test item administration on developmental milestones. There were no test item-related macroscopic necropsy findings for the parental females or their litters. Females from the 160 mg/kg/day group started the F1 generation phase of the study lighter than Controls and mean body weight gains remained lower until the start of the gestation period; during gestation, group mean body weight gain was similar to Controls. Body weight gains for males at all dose levels and for females in the maternal groups given 40 or 80 mg/kg/day were similar to Controls. There were no effects on the development, behaviour or reproductive function of the F1 generation following maternal administration of sodium tungstate. 

 

The administration of sodium tungstate to pregnant Crl:CD(SD) rats at dose levels of 40, 80 or 160 mg/kg/day once daily from Day 6 of gestation to Day 20 of lactation, inclusive, was generally well tolerated, with only non-adverse body weight changes for maternal animals.  For the F1 generation, although animals from the maternal group given 160 mg/kg/day were slightly lighter than Controls, there were no adverse effects on growth, development, behaviour or reproductive performance.

 

Cohort 2A - Neurobehavioral/ Testing as Adults (F1 Generation) (ICH Segment III)

Learning and memory tests were conducted during Weeks 1 and 2 (session 1 and 2) of the F1 maturation phase, each selected animal was tested for learning and memory using a water-filled E-maze, monitored by an automated video-tracking system.  All groups were tested during Week 1 for learning ability.  As there was no test-item related effect on learning, during Week 2 only Groups 1 and 4 were tested for memory. The auditory function During Week 0 of the F1 maturation phase, the auditory function of each selected animal in Groups 1 and 4 was assessed using the auditory startle test.  Motor activity During Week 0 of the F1 maturation phase, the motor activity of each selected animal in Groups 1 to 4 was assessed using an automated, infra-red beam activity monitoring system. The following observations were reported:

1) Learning and memory: There was no effect of maternal administration of sodium tungstate on learning and memory of the F1 generation as assessed by the E-maze test.

2) Auditory function: There was no effect of maternal administration of sodium tungstate on the hearing ability of the F1 generation; all animals in Groups 1 and 4 passed the auditory startle reflex assessment.  

3) Motor activity: There was no effect of maternal administration of sodium tungstate on motor activity. During the first 10 minutes of the monitoring females at all maternal doses and males maternally given 80 or 160 mg/kg/day travelled a greater distance and rested less than the Controls, although there was no dose-relationship.  This is considered not to be related to maternal sodium tungstate administration.

Cohort 2B (F1a Generation) - Developmental Neuro-Toxicity (DNT)

F1 Pups were monitored for the following developmental milestones:

- Ears open - examined daily until occurrence.

- Eyes open - examined daily from Day 11 of age until occurrence

- Static righting reflex - examined on Day 5 of age.

- Startle response - examined on Day 15 of age.

- Pupillary light reflex - examined on Day 21 of age

There was no effect of maternal administration of sodium tungstate on pup development, as assessed by pinna detachment, eyelid separation, static righting reflex, startle response or pupillary light reflex.

Conclusion: There is sufficient toxicological information from the rat Segment III study conducted following ICH guidelines (Sequani et al, 2016) and McInturf et al (2007, 2008 & 2011) to assess the developmental neurotoxicity of sodium tungstate and based on this evidence, the addition of Cohort 2A and 2B in an EOGRT study is not justified.

Cohort 3 - Developmental Immunotoxicity (DIT)

There is no developmental immunotoxicity (DIT) study on sodium tungstate conducted under OECD guidelines. An adaptation for this endpoint has been conducted based on a one-generation mice study (Osterburg et al, 2014) on sodium tungstate that investigated the longer-term effects of tungstate exposure on the DIT response of mice after activation of the immune system with Staphylococcal enterotoxin B (SEB).

Osterburg et al (2014) tested if exposure to sodium tungstate can result in an immune effect in a one-generation (one-gen) model and intraperitoneally injected with Staphylococcal enterotoxin (SEB). For this, parental male and female mice were orally exposed (via drinking water) to 0, 2, 62.5, 125, 200 mg sodium tungstate/kg bw/day. Both P and F1 mice were maintained on these doses for the course of the study. These tungstate doses were selected based on previous work by McInturf et al (2011) that used similar doses in rats.

