Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

No fertility, reproductive, or developmental toxicity data of sufficient quality are available for tungsten carbide (target substance). However, reproductive toxicity data are available for sodium tungstate (source substance), which are used for read-across. Due to lower water solubility and lower toxicity for the target substance compared to the source substance, the resulting read-across from the source substance to the target substance is appropriate as a conservative estimate of potential toxicity for this endpoint. In addition, read-across is appropriate because the classification and labelling is more protective for the source substance than the target substance, the PBT/vPvB profile is the same, and the dose descriptors are, or are expected to be, lower for the source substance. For more details, refer to the attached description of the read-across category approach or see Annex 3 of the CSR.


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


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


 






















 REACH Requested Test (OECD TG 443)



 Studies Requesting Adaptation


Cohort 1A Segment I - ICH Testing Guideline  (4.1.1. Fertility Study)

Cohort 2A


Cohort 2B



Segment III - ICH Testing Guideline (4.1.2. Pre- and postnatal study)


 


McInturf et al (2007, 2008 & 2011)
Cohort 3Osterburg et al (2014)

 


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


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


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


 


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


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


 


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


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


OECD TG 443 developmental neurotoxicity (DNT) in addition to brain histopathology include auditory startle, functional observation, motor activity endpoints. ICH Segment III includes P1 generation learning and memory, auditory function and motor activity, and in F1 offspring 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 femals at all maternal doses and males maternally given 80 or 160 mg/kg/day travelled a greater distance and rested less than the Controls, although there was no dose-relationship.  This is considered not to be related to maternal sodium tungstate administration.


 


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


F1 Pups were monitored for the following developmental milestones:


- Ears open - examined daily until occurrence.


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


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


- Startle response - examined on Day 15 of age.


- Pupillary light reflex - examined on Day 21 of age


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


 


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


 Cohort 3 - Developmental Immunotoxicity (DIT)


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


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


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


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


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


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


No significant changes in any of the parameters measured in response to tungstate, except for the percent monocytes. There were fewer lymphocytes in the F1 generation compared to the P generation (p<0.023), but this was not dose-related. Additionally, the red blood cell distribution width (RDW) was higher in the P generation vs the F1 pups (p<0.004). The percentage of monocytes was dose-dependently lower at higher concentrations when compared to control (p<0.003). Other parameters suggest a dose-dependent trend (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:
fertility, other
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
Study conducted only on female rats. Reproductive toxicity indicators included estrogen, progesterone and prolactin receptors, luteinizing hormone, progesterone, and follicle-stimulating hormone serum concentrations.
Justification for type of information:
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: Tungsten carbide
3. CATEGORY APPROACH JUSTIFICATION: See Annex 3 in CSR
4. DATA MATRIX: See Annex 3 in CSR
Reason / purpose for cross-reference:
reference to other study
Remarks:
Same authors published reproductive study in males
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
GLP compliance:
not specified
Remarks:
Study has adequate and reliable coverage of the key parameters foreseen to be investigated in the corresponding test methods and adequate and reliable documentation of the study is provided
Limit test:
yes
Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals or test system and environmental conditions:
Adult female Wistar rats (200 g) were kept under a constant 12-hour light-dark cycle and were allowed to eat and drink ad libitum.
Route of administration:
oral: drinking water
Vehicle:
other: 0.9% NaCl
Details on exposure:
The treatment was carried out for 12-weeks. During this period, glycemia and body weight were measured regularly.
Details on mating procedure:
To evaluate reproductive performance, after 10 weeks of treatment, individual females were placed in a cage with one healthy adult male (body weight: 250 g). The animals were kept together overnight, and then separated the following morning. Immediately after each separation, a vaginal examination and scrape were carried out to determine if sexual intercourse had occurred. When intercourse was positive (presence of a vaginal tap and/or spermatozoa), the night/day routine was discontinued and the female was housed individually during the estimated period of gestation. When females showed no sexual contact with the male during 9 consecutive days, they were not used for further mating and were labelled ‘unable’, whereas those that showed sexual activity were labelled ‘able’.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
12-weeks
Frequency of treatment:
Daily via drinking water
Dose / conc.:
2 000 mg/L drinking water
Remarks:

nominal in water
No. of animals per sex per dose:
24 female rats per group.
Control animals:
yes, concurrent vehicle
Details on study design:
All the animals were anesthetized with diethyl ether and sacrificed by decapitation.
Parental animals: Observations and examinations:
- Histological analyses of ovaries
- Wester blot analyses for estrogen receptor, progesterone receptor, insulin receptor and FSH receptor.
Litter observations:
At parturition, litter size was determined
Postmortem examinations (parental animals):
Blood was collected immediately to measure serum parameters. All the animals were then anesthetized with diethyl ether and killed by decapitation. Ovaries horns were prepared in two ways. First, part of the tissues were immediately fixed in 3% formaldehyde in a buffer solution containing 54 mM NaH2PO4 and 28 mM Na2HPO4 (pH 7.4) at 4°C (buffered formaldehyde). This material can be stored for up to 5 weeks. These tissues were used for optical microscope histology. The rest of the tissues were immediately frozen in liquid N2 and stored at -90°C until Western blot and semi-quantitative RT–PCR analyses were performed.
Statistics:
All values are means + SEM. Statistical comparisons of the means were performed by analysis of variance, followed by the Student– Neumann–Keuls test. This test makes multiple pairwise comparisons, where the cut-off point to which comparisons are made changes between them. Thus, this methodology shows greater sensitivity than other similar multiple pairwise comparison tests, such as the Student’s t-test. A P-value of (0.05 was considered to be statistically significant.
Reproductive indices:
The reproductive performance of the rats was measured as the proportion of ‘able’ females to the total number tested (the percentage of positive vaginal scrapes, i.e. those with presence of spermatozoa, with respect to the total number of vaginal scrapes performed in one experimental group). This parameter was named as ‘mating index’. The number of vaginal scrapes varied depending on the time required by animals to show positive intercourse. Furthermore, we also calculated the percentage of parturitions with respect to the number of positive scrapes. This parameter was named ‘fertility’. These definitions do not necessarily coincide with the common definitions for mating index, fertility and prolificacy published elsewhere.
Offspring viability indices:
The mean litter size was also calculated and was defined as ‘prolificacy’. These definitions do not necessarily coincide with the common definitions for mating index, fertility and prolificacy published elsewhere.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Description (incidence):
At the end of the experiment, all the rats in the tugstate treated healthy (TH) rats were alive.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Tungstate treatment also caused a decrease in the body weight gain of healthy rats
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Tungstate treatment did not modify daily food intake in rats.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Tungstate treatment did not modify daily water consumption in healthy rats
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Tungstate treatment did not modify serum levels of glucose, ALT activity, insulin, progesterone, FSH and LH in healthy rats
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Histological analyses of ovaries did not show evident differences between groups. Thus, active ovaries showing evolutive follicles, corpora lutea and follicular glands were detected in all
groups. Ovaries were generally covered by a significant amount of adipose tissue.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
- Tungstate did not significantly affect LH receptor mRNA content in healthy rats
- Tungstate slightly decreases western blot band intensity of GLUT 3 hexose expression in ovaries of healthy rats.
- No differences in the expression of the estrogen receptor in tungstate treated healthy rats.
- Tungstate does not affect the amount of ovarian prolactin receptor mRNA.
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Description (incidence and severity):
Tungstate treatment did not modify Leydig cell function in healthy rats
Reproductive performance:
no effects observed
Description (incidence and severity):
Tungstate administration did not alter the reproductive performance of healthy female rats.
- All the rats in the conrol and tretated tungstate group survived.
- Tungstate exposed rats have lower body weight gain than control animals.
- Tungsten treatment had no effect on serum glucose, insulin, alanine aminotransferase, luteinizing hormone, progesterone, and follicle-stimulating hormone concentrations.
- Tungstate treatment did not affect any reproductive parameter in healthy rats.
- Tungsten treatment does not affect the expression of estrogen, progesterone and prolactin receptors as determined by western blotting.
Key result
Dose descriptor:
NOAEL
Effect level:
> 2 000 mg/L drinking water
Based on:
test mat.
Sex:
female
Basis for effect level:
reproductive performance
Clinical signs:
not examined
Mortality / viability:
not examined
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Tungsten treatment did not alter prolifacy (litter size)
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Reproductive effects observed:
no
Conclusions:
Oral administration (2 mg/ml in drinking water) of sodium tungstate to adult female for 12 weeks did not show hypoglycemia, did not alter the reproductive performance of healthy females or any alteration in estrogen, progesterone and prolactin receptors. In adddition, tungsten treatment had no effect on luteinizing hormone, progesterone, and follicle-stimulating hormone serum concentrations. Sodium tungstate administered orally did not alter the reproductive performance of healthy females.
Executive summary:

No fertility, reproductive, or developmental toxicity data of sufficient quality are available for tungsten carbide (target substance). However, reproductive toxicity data are available for sodium tungstate (source substance), which are used for read-across. Due to lower water solubility and lower toxicity for the target substance compared to the source substance, the resulting read-across from the source substance to the target substance is appropriate as a conservative estimate of potential toxicity for this endpoint. In addition, read-across is appropriate because the classification and labelling is more protective for the source substance than the target substance, the PBT/vPvB profile is the same, and the dose descriptors are, or are expected to be, lower for the source substance. For more details, refer to the read-across category approach included in the Category section of this IUCLID submission on Annex 3 in the CSR. 

Endpoint:
developmental immunotoxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
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: Tungsten carbide
3. CATEGORY APPROACH JUSTIFICATION: See Annex 3 in CSR
4. DATA MATRIX: See Annex 3 in CSR
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
GLP compliance:
not specified
Limit test:
no
Justification for study design:
- Other considerations, eg on choice of species, strain, vehicle and number of animals [if applicable]: This study was conducted in the generic, tumor-resistant C57BL/6 mouse to represent a normal human population
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Acros Organics, 99 + %, ACS, Lot A0260722, IN, USA.
Species:
mouse
Strain:
C57BL
Remarks:
C57BL/6J male and female mice
Details on species / strain selection:
Purchased through an IACUC approved protocol.
Sex:
male/female
Route of administration:
oral: drinking water
Vehicle:
water
Duration of treatment / exposure:
Prenatal exposures
Frequency of treatment:
Daily
Details on study schedule:
Inoculatrion of Respiratory Syncytial Virus (RSV) within 2 weeks of weaning
Dose / conc.:
15 mg/L drinking water
No. of animals per sex per dose:
A total of 8–10 mouse pups were obtained for each group
Control animals:
yes, concurrent vehicle
Litter observations:
PARAMETERS EXAMINED

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: At 21–35 days of age the mouse pups were lightly anesthetized and the nasal cavity inoculated with 10 ll of human RSV in medium for a total exposure of 1x10Exp6 pfu.
Postmortem examinations (offspring):
- Peripheral hematology was evaluated utilizing complete blood counts.
- Spleen tissue was massed and splenic ratio calculated as spleen mass per body mass. Spleens and femurs from each group were preserved
Statistics:
When a population is nonparametric due to outliers and has a small n-value, a median is less sensitive to these outlier values and therefore, provides a better representation of the data. Statistical analysis was performed with a one-tailed Mann–Whitney test utilizing Minitab version 15.1.1.0. Data are presented as medians with interquartile ranges and outliers indicated. Significance was accepted when p≤0.05.
Clinical signs:
not specified
Mortality / viability:
not specified
Body weight and weight changes:
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
NEUTROPHILIA
- Na2WO4-only, RSV-only and Na2WO4+ RSV demonstrated significantly elevated neutrophil counts as compared to the longitudinal controls (p = 0.0162, 0.0081, 0.0059 for RSV-only, Na2WO4-only and Na2WO4 + RSV, respectively). However, the first and third quartiles for all experimental groups were within the normal range (0.1–2.4 k/ul) except for RSV-only whose third quartile was 2.76 k/ul. Additionally, Na2WO4+ RSV possessed an extreme outlier. No significant differences were demonstrated between RSV-only, Na2WO4-only and Na2WO4+ RSV.
- The percentage of neutrophils contributing to the total WBC count was significantly greater for all three exposure groups as compared to the controls. However, the first and third quartiles for all experimental groups were within the normal range (6.6–38.9%) except for RSV-only whose third quartile was 44.67%. Additionally, Na2WO4+ RSV possessed an extreme outlier for whom 76.3% of the WBC was neutrophils. No significant differences.

TOTAL WHITE BLOOD CELL COUNTS
While the percentage