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 fused tungsten carbide (target substance). However, reproductive toxicity data are available for sodium tungstate (source substance), which will be 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 attached description of the read-across approach.

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 Fertlity 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 exposedfrom Day 6 to Day 17 of gestation. Exposure to50 or 100 mg/kg/day was well tolerated the with no significant with no 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 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 (207, 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 2010) 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 either 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 (e.g. 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 libetum) 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 libetum). 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.

Link to relevant study records

Referenceopen allclose all

Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
Experiment start date (Animal arrival): 15 October 2015; Completion date of experimental phase (Last day of necropsy): 01 December 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
1. HYPOTHESIS FOR THE CATEGORY 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: Fused tungsten carbide
3. CATEGORY APPROACH JUSTIFICATION: See Annex 3 in CSR
4. DATA MATRIX: See Annex 3 in CSR
Reason / purpose:
read-across: supporting information
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Principles of method if other than guideline:
ICH Harmonised Tripartite Guideline: Detection of Toxicity to Reproduction for Medicinal Products & Toxicity to Male Fertility S5 (R2): finalised (Step 4) November 2005
GLP compliance:
yes
Limit test:
no
Justification for study design:
Dose levels were selected in consultation with the Sponsor on the basis of results from an embryo-foetal development study performed at Sequani (Sequani Study Number: OHS0005)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 1504246000
- Expiration date of the lot/batch: 05 May 2018
- Purity: 93.2%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The test item was stored at room temperature, protected from light and moisture.
- Stability under test conditions: Test item formulations at concentrations of 0.5 mg/mL to 50 mg/mL in the vehicle, spanning those used in this study (8 to 32 mg/mL), have been shown to be stable for up to 30 days at room temperature, when stored refrigerated and when frozen (approximately -80 ºC).

Species:
rat
Strain:
other: Crl:CD(SD) strain
Details on species / strain selection:
The rat is a suitable rodent species, acceptable to regulatory authorities and for which extensive background data are available. The Sprague Dawley rat is commonly used in reproduction studies because of the good fertility and fecundity of the strain.
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent, CT9 4LT, England
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: The animals were six to seven weeks of age on arrival and on examination were found to be healthy.
- Weight at study initiation: On the first day of dosing the males weighed 295 to 365 g and the females weighed 185 to 244 g.
- Housing:
- Diet (e.g. ad libitum): A pelleted rodent diet, VRF1 (manufactured by SDS) supplied by Charles River (UK) Limited, Margate, Kent, CT9 4LT, England, and mains tap water (in bottles) were freely available.
- Water (e.g. ad libitum): Tap water (in bottles) were freely available
- Acclimation period:
ENVIRONMENTAL CONDITIONS
- Temperature (°C): The target ranges for temperature were 19 °C to 23 °C.
- Humidity (%): The target ranges for humidity were 40 % to 70 %.
- Air changes (per hr): Room was air-conditioned.
- Photoperiod (hrs dark / hrs light): The study room was illuminated by fluorescent light set to give a cycle of 12 hours light and 12 hours dark.
IN-LIFE DATES: From: 15 October 2015 To: 01 December 2015
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
purified
Details on exposure:
Animals were dosed once daily, by gavage, using a rubber catheter and disposable syringe at a constant dose volume of 5 mL/kg body weight. Males were dosed for 14 days before and during pairing and until the day before necropsy. Females were dosed for 14 days before, during pairing and until Day 6 of gestation, inclusive. Individual doses were adjusted according to the most recent body weight.
Details on mating procedure:
After the pre-pairing dosing period, each female was paired with a male from the same group for up to 10 days. If a female is not mated within 10 days, the male was removed and another from the same group, that has previously mated, was substituted. This second pairing was continue for up to a further 5 days. Females with no evidence of mating after this second pairing period were sent for necropsy at the end of the pairing period. On confirmation of mating, the male was returned to the group cages and the female was caged individually. The male was allso returned to the group cage at the end of the pairing period.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis were conducted at Kymos Pharma Services using a validated method (SOP code C003-MP0023)
Duration of treatment / exposure:
The males were dosed once daily for 14 days before and during pairing until the day before necropsy. The females were dosed for 14 days before and during pairing and then until Day 6 of gestation, inclusive (Day 0 of gestation is the day of a sperm positive vaginal smear), or until the day before necropsy for unmated females.
Frequency of treatment:
Animals were dosed daily, males for a period of 14-days, and females for a period of 20 days.
Details on study schedule:
The males were dosed once daily for 14 days before and during pairing until the day before necropsy. The females were dosed for 14 days before and during pairing and then until Day 6 of gestation.
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Vehicle control
Dose / conc.:
40 mg/kg bw/day (actual dose received)
Dose / conc.:
80 mg/kg bw/day (actual dose received)
Dose / conc.:
160 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
There were 40 animal per group (20 males and 20 females)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
- Rationale for animal assignment (if not random):
- Fasting period before blood sampling for clinical biochemistry:
- Other:
Positive control:
Not applicable
Parental animals: Observations and examinations:
Animals were examined twice daily for mortality and morbidity, daily from the start of treatment for clinical signs of toxicity and changes in behaviour and were given a detailed clinical examination weekly.
Oestrous cyclicity (parental animals):
For 10 days before the start of the pairing period, vaginal smears were taken daily by lavage. During the pairing period, vaginal smears were taken daily, by lavage, until mating was confirmed by sperm being found in the smear.

The smear was examined under light microscopy and the stage of the oestrous cycle was determined by the type of cell present. The number of copulation plugs was also recorded to give an assessment of the mating activity of the animals.
Sperm parameters (parental animals):
When conducted, seminal fluid from the epididymis of all males was collected at scheduled necropsy and assessed for sperm concentration and motility using the Hamilton Thorne IVOS CASA system.
Postmortem examinations (parental animals):
- Females were killed on Day 13 of gestation. The males were killed approximately two weeks after completion of the mating period and a necropsy was performed.. The thoracic and abdominal cavities were opened by a ventral mid-line incision and the major organs were examined. Organs or tissues showing macroscopic abnormalities were removed and retained in fixative.
- The ovaries, testes, epididymides, prostate and seminal vesicles (including coagulating gland) were removed and the ovaries and testes were weighed. - - Pregnancy status was checked and the uterus of any apparently non-pregnant female was stained with ammonium sulphide to confirm pregnancy status.
Postmortem examinations (offspring):
For all pregnant females, the number of corpora lutea and the number and distribution of implantations in each uterine horn were recorded. Implantations were classified as early intrauterine deaths, dead embryos or live embryos. The implantations were numbered separately for the right and left horns. Numbering was sequential, commencing at the ovarian end through to the cervix.
Statistics:
Comparisons: Group 1 against Groups 2, 3 and 4
Statistical tests and parameters: Data was processed to give group mean values and standard deviations, where appropriate. Where the data allow, the following methods were used for statistical analysis. Depending on the nature of the data set to be analysed, appropriate tests were applied as indicated in the table below. Where parametric tests may be appropriate they were preceded by a check for homogeneity of variance using the Levene test and, where available, the Shapiro-Wilks test for normality. If either of these two assumptions fails a log transformation was applied before retesting. If the transformation fails, appropriate non-parametric tests was applied.
Probability values of less than 5 % were regarded as providing sufficient evidence to reject the null hypothesis and therefore statistical significance was identified at the p<0.05 level. For illustrative purposes, significance levels of p<0.01 and p<0.001 were also noted.
Reproductive indices:
1) Copulation index (%) = (no. of animals mated/no. of animals paired) x 100
2) Female Fertility index (%) = (no. of pregnant females/no. of females paired) x 100
3) Male Fertility index (%) = (no. of males siring one or more pregnancies/ no. of males paired) x 100
4) Fecundity index (%) female = no. of pregnant females no. of females mated x 100
5) Pre-implantation loss (%) = [(no. of corpora lutea –no. of implantation sites)/no. of corpora lutea] x 100
6) Post implantation loss (%) = [(no. of implantation sites –no. of live embryos)/no. of implantation sites] x 100
Clinical signs:
no effects observed
Mortality:
no mortality observed
Description (incidence):
There was no mortality or clinical signs associated with sodium tungstate. There was only one death: Control Male 10 was euthanised on Day 17 of the study following a hind limb injury which was unrelated to sodium tungstate.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
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 significantly lower than Controls (- 8.2 %; p<0.01). In females given 160 mg/kg/day, mean body weight gains were also lower than Controls during the dosing period but, this was 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.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Male and female food intake was unaffected by sodium tungstate.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
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.
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed
Description (incidence and severity):
There was no effect of sodium tungstate on fertility or mating; all animals mated and only one Control female (Animal 86) was not pregnant.
Pregnancy data were similar in all groups, with no adverse effect on the mean numbers of corpora lutea, implantations, the incidence of pre- or post-implantation loss or on the number of live embryos.
Key result
Dose descriptor:
NOAEL
Effect level:
> 160 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive function (oestrous cycle)
reproductive performance
Key result
Critical effects observed:
no
Key result
Remarks on result:
not measured/tested
Key result
Reproductive effects observed:
no
Conclusions:
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.
Endpoint:
extended one-generation reproductive toxicity - with developmental neurotoxicity (Cohorts 1A, 1B without extension, 2A and 2B)
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
Experiment start date (Animal arrival): 15 October 2015 - Completion date of experimental phase (Last day of data collection) 12 February 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
1. HYPOTHESIS FOR THE CATEGORY 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: Fused tungsten carbide
3. CATEGORY APPROACH JUSTIFICATION: See Annex 3 in CSR
4. DATA MATRIX: See Annex 3 in CSR
Reason / purpose:
read-across source
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Deviations:
no
Principles of method if other than guideline:
ICH Harmonised Tripartite Guideline: Detection of Toxicity to Reproduction for Medicinal Products & Toxicity to Male Fertility S5 (R2): finalised (Step 4) November 2005.
GLP compliance:
yes
Limit test:
no
Justification for study design:
SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS [please address all points below]:

- Premating exposure duration for parental (P0) animals
- Basis for dose level selection : Dose levels were selected in consultation with the Sponsor on the basis of results from an embryo-foetal development study performe
- Inclusion/exclusion of extension of Cohort 1B
- Termination time for F2
- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B
- Inclusion/exclusion of developmental immunotoxicity Cohort 3
- Route of administration
- Other considerations, eg on choice of species, strain, vehicle and number of animals [if applicable]
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 1504246000
- Expiration date of the lot/batch: 05 May 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Test item formulations at concentrations of 0.5 mg/mL to 50 mg/mL in the vehicle, spanning those used in this study (8 to 32 mg/mL), have been shown to be stable for up to 30 days at room temperature, when stored refrigerated and when frozen (approximately -80 ºC).

Species:
rat
Strain:
other: Crl: CD(SD)
Details on species / strain selection:
The rat is a suitable rodent species, acceptable to regulatory authorities and for which extensive background data are available
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent, CT9 4LT, England
- Age at study initiation: The animals were 9 to 10 weeks of age on arrival.
- Weight at study initiation: (On the first day of dosing the females weighed 182 g to 293 g.
- Housing: The parental females were housed individually in grid-floor cages suspended over paperlined trays. Towards the end of gestation (from Day 15 ± 1 of gestation), and with the litter during lactation, females were housed in solid-floor cages with appropriate bedding provided. The weaned, selected F1 generation animals were housed in fives, by sex, in grid-floor cages suspended over paper-lined trays until mating. For mating of the F1 generation, one male and one female from the same group (not siblings) were housed together in grid-floor cages suspended over paper-lined trays. On confirmation of mating, the males were returned to the group cages and the females were housed in grid-floor cages, up to five females per cage.
- Diet (e.g. ad libitum): A pelleted rodent diet, VRF1 (manufactured by SDS) supplied.
- Water (e.g. ad libitum): tap water (in bottles) were freely available
- Acclimation period: After at least two days acclimatisation, the animals were re-examined and confirmed to be suitable for us.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): The target ranges for temperature were 19 °C to 23 °C.
- Humidity (%): The target ranges for humidity were 40 % to 70 %.
- Air changes (per hr): Room was air-conditioned.
- Photoperiod (hrs dark / hrs light): The study room was illuminated by fluorescent light set to give a cycle of 12 hours light and 12 hours dar.
IN-LIFE DATES: Experiment start date (Animal arrival) 15 October 2015; Completion date of experimental phase (Last day of data collection) 12 February 2016.
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was formulated and used within the known stability period, for each group separately, as a solution in purified water.

Details on mating procedure:
Each female had been mated with a sexually mature male of the same strain. The day on which mating was detected was designated Day 0 of gestation.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
All test item formulations were analysed to assess achieved concentrations of sodium tungstate, using method C003-MP0023.
Duration of treatment / exposure:
Animals were dosed from Day 6 of gestation to Day 20 of lactation
Frequency of treatment:
Animals were dosed once daily, by oral gavage
Details on study schedule:
F1 parental animals not mated until 3 weeks after selected from the F1 litters: From Day 21 of age, after the F1 generation were weaned, 20 male and 20 female pups per group were randomly selected (at least one of each sex from each of the first 20 weaned litters, where possible) for rearing to sexual maturity. Where there were fewer than 20 weaned litters for selection of a particular sex, a random selection was made of those available litters and two pups per sex in the first instance were taken from selected litters to ensure a full complement of 20 males and 20 females per group. Pups were selected on a total randomisation basis.
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
40 mg/kg bw/day (actual dose received)
Dose / conc.:
80 mg/kg bw/day (actual dose received)
Dose / conc.:
160 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
P0 - Twenty two mated female Crl: CD(SD) rats per dose.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected based on results from an embryo-foetal development previously study.
- Rationale for animal assignment (if not random): Parental females were allocated to group using a stratified randomisation procedure based on individual body weights recorded on Day 0 of gestation (making sure that females mated with the same male were spread across the groups).
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Animals were examined twice daily for mortality and morbidity, daily from the start of dosing for clinical signs of toxicity or changes in behaviour, and were given a detailed clinical examination weekly.

BODY WEIGHT: Yes
- Day 0 gestation body weights were recorded by the animal supplier. At Sequani, body weights were recorded daily from Day 5 of gestation, throughout gestation and then on Days 0 (where required for dose calculation), 1, 4, 7, 10, 14, 17 and 21 of lactation.

FOOD CONSUMPTION AND COMPOUND INTAKE:
The amount of food consumed by each female was recorded over Days 6 to 9, 9 to 12, 12 to 15 and 15 to 20 of gestation and over Days 1 to 4, 4 to 7, 7 to 10 and 10 to 14 of lactation. Food intake was not recorded during late lactation as the pups also start to eat diet.
Oestrous cyclicity (parental animals):
F1 - During the pairing period, vaginal smears were taken daily, by lavage, until mating was confirmed by sperm in the smear or by other evidence of mating (in consultation with the Study Director). The number of ejected copulation plugs was also recorded to give an assessment of the mating activity of the animals.
Litter observations:
STANDARDISATION OF LITTERS
On Day 4 of lactation, the size of each litter was adjusted by eliminating extra pups to yield, as nearly as possible, 4 males and 4 females. Litters of fewer than 8 pups were not altered. Pups were selected on a total randomisation basis.

PARAMETERS EXAMINED
The following parameters were examined in F1A offspring:
The total litter size was recorded after completion of parturition and daily thereafter. The number of each sex was recorded daily, from Day 1 of lactation up to and including Day 21 of lactation. The thoracic and abdominal cavities were opened by a ventral mid-line incision and the major organs examined. All pups were examined for malformations.

GROSS EXAMINATION OF DEAD PUPS: Yes
For all animals, including early decedents, the thoracic and abdominal cavities will be opened by a ventral mid-line incision and the major organs examined. Organs or tissues showing any macroscopic abnormalities.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: Yes

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

P1 (F1 Generation) Females -
- Learning and memory 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 (Ethovision®). 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.
- 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 (Motormonitor™).

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: The males will be killed approximately 2 weeks after completion of the mating period and a necropsy performed.
- Maternal animals: The P generation was killed on Day 21 of lactation after weaning of their litters or on Day 26 post-mating, if not apparently pregnant or had not littered. F1 mated females were killed on Day 13 of gestation (or on an estimated Day 13 of gestation for undetected matings). Non-mated females were killed 14 days after the end of the pairing period.

GROSS NECROPSY
For all animals, including early decedents, the thoracic and abdominal cavities were opened by a ventral mid-line incision and the major organs examined.

HISTOPATHOLOGY / ORGAN WEIGHTS
Histopathological examination of organs or tissues with macroscopic abnormalities was undertaken if considered necessary

Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 4 days of lactation.
- These animals were subjected to postmortem examinations (macroscopic) as follows:

GROSS NECROPSY
- The thoracic and abdominal cavities were opened by a ventral mid-line incision and the major organs examined.

HISTOPATHOLOGY / ORGAN WEIGHTS
- Histopathological examination of organs or tissues with macroscopic abnormalities was undertaken if considered necessary
Statistics:
Data were processed to give group mean values and standard deviations, where appropriate. Where the data allowed, the following methods were used for statistical analysis, comparing Groups 2, 3 and 4 against Group 1. Depending on the nature of the data set that was to be analysed, appropriate tests were applied, as indicated in the table below. Where parametric tests were appropriate they were preceded by a check for homogeneity of variance using the Levene test and, where available, the Shapiro-Wilks test for normality. If either of these two assumptions failed, a log transformation was applied before retesting. If the transformation failed, appropriate nonparametric tests were applied. Probability values of less than 5 % were regarded as providing sufficient evidence to reject the null hypothesis and therefore statistical significance was identified at the p<0.05 level. For illustrative purposes, significance levels of p<0.01 and p<0.001 were also noted.
Reproductive indices:
Parental:
- Gestation Index (%) = (no. of pregnant females with live pups born/no. of pregnant females) x 100
- Post-implantation loss (%) = (no. of implantation sites - no. of live embryos)/no. of implantation sites x 100

F1 Generation - Parental
- Female copulation index (%) = (no. of females mated/no. of females paired) x 100
- Male copulation index (%) = (no. of males mated/no. of males paired) x 100
- Female fertility index (%) = (no. of pregnant females/no. of females paired) x 100
- Male fertility index (%) = (no. of males siring one or more pregnancies/no. of males paired) x 100
- Pre-implantation loss (%) = [(no. of corpora lutea - no. of implantation sites)/no. of corpora lutea] x 100
- Post-implantation loss (%) = [(no. of implantation sites - no. of live embryos)/no. of implantation sites] x 100
Offspring viability indices:
- Sex ratio (%) = no. of male pups total no. of pups x 100
- Live birth index (%) = (no. of pups born alive total/no. of pups born) x 100
- Viability index 1 (%) = (no. of pups alive on Day 4 of age before culling/no. of pups born alive) x 100
- Viability index 2 (%) = (no. of pups alive on Day 7 of age/no. of pups alive on Day 4 of age after culling) x 100
- Viability index 3 (%) = (no. of pups alive on Day 14 of age/no. of pups alive on Day 7 of age) x 100
- Viability index 4 (%) = (no. of pups alive on Day 21 of age/no. of pups alive on Day 14 of age) x 100
- Lactation index (%) = (no. of pups alive on Day 21 of age/no. of pups on Day 4 of age after culling) x 100
- Cumulative survival index (%) = (no. of pups alive Day 21 of age/no. of pups Day 4 of age after culling) x (no. of pups alive Day 4 of age before culling/ total no. of pups born) x 100
Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs associated with sodium tungstate.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were no deaths associated with sodium tungstate. There were three decedents detailed as below: Female 28 given 40 mg/kg/day was euthanised on Day 15 of lactation following a dosing trauma which was unrelated to the test item. Female 73 given 160 mg/kg/day was euthanised on Day 22 of gestation due to dystocia. Dystocia is seen occasionally in this strain of rat and as the remaining females in this group littered successfully, this was considered not to be test item-related. Female 72 given 160 mg/kg/day was found dead on Day 21 of gestation with no evidence at necropsy to confirm cause of death. Although this death was in the high dose group, due to the lack of clinical signs seen in other animals it was considered to be unrelated to test item.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Maternal body weight gains for females given 160 mg/kg/day were lower than Controls (p<0.001) during gestation; however, although body weights were lower at the start of the lactation period, these females gained more weight (p<0.001) than Controls over the whole lactation period. During gestation and lactation, body weight gains for females given 40 or 80 mg/kg/day were similar to Controls.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There was no effect of sodium tungstate on food intake at any dose level during gestation or lactation.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed
Description (incidence and severity):
The duration of gestation in all groups given sodium tungstate was similar to that of the Controls and there were no effects on parturition that were considered to be related to sodium tungstate.
There were 21, 19, 21 and 20 females that littered in the Control group and groups given 40, 80 or 160 mg/kg/day, respectively. Control Female 2, Females 24, 36 and 38 given 40 mg/kg/day and Female 61 given 80 mg/kg/day were all not pregnant and Female 88 was sent to necropsy on Day 4 of lactation due to total litter loss.
The mean number of pups born was similar in all groups and there was no effect on pup survival from birth to weaning. Female 88 given 160 mg/kg/day had lost its entire litter by Day 4 of lactation, the pups were either found dead or had no milk in the stomach, which suggests the dam was not lactating properly. Although this was a high dose female, total litter death is seen occasionally in this strain of rat and therefore this was considered not to be test item-related. 4
Key result
Dose descriptor:
NOEL
Remarks:
P0 females
Effect level:
80 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:
160 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical signs
mortality
food consumption and compound intake
reproductive performance
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs that were associated with maternal administration of sodium tungstate.
Mortality:
no mortality observed
Description (incidence):
There were no deaths associated with maternal administration of sodium tungstate.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
During the first week of the F1 generation period, females maternally given 160 mg/kg/day had lower group mean body weights and body weight gains when compared with Controls, achieving statistical significance (p<0.05). Female body weight gains were lower throughout the pre-pairing period (p<0.05, p<0.01) and although group mean body weights were lower than Controls during gestation, body weight gains were similar to Controls. At this maternal dose, males also had lower body weight gains than Controls (p<0.05) during the first week of the F1 generation; thereafter body weight gains were generally similar to Controls. Group mean body weights and body weight gains for both sexes in the maternal groups given 40 or 80 mg/kg/day were similar to Controls.
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:
no effects observed
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
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.
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.
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
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no macroscopic necropsy findings that were considered to be related to maternal administration of sodium tungstate.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
There was no effect of maternal administration of sodium tungstate on female sexual development. There was no effect of maternal administration of sodium tungstate on the pre-coital interval.
Reproductive function: sperm measures:
not specified
Description (incidence and severity):
Two males maternally given 40 mg/kg/day, Males 123 and 126, and one male maternally given 160 mg/kg/day, Male 169, did not mate. At necropsy there were no abnormalities noted in the reproductive organs of these males.
Reproductive performance:
no effects observed
Description (incidence and severity):
There was no effect of maternal administration of sodium tungstate on the copulation or fertility indices. One female in the group maternally given 160 mg/kg/day was not pregnant, which was considered not to be related to sodium tungstate.
Key result
Dose descriptor:
NOEL
Effect level:
80 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
NOAEL
Effect level:
160 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical signs
mortality
reproductive function (oestrous cycle)
reproductive performance
Key result
Dose descriptor:
NOAEL
Effect level:
160 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
gross pathology
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs that were associated with maternal administration of sodium tungstate.
Mortality / viability:
no mortality observed
Description (incidence and severity):
There were no deaths that were associated with maternal administration of sodium tungstate.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean pup body weights in the group maternally given 160 mg/kg/day, were slightly lower than Controls on Day 1 of age. Group mean body weights on Day 4 and 7 of age were statistically significantly lower than Controls (p<0.05) so that overall group mean body weight gain over Day 1 to 21 of age was slightly lower than Controls; this is considered to be related to maternal sodium tungstate administration. There was no adverse effect on pup body weight or body weight gain at 40 or 80 or 160 mg/kg/da
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
Anogenital distance (AGD):
not specified
Nipple retention in male pups:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no macroscopic necropsy findings in the offspring that were attributed to the maternal administration of sodium tungstate.
Histopathological findings:
not specified
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
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
Developmental immunotoxicity:
not specified
Key result
Dose descriptor:
NOEL
Generation:
F1 (cohort 1A)
Effect level:
160 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
sexual maturation
clinical signs
mortality
gross pathology
Key result
Dose descriptor:
NOAEL
Generation:
F1 (cohort 2A)
Effect level:
160 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Behavior (funtional observations)
Key result
Dose descriptor:
NOEL
Generation:
F1 (cohort 2B)
Effect level:
160 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
developmental neurotoxicity
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Conclusions:
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.

On this basis, the No Observed Adverse Effect Level (NOAEL) for embryonic, foetal and post-natal development was considered to be 160 mg/kg/day.
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, i.e. 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

Segment II - Embryo-Foetal Development Study

No developmental toxicity data of sufficient quality are available for fused tungsten carbide (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.

A test adaptation is requested as no rat and/or rabbit OECD TG 414 study is available on sodium tungstate. Instead, we are submitting an Embryo-Foetal Development (ICH Segment II) study on one species (rat) on sodium tungstate 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 study was 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-inperiod, 30subjectswere 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.

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.  

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 on one species. Under this resubmission update new ICH Segment II rat study (never submitted previously) is included with the requested skeletal malformation information in one species satisfying the OECD 414 (first species) requirement.

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
Study period:
Experiment start date (Animal Arrival) - 07 April 2015 to Completion date of experimental phase (Foetal pathology): 23 April 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
1. HYPOTHESIS FOR THE CATEGORY 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: Fused tungsten carbide
3. CATEGORY APPROACH JUSTIFICATION: See Annex 3 in CSR
4. DATA MATRIX: See Annex 3 in CSR
Reason / purpose:
read-across: supporting information
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
ICH Harmonised Tripartite Guideline: Detection of Toxicity to Reproduction for Medicinal Products & Toxicity to Male Fertility S5 (R2): finalised (Step 4) November 2005.
Deviations:
no
Principles of method if other than guideline:
ICH Harmonised Tripartite Guideline: Detection of Toxicity to Reproduction for Medicinal Products & Toxicity to Male Fertility S5 (R2): finalised (Step 4) November 2005.
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 2214
- Expiration date of the lot/batch: 01 March 2017
- Purity test date: 85.4%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The test item will be stored at room temperature, protected from light.
- Stability under test conditions: Test item formulations at concentrations of 0.5 mg/mL to 50 mg/mL in vehicle have been shown to be stable for up to 14 days at room temperature, when stored refrigerated and when frozen (approximately -80 ºC) (Kymos Reference S15/341-KE). On this basis, formulations, including Control, were stored refrigerated prior to use.
Species:
rat
Strain:
other: Crl:CD (SD
Remarks:
Charles River (UK) Limited, Margate, Kent, CT9 4LT, England.
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent, CT9 4LT, England.
- Age at study initiation: 9 to 10 weeks
- Weight at study initiation: On the first day of dosing they weighed 213 to 274 g.
- Housing: Females were housed individually in grid-floor cages over paper lined trays.
- Diet and water (e.g. ad libitum): A pelleted rodent diet, VRF1 (manufactured by SDS) supplied by Charles River (UK) Limited, Margate, Kent, CT9 4LT, England, and mains tap water (in bottles) will be freely available.
- Acclimation period: The animals were acclimatised within the study room for at least two days after arrival. Towards the end of this period the animals were re-examined to confirm their suitability for use.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Target ranges 19 °C to 23 °C
- Humidity (%): Target ranges 40 % to 70 %,
- Air changes (per hr): Room was air-conditioned
- Photoperiod (hrs dark / hrs light): The room was illuminated by fluorescent light set to give a cycle of 12 hours light and 12 hours dark.

IN-LIFE DATES: From: To: 07 April 2015-23 April 2015
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was formulated for dosing as a solution in the vehicle (purified water). Separate formulations were prepared for each dose level. The weighed quantity of test item was mixed in the appropriate quantity of vehicle. The formulations were prepared within the known stability period (14 days).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Formulations for use on the first day and towards the end of dosing was analysed to determine their achieved concentrations. Vehicle (for Controls) was also be analysed on these occasions to confirm the absence of the test item. Analysis was conducted at Kymos Pharma Services using a validated method (code C003-MP0023).
Details on mating procedure:
The females were obtained from the supplier timed-mated and were by Day 1 to Day 4 of gestation on arrival. For mating, each female were paired with a sexually mature male of the same strain. The day on which mating is detected was designated Day 0 of gestation.
Duration of treatment / exposure:
Animals wiere dosed once daily, from Day 6 of gestation to Day 17 of gestation inclusive. Control animals received the vehicle only, following the same regimen as the other groups.
Frequency of treatment:
Animals were dosed once daily
Duration of test:
From Day 6 of gestation to Day 17 of gestation inclusive
Dose / conc.:
0 mg/kg bw/day
Remarks:
Vehicle control
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
20 females per group per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels have been selected after examining existing toxicity data and on the basis of results from a preliminary dose range finding study (Sequani Study Number: OHS0004).
- Rationale for animal assignment (if not random): Allocation to groups wiere performed using a stratified randomisation procedure based on body weight ranges recorded on arrival.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
Animals will be examined twice daily for mortality and morbidity. During the treatment period each animal was routinely checked pre-dose and soon after completion of dosing. On week days, additional observations were made approximately 1 hour after dosing and approximately 4 hours after dosing or at the end of the working day (whichever is sooner).

DETAILED CLINICAL OBSERVATIONS: Yes
All animals will be examined daily for clinical signs of toxicity or changes in behaviour and appearance from the start of treatment.

BODY WEIGHT: Yes
Day 0 of gestation body weight will be recorded by the supplier. At Sequani, body weights will be recorded daily from Day 5 to 18 of gestation, inclusive, then again on Day 20 of gestation.

FOOD CONSUMPTION: Yes
The amount of food consumed by each animal will be recorded over Days 6 to 9, 9 to 12, 12 to 15, 15 to 18, and 18 to 20 of gestation.


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #: Females were killed on Day 20 of gestation
- Organs examined: The animals were weighed, the thoracic and abdominal cavities were opened by a ventral mid-line incision and the major organs were examined. Gravid uterus and placenta weights were recorded and organs or tissues showing any macroscopic abnormalities were removed and retained in fixative

Ovaries and uterine content:
For pregnant females the following observations will be made: 1. Number of corpora lutea 2. Number and distribution of implantations in each uterine horn, classified as early intrauterine deaths, late intrauterine deaths, dead foetuses or live foetuses 3. Gravid uterus weight 4. Placental weight
The implantations are numbered separately for the right and left horns. Numbering is sequential, commencing at the ovarian end through to the cervix. The live foetuses and their placentae will be removed and the uterus and ovaries will be retained in neutral buffered formaldehyde. Gravid uterus and placenta weights will be recorded for pregnant females killed on Day 20 of gestation only.
Fetal examinations:
Live foetuses will be killed by rapid cooling followed by immersion in fixative. The following observations will be made for live foetuses killed on Day 20 of gestation: 1. Foetal weights; 2. Foetal sexes, and 3. Foetal abnormalities (to include external, fresh visceral, fixed visceral and/or skeletal examinations)
Statistics:
Comparisons: Group 1 against Groups 2, 3 and 4.

Statistical tests and parameters: Data was processed to give group mean values and standard deviations, where appropriate. Where the data allow, the following methods wiere used for statistical analysis. Depending on the nature of the data set to be analysed, appropriate tests were applied. Where parametric tests may be appropriate they preceded by a check for homogeneity of variance using the Levene test and, where available, the Shapiro-Wilks test for normality. If either of these two assumptions fails a log transformation was applied before retesting. If the transformation fails, appropriate non-parametric tests was applied.

Proportions of foetuses affected were treated as continuous nonparametric data, using one-sided step-wise Jonckheere Tests. Probability values of less than 5 % were regarded as providing sufficient evidence to reject the null hypothesis and therefore statistical significance was identified at the p<0.05 level. For illustrative purposes, significance levels of p<0.01 and p<0.001 also were noted.
Indices:
1) Pre-implantation loss (%) = [(no. corpora lutea – no. implantation sites)/ number of corpora lutea] x 100
2) Post-implantation loss (%) = [(no. implantation sites – no. live foetuses)/no. of implantation sites] x 100

Mean foetal body weights will be calculated separately by sex for each litter; group mean body weights will be calculated (separately by sex) from the litter means. The percentage of foetuses in each litter exhibiting each classification of abnormality was calculated; group mean percentages was calculated from the litter percentages. The percentage of male foetuses, out of the total number of foetuses, was calculated for each litter.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Most animals given 250 mg/kg/day had decreased activity by four hours after dosing on Day 6 of gestation, however, this was an isolated occasion. Treatment-related adverse clinical signs, with decreases in activity seen between one and four hours after dosing on Days 6 and/or 7 of gestation considered to be transient.
Mortality:
mortality observed, treatment-related
Description (incidence):
Female 28 (250 mg/kg/day) was killed on Day 9 of gestation due to marked body weight loss (-14 %) from the start of dosing; this animal also ate notably less than Controls (-51 %). There were no deaths at 50 or 100 mg/kg/day.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Female 28 (250 mg/kg/day) was killed on Day 9 of gestation due to marked body weight loss (-14 %) from the start of dosing; this animal also ate notably less than Controls (-51 %). For the remaining animals in this group, although food intake was comparable with Controls, marked body weight losses were also apparent from Day 8 of gestation (up to 12 %), such that by Day 11 of gestation, this dose level was considered unsuitable for future use and the remaining animals in this group killed. There was no effect of sodium tungstate on body weight gain at 50 or 100 mg/kg/day.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
There was no effect of sodium tungstate on body weight gain or food intake at 50 or 100 mg/kg/day.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Female 28 (250 mg/kg/day) was killed on Day 9 and macroscopic necropsy identified a pale corticomedullary junction in each kidney. At necropsy, all animals, presented no macroscopic abnormalities in animals exposed to 50 or 100 mg/kg/day.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
At 50 or 100 mg/kg/day, there were no effects on the uterine/implantation data. At 100 mg/kg/day the incidence of pre- and post-implantation losses were similar to Controls.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Description (incidence and severity):
In the group given 100 mg/kg/day, mean number of live foetuses was lower than Controls, however, this reflected a lower mean number of corpora lutea as the incidence of pre- and post- implantation losses were similar to Controls; therefore, this difference was unrelated to sodium tungstate.
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
At necropsy, all animals, except Female 26, were pregnant. All females in the groups given 0, 50 or 100 mg/kg/day were pregnant.
Key result
Dose descriptor:
NOEL
Effect level:
ca. 100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
LOAEL
Effect level:
250 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
clinical signs
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
No changes on group mean values of Litter weights (g) / foetal data
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
In the group given 100 mg/kg/day, mean number of live foetuses was lower than Controls, however, this reflected a lower mean number of corpora lutea as the incidence of pre- and post-implantation losses were similar to Controls; therefore, this difference was unrelated to sodium tungstate
Changes in sex ratio:
no effects observed
Description (incidence and severity):
At 50 or 100 mg/kg/day there was no adverse effect on sex ratio.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Mean foetal weight was marginally higher than Controls in the group given 100 mg/kg/day, but was attributed to the smaller litter size and not an effect of sodium tungstate.
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Description (incidence and severity):
Maternal treatment at 50 or 100 mg/kg/day was not associated with any external foetal abnormalities, with only one foetus in the group given 100 mg/kg/day having the minor abnormality of a filamentous tail tip. As no other foetuses had this or similar abnormalities, this abnormality was considered unrelated to Sodium Tungstate
Skeletal malformations:
not specified
Visceral malformations:
not specified
Key result
Dose descriptor:
NOEL
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
external malformations
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Conclusions:
Administration of 50 or 100 mg/kg/day sodium tungstate, once daily, by oral gavage to the rat, from Day 6 to Day 17 of gestation, was well tolerated, with no maternal or embryo-foetal toxicity. Administration of 250 mg/kg/day sodium tungstate elicited marked maternal toxicity which necessitated the termination of the group by Day 11 of gestation. Consequently, a dose level below 250 mg/kg/day is considered a suitable high dose level for the subsequent pivotal embryo-foetal development study.
Endpoint:
developmental toxicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
Experiment start date (Animal Arrival) - 30 July 2015 to Completion date of experimental phase (Foetal pathology): 09 October 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
1. HYPOTHESIS FOR THE CATEGORY 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: Fused tungsten carbide
3. CATEGORY APPROACH JUSTIFICATION: See Annex 3 in CSR
4. DATA MATRIX: See Annex 3 in CSR
Reason / purpose:
read-across: supporting information
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
ICH Harmonised Tripartite Guideline: Detection of Toxicity to Reproduction for Medicinal Products & Toxicity to Male Fertility S5 (R2): finalised (Step 4) November 2005.
Deviations:
no
Principles of method if other than guideline:
ICH Harmonised Tripartite Guideline: Detection of Toxicity to Reproduction for Medicinal Products & Toxicity to Male Fertility S5 (R2): finalised (Step 4) November 2005.
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 1504246000
- Purity test date: 93.1%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The test item will be stored at room temperature, protected from light.
- Stability under test conditions: Test item formulations at concentrations of 0.5 mg/mL to 50 mg/mL in vehicle have been shown to be stable for up to 14 days at room temperature, when stored refrigerated and when frozen (approximately -80 ºC) (Kymos Reference S15/341-KE). On this basis, formulations, including Control, were stored refrigerated prior to use.
Species:
rat
Strain:
other: Crl:CD (SD
Remarks:
Charles River (UK) Limited, Margate, Kent, CT9 4LT, England.
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent, CT9 4LT, England.
- Age at study initiation: 9 to 10 weeks
- Housing: Females were housed individually in grid-floor cages over paper lined trays.
- Diet and water (e.g. ad libitum): A pelleted rodent diet, VRF1 (manufactured by SDS) supplied by Charles River (UK) Limited, Margate, Kent, CT9 4LT, England, and mains tap water (in bottles) will be freely available.
- Acclimation period: The animals were acclimatised within the study room for at least two days after arrival. Towards the end of this period the animals were re-examined to confirm their suitability for use.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Target ranges 19 °C to 23 °C
- Humidity (%): Target ranges 40 % to 70 %
- Air changes (per hr): Room was air-conditioned
- Photoperiod (hrs dark / hrs light): The room was illuminated by fluorescent light set to give a cycle of 12 hours light and 12 hours dark.

IN-LIFE DATES: From: To:
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was formulated for dosing as a solution in the vehicle (purified water). Separate formulations were prepared for each dose level. The weighed quantity of test item was mixed in the appropriate quantity of vehicle. The formulations were prepared within the known stability period (14-days).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Formulations for use on the first day and towards the end of dosing was analysed to determine their achieved concentrations. Vehicle (for Controls) was also be analysed on these occasions to confirm the absence of the test item. Analysis was conducted at Kymos Pharma Services using a validated method (code C003-MP0023).
Details on mating procedure:
The females were obtained from the supplier timed-mated and were by Day 1 to Day 4 of gestation on arrival. For mating, each female were paired with a sexually mature male of the same strain. The day on which mating is detected was designated Day 0 of gestation.
Duration of treatment / exposure:
Animals wiere dosed once daily, from Day 6 of gestation to Day 17 of gestation inclusive. Control animals received the vehicle only, following the same regimen as the other groups.
Frequency of treatment:
Animals were dosed once daily
Duration of test:
From Day 6 of gestation to Day 17 of gestation inclusive
Dose / conc.:
0 mg/kg bw/day
Remarks:
Vehicle control
Dose / conc.:
40 mg/kg bw/day (actual dose received)
Dose / conc.:
80 mg/kg bw/day (actual dose received)
Dose / conc.:
160 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
20 females per group per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels have been selected after examining existing toxicity data and on the basis of results from a preliminary dose range finding study (Sequani Study Number: OHS0004).
- Rationale for animal assignment (if not random): Allocation to groups wiere performed using a stratified randomisation procedure based on body weight ranges recorded on arrival.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
Animals will be examined twice daily for mortality and morbidity. During the treatment period each animal was routinely checked pre-dose and soon after completion of dosing. On week days, additional observations were made approximately 1 hour after dosing and approximately 4 hours after dosing or at the end of the working day (whichever is sooner).

DETAILED CLINICAL OBSERVATIONS: Yes
All animals will be examined daily for clinical signs of toxicity or changes in behaviour and appearance from the start of treatment.

BODY WEIGHT: Yes
Day 0 of gestation body weight will be recorded by the supplier. At Sequani, body weights will be recorded daily from Day 5 to 18 of gestation, inclusive, then again on Day 20 of gestation.

FOOD CONSUMPTION: Yes
The amount of food consumed by each animal will be recorded over Days 6 to 9, 9 to 12, 12 to 15, 15 to 18, and 18 to 20 of gestation.


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #: Females were killed on Day 20 of gestation
- Organs examined: The animals were weighed, the thoracic and abdominal cavities were opened by a ventral mid-line incision and the major organs were examined. Gravid uterus and placenta weights were recorded and organs or tissues showing any macroscopic abnormalities were removed and retained in fixative

Ovaries and uterine content:
For pregnant females the following observations will be made: 1. Number of corpora lutea 2. Number and distribution of implantations in each uterine horn, classified as early intrauterine deaths, late intrauterine deaths, dead foetuses or live foetuses 3. Gravid uterus weight 4. Placental weight
The implantations are numbered separately for the right and left horns. Numbering is sequential, commencing at the ovarian end through to the cervix. The live foetuses and their placentae will be removed and the uterus and ovaries will be retained in neutral buffered formaldehyde. Gravid uterus and placenta weights will be recorded for pregnant females killed on Day 20 of gestation only.
Fetal examinations:
Live foetuses will be killed by rapid cooling followed by immersion in fixative. The following observations will be made for live foetuses killed on Day 20 of gestation: 1. Foetal weights; 2. Foetal sexes, and 3. Foetal abnormalities (to include external, fresh visceral, fixed visceral and/or skeletal examinations)
Statistics:
Comparisons: Group 1 against Groups 2, 3 and 4.

Statistical tests and parameters: Data was processed to give group mean values and standard deviations, where appropriate. Where the data allow, the following methods wiere used for statistical analysis. Depending on the nature of the data set to be analysed, appropriate tests were applied. Where parametric tests may be appropriate they preceded by a check for homogeneity of variance using the Levene test and, where available, the Shapiro-Wilks test for normality. If either of these two assumptions fails a log transformation was applied before retesting. If the transformation fails, appropriate non-parametric tests was applied.

Proportions of foetuses affected were treated as continuous nonparametric data, using one-sided step-wise Jonckheere Tests. Probability values of less than 5 % were regarded as providing sufficient evidence to reject the null hypothesis and therefore statistical significance was identified at the p<0.05 level. For illustrative purposes, significance levels of p<0.01 and p<0.001 also were noted.
Indices:
1) Pre-implantation loss (%) = [(no. corpora lutea – no. implantation sites)/ number of corpora lutea] x 100
2) Post-implantation loss (%) = [(no. implantation sites – no. live foetuses)/no. of implantation sites] x 100

Mean foetal body weights will be calculated separately by sex for each litter; group mean body weights will be calculated (separately by sex) from the litter means. The percentage of foetuses in each litter exhibiting each classification of abnormality was calculated; group mean percentages was calculated from the litter percentages. The percentage of male foetuses, out of the total number of foetuses, was calculated for each litter.
Clinical signs:
no effects observed
Description (incidence and severity):
There was no clinical signs associated with sodium tungstate.
Mortality:
no mortality observed
Description (incidence):
There was no moratlity associated with sodium tungstate.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
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. Whilst this initial body weight effect resulted in a statistically significantly lower body weight gain over the dosing period (p<0.05), absolute body weight on Day 20 of gestation was comparable with Controls, including that corrected for the gravid uterus weight. Body weight was unaffected in the groups given 40 or 80 mg/kg/da
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There was no effect of sodium tungstate on food intake.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no macroscopic abnormalities associated with sodium tungstate
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There was no adverse effect of sodium tungstate on the uterine/implantation data
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
All females were pregnant with the exception of Animal 53 given 80 mg/kg/day. There was no adverse effect of Sodium Tungstate on the uterine/implantation data.
Key result
Dose descriptor:
NOEL
Effect level:
ca. 80 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
LOAEL
Effect level:
ca. 160 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
No changes on group mean values of Litter weights (g) / foetal data
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There was no effect of sodium tungstate on the sex ratio
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
No changes on group mean values of Litter weights (g) / foetal data
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
The overall incidence of minor foetal abnormalities was significantly higher than Controls in litters given 160 mg/kg/day (p<0.05). This was largely attributable to an increase in the number of foetuses which showed irregular palate ridging and incomplete nasal ossification; both incidences were above the background data ranges. Foetuses from litters given 80 mg/kg/day also showed slightly higher incidences of these abnormalities, although these did not achieve statistical significance and were only marginally higher than the background data ranges.

Changes in the palate rugae seen on this study are considered likely to be related to Sodium Tungstate given the dose response and increase in litter incidence (25 foetuses from 16 litters in the group given 160 mg/kg/day compared with 7 foetuses from 6 litters in the Control group). However, in the absence of any attendant major abnormalities, or other signs of generalised disturbance in foetal development, the changes seen at the dose levels on this study are considered not adverse. In addition to the changes in the extent of nasal ossification, there was also a slight increase in the number of foetuses from litters given 160 mg/kg/day which showed the minor defects of incomplete ossification of the caudal vertebral centra and/or hyoid bone of the skull. These incidences however, were either within, or only just outside, the background data ranges. Foetuses from litters given 80 or 160 mg/kg/day also had an increase in the variant finding of ossified cervical vertebral centra. Whilst changes in the extent of ossification typically reflect a slight developmental delay, due to the propensity for repairing by postnatal skeletal remodelling, these changes are considered not adverse.
Visceral malformations:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
160 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
skeletal malformations
Key result
Dose descriptor:
NOEL
Effect level:
ca. 40 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
skeletal malformations
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Conclusions:
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 generally 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 changes were considered not adverse.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
160 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
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, 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 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 (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.

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 (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 potential adverse results from this sodium tungstate study is reported in three separate documents [Mclnturf et al (2007, 2008, 20110] 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.

Justification for classification or non-classification

No reproductive/developmental studies are available for the fused tungsten carbide. However, data were available on sodium tungstate, which were used for read-across. However, sodium tungstate tripartite studies (Fertility and Early Embryonic Development, Embryo-Foetal Development and Pre- and Post-Natal Development studies) conducted under ICH Testing Guideline reported no significant effects. Therefore, based on the lack of reproductive and/or developmental effects observed, no classification is warranted.