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, approximately 19 weeks after initiation of the study. The F1 generation was exposed to tungstate for a further 90-days after weaning and then necropsied. Mice were housed singly during the course of the study and pair mated for breeding. After confirmation of pregnancy, males were removed. During all phases of the one-gen study mice were kept on the appropriate tungstate dose (Osterburg et al, 2014).

OECD TG 443 DIT endpoints include splenic lymphocyte subpopulation analysis, lymph node weight and histopathology, primary IgM antibody response to a T cell dependent antigen. Osterburg et al (2014) DIT study covered splenic lymphocyte subpopulation analysis by flow cytometry on whole blood and splenocyte single cell suspensions to collect typical immunological populations. No IgM antibody response was measured against an antigen, instead the cytotoxic response of T-cells (TCTL; CD3 + CD8 + CD71+) and helper T-cells (TH; CD3+ CD4 + CD71+) were determined from spleens of mice after treatment with Staphylococcal enterotoxin (a superantigen with the ability to bind to class II MHC molecules on antigen presenting cells and stimulate large populations of T cells that share variable regions on the β chain of the T-cell receptor).

Osterburg et al (2014) results showed no statistically significant changes in body weight due to any tungstate dose levels. The 200 mg/kg bw/day males in the P generation show a consistent trend towards decreased weights. This observation, however, was not statistically significant. Additionally, no statistically significant changes in the number of live births, litter size, or sex ratio at any dose of tungstate tested.

Complete blood counts and hematological parameters from the blood of animals at necropsy were performed. With two exceptions (monocyte% and red blood cell distribution width), there were no statistical differences in the data between P and F1.

No significant changes in any of the parameters measured in response to tungstate, except for the percent monocytes. 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 (eg hematocrit); however, these trends were not statistically significant.

Tungstate-dependent changes were only observed in the spleens of animals. Furthermore, any statistically significant differences between the innate or immune responses of P and F1 mice were not noted. One-gen tungstate exposures resulted in reduced quantities of CD71+TH cells in the P and F1 mice for the 200 mg/kg/day dose group compared to the control groups. No statistically significant differences were noted in the overall quantity of CD3+CD4+TH cells, as well no statistically significant differences in quantities of CD3+ CD8+ cells. The cytotoxic CD3+CD8+CD71+cells in the P and F1 mice were decreased in SEB-challenged groups in 200 mg/kg/day group.

Among cytokines measured in plasma, the only significant change was a dose-dependent quantitative decrease in interferon IFNγ levels in SEB-treated mice. Although not statistically significant, the F1 mice had an overall reduced IFNγ response, especially at the 62.5 mg/kg/day dose, compared to their parents (Osterburg et al, 2014).

A supporting study (Fastje et al, 2012) attempted to determine if sodium tungstate can cause a DIT response. Mice were exposed in utero by parental inhalation or ingestion of an estimated dose of 1 and 4 mg/kg bw/day, respectively; and inoculated with respiratory syncytial virus (RSV) (within 2 weeks of weaning). The dams were exposed to sodium tungstate through water (15 ppm, ad libitum) and aerosol. During the 45-min, 5 days/week aerosol exposures, female mice were exposed to a 187 g/L solution nose-only for 1 week prior to conception and 3 weeks of gestation until parturition halted exposures. Pups were weaned onto tungstate-spiked water (15 ppm, ad libitum). At 21–35 days of age the mouse pups were lightly anesthetized and the nasal cavity inoculated with human RSV. Peripheral hematology was evaluated utilizing complete blood counts. Spleen tissue was massed and splenic ratio calculated as spleen mass per body mass.

Results showed that controls and tungstate only-treated mice did not exhibit pathological indicators. RSV inoculation within 2 weeks of weaning was associated with a neutrophil shift. When the RSV inoculation was combined with exposure to tungstate (Na2WO4+ RSV), significant splenomegaly resulted in addition to other hematological pathologies which were not significant. Exposure to Na₂WO₄+ RSV resulted in hematological/immunological disease, the nature of which was inconclusive (Fastje et al, 2012).

Overall, a DIT response was confirmed by two in utero exposure studies when tungstate is co-exposed with an immune-stimulating agent such as RSV (Fastje et al, 2012) or SEB (Osterburg et al, 2014). When tungstate was only co-exposed with an immune-stimulating agent an enlarge spleen or immunosuppression can be observed. The dose required for immune-suppression in the SEB model is 200 mg/kg/day. In contrast, data indicates little to no effect of tungstate at any dose on groups of mice exposed only to tungstate.

 

Conclusion: There is sufficient developmental immunotoxicity information published by Osterburg et al (2014) that demonstrated that sodium tungstate causes at 200 mg/kg bw/day a modulation of the normal cell-mediated immune in pups as a response only when tungstate is co-exposed with an immune-stimulating agent (such as SEB or RSV). Based on this evidence, the addition of Cohort 3 in an EOGRT study is not justified.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

160 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Study conducted under recognised international testing guidelines and following good laboratory principles (GLP)

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Additional information on Reproductive Toxicity:

Several 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 toxicity is also covered by McInturf et al (2007, 2008, and 2011) gavage study together with the US NTP perinatal drinking water study.

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.

1. Ballester et al (2005, 2007) Fertility Studies

Male rats (15 animals per group) were given 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 rats were 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 smears were performed to determine whether overnight mating had occurred. The reproductive performance of the male rats was determined by calculating the percentage of fertile males 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, however did not: 1) alter the reproductive performance of healthy animals; 2) modify the appearance of number of Leydig cells, or 3) change the serum levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH) and testosterone.

Female rats were given 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 smears were carried out to determine if mating had occurred. When vaginal smears were positive (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 CO2 inhalation.

The reproductive performance of the rats was measured as the proportion of inseminated females to the total number tested (the percentage of positive vaginal smears, ie those with presence of spermatozoa, with respect to the total number of vaginal smears performed in one experimental group). This parameter was named as ‘mating index’. The number of vaginal smears varied depending on the time required by animals to show positive mating. Furthermore, the percentage of parturitions was calculated 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 rats tungstate treatment caused a decrease in the body weight gain but did not modify daily food and water consumption. Tungstate treatment did not modify alanine aminotransferase (ALT) activity, progesterone, FSH or LH. In addition, tungstate treatment did not affect any reproductive parameter or affected the expression of the estrogen receptor. However, in ovaries tungstate treatment had a considerable effect on the expression of the progesterone receptor. In contrast, the uterine expression of the progesterone receptor was not affected by tungstate treatment.

2. McInturft et al (2007, 2008 & 2011) - Reproduction/Developmental Toxicity Screening 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/day, 125 mg/kg bw/day ay or 5 mg/kg bw/day 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/day) significantly decreased body-weight gain in the P0 males and gestational weight gain (about 20% decrease) as well as increasing gestational length (1.2 days) in the dams. At 250 mg/kg bw/day 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). 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 P0 males 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).

3. US National Toxicology Program (US NTP) - Perinatal Study

The US National Toxicology program (US NTP) has conducted a drinking water doses of 0, 125, 250, 500, 1000, or 2000 mg/L (an estimated oral dose between 16-247 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 the US NTP website. Furthermore, at the 2012 Annual Meeting of the Society of Toxicology, a Scientific Poster was presented. The following preliminary results showed:

A) P0 (First Parental animals)

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

·      Reduced gestational body weigh were observed in 1000 and 2000 mg/L at gestational days 7 -21 days.

·      Lactational body weights were decreased at 1000 and 2000 mg/L at lactational days 1-21.

·      Lactational water consumptions were decreased at 1000 and 2000 mg/L at lactational days 7 -21 days.

·      No statistically differences in the number of males per litter.

·      No statistically differences in number of females mated, females pregnant and females littering.

·      No statistically differences in the percent of pregnant females/mated, percent of littered females/mated, percent of littered females/pregnant

·      No statistically differences in gestational length

 

B) F1- Generation

·      At PND 1:

o  Male - Pup weights of the 2000 mg/L group were statistically reduced.

o  Combined pup weights (male + female) of the 2000 mg/L group were statistically reduced.

o  At 2000 mg/L the adjusted pup male and male+female body weights were statistically reduced.

o  At PND 21 male, female, and male+female pups body weights were statistically reduced at the 2000mg/L group.

·      At PND 1 no differences in total pup lived and dead, live males, live females, live pups (male andfemale), dead male, dead female pups, or total dead pups, total dead per litter were observed.

·      At PND 21 no statistically differences in live pups.

·      No differences in the dead/litter at PND 1-4 and 4-21.

·      No differences in survival at PND 1-4 and 4-21.

·      No changes on testis weights, however epididymitis and cauda epididymitis weights were statistically reduced in male pups at the 2000 mg/L.

Effects on developmental toxicity

Description of key information

No developmental toxicity data of sufficient quality are available for tungsten (target substance). However, developmental (teratogenic) toxicity data are available for sodium tungstate (source substance), which are used for read-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 in the Category section of this IUCLID submission or Annex 3 in the CSR.

 

Segment II - Embryo-Foetal Development Study

A test adaptation is requested as no rat and/or rabbit OECD TG 414 study is available on sodium tungstate. Instead, we are submitting two separate Embryo-Foetal Development (ICH Segment II) studies on two species (rat and rabbit) on sodium tungstate studies conducted according to ICH's Harmonised Tripartite Guideline on Detection of Toxicity to Reproduction for Medicinal Products & Toxicity to Male Fertility S5 (R2) [finalised (Step 4) November 2005] and following GLP.

 

These ICH studies were completed in 2010 following the 2005’s adopted testing guidelines, which did not require anogenital distance (AGD) measurement in fetuses, testosterone levels in male pups, or thyroid hormone analysis in dams and pups as part of the study design (parameters included in the recently 2018 adopted OECD 414 testing guideline). 

 

The thyroid toxicity weight of evidence did not find adverse effects in adult rodents and humans:

·      US NTP sponsored studies reported no adverse effects on parathyroid and thyroid glands of female and male rats and mice exposed to sodium tungstate via drinking water at the highest dose of 2000 mg/kg bw/day (equivalent to 180 mg/kg bw/day in rats, and 500 mg/kg bw/day in mice).

·      McCain et al (2015) reported no adverse effects on thyroid and parathyroid glands in male and female rats exposed for 90-days to sodium tungstate (up to 200 mg/kg bw/day) via oral gavage.

·      Hanzu et al (2010) conducted a prospective, randomized, placebo-controlled, double-blind, proof-of-concept studyin humans (n=30). Following a 2-week lead-in period, 30 subjects were randomized to receive either sodium tungstate (n=16;100 mg/12h) or placebo (n=14) for 6 weeks. No differences in thyroid hormones [thyroid stimulating hormone (TSH), free triiodothyronine (T3) and thyroxine (T4)] were found.

 

A. First Species- Rat Embryo-Foetal Development

The effects of sodium tungstate on embryonic and foetal development were assessed via oral gavage to the rat, daily during the period of organogenesis.  Four groups of 20 time-mated, female rats of the Crl:CD (SD) strain were given 0 (purified water), 40, 80 or 160 mg/kg/day sodium tungstate (doses selected based on a dose range finding study which tested doses of 0, 50, 100, and 250 mg/kg/day ), once daily, by oral gavage, from Day 6 to Day 17 of gestation, inclusive.  The dose volume was 5 mL/kg body weight.   For all animals, clinical observations, body weights and food intake were recorded.  Blood samples for toxicokinetic evaluation were obtained on Days 6 and 17 of gestation.  Animals were killed on Day 20 of gestation and the progress and outcome of pregnancy were assessed.  Maternal dead body weight, gravid uterus weight and placenta weights were also recorded.  Foetuses were removed from the uterus, weighed, their sex was determined, and they were examined for external, visceral and skeletal abnormalities.

There was no mortality or clinical signs associated with sodium tungstate.  Females given 160 mg/kg/day gained less weight up to Day 15 of gestation but thereafter, mean weight gain was similar to, or greater than, Controls and consequent absolute body weight on Day 20 of gestation was comparable with Controls.  Body weight was unaffected at all other dose levels and there was no effect of sodium tungstate on food intake.  All females except one in the group given 80 mg/kg/day, were pregnant with live foetuses.  There was no adverse effect of sodium tungstate on the uterine/implantation or foetal data and no test item-related macroscopic abnormalities at necropsy. Sodium tungstate was not associated with any major foetal abnormalities.  Although there were increases in irregular palate rugae and changes in the extent of ossification in foetuses from litters given 80 or 160 mg/kg/day, these minor changes were considered not adverse.

Administration of sodium tungstate by oral gavage, once daily from Days 6 to 17 of gestation to Crl:CD(SD) rats at 40, 80 or 160 mg/kg/day was well tolerated.  Maternal effects (initial body weight change) were evident at 160 mg/kg/day and minor foetal abnormalities were apparent at 80 or 160 mg/kg/day; however, these sodium tungstate related changes were considered not adverse.

 

B. Second Species - Rabbit Embryo-Foetal Development

The effects of sodium tungstate on the embryonic and foetal development were assessed when administered orally, by gavage, to the pregnant rabbit, daily during the period of organogenesis.  

Doses for confirmatory study were selected based on a 7-day maximum tolerable dose (MTD) study. Animals dosed 200-300 mg/kg/day showed progressive weight loss suggesting that the oral MTD oral dose of sodium tungstate in the non-pregnant rabbit was less than 200 mg/kg/day.  Doses of 100 or 50 mg/kg/day were generally well tolerated with only small fluctuations in body weight. A high dose level between 100 and 200 mg/kg/day is concluded to be appropriate for the subsequent rabbit studies.

Four groups of 22 sexually mature timed-mated female New Zealand White rabbits were given 0 (purified water), 10, 30 or 100 mg/kg/day sodium tungstate (doses selected based on a dose range finding study which tested doses of 0, 30, 100, and 150 mg/kg/day) once daily from Day 6 to Day 18 of gestation, inclusive by oral gavage. Body weights, food intake and clinical observations were recorded.  Females were killed on Day 28 of gestation and a gross macroscopic necropsy performed.  The progress and outcome of pregnancy were assessed and maternal dead body weight, gravid uterus and placenta weights recorded.  The foetuses were removed from the uterus, weighed, sexed and examined for external, visceral and skeletal abnormalities.  There were no deaths or clinical signs considered to be related to sodium tungstate administration.   In the group given 100 mg/kg/day, mean body weight gain was significantly lower than that of the Controls during the dosing period.  Body weights and body weight gains were similar to Controls for animals given 10 or 30 mg/kg/day sodium tungstate.  Group mean food intake was similar in all groups throughout the study. There were 22, 19, 21 and 20 females with live foetuses on Day 28 of gestation in the groups given 0, 10, 30 or 100 mg/kg/day, respectively.  There was no effect of sodium tungstate on the uterine/implantation or foetal data. There were no test item-related major foetal abnormalities and the incidences of minor and variant foetal abnormalities were similar in all groups. Administration of sodium tungstate once daily by oral gavage from Day 6 of gestation until Day 18 of gestation to New Zealand White rabbits at dose levels of 10, 30 or 100 mg/kg/day was well tolerated with reductions in body weight gain at 100 mg/kg/day only.  There was no effect on the pregnancy data and no foetal abnormalities considered to be related to administration of sodium tungstate. Based on the above findings the No Observed Effect Level (NOEL) for maternal toxicity was considered to be 30 mg/kg/day and the No Observed Adverse Effect Level (NOAEL) was considered to be 100 mg/kg/day. The NOEL and the NOAEL for embryo-foetal development were considered to be 100 mg/kg/day.

Conclusion: The major ECHA criticism on the previous weight of evidence submitted to fulfill the developmental toxicity endpoint was the lack of skeletal malformation information as well as a second species (rabbit) study. Under this submission update new ICH Segment II rat and rabbit studies (never submitted previously) are included with the requested skeletal malformation information in two species satisfying the OECD 414 (first and second species) requirement.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subchronic

Species:

rabbit

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Additional information on Neurotoxicity and Developmental Neuro-Toxicity (DNT)

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, 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 results presented, and study design selected and the interpretation of the results in regulatory context. are further specified below.

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 (eg 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.

 

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
Water maze (dams)	No effect	No effect
Acoustic startle PPI (dams)	No effect	No effect
Spontaneous 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 (eg “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 potential adverse results from this sodium tungstate study is reported in three separate documents [Mclnturf et al (2007, 2008, 2011)] 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 could be considered prof of an adverse influence of tungstate on adult behavior, early pup behavior or an indicator of developmental toxicity and/or neurotoxicity in rats.

 

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

 

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 Test https://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

Justification for classification or non-classification

No reproductive/developmental studies are available for tungsten metal. However, data were available on sodium tungstate, which are 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 sodium tungstate is a reproductive/developmental (teratogenic) toxicant. Furthermore, based on bioaccessibility studies of tungsten metal that reported a 0.23% bioelution of tungsten after 5-hrs. incubation in gastric fluid, the equivalent amount of tungsten metal to reach NOAEL doses is substantially higher than 1,000 mg/kg/day (limit dose). Therefore, based on the lack of reproductive and/or developmental effects observed, no classification is warranted for tungsten metal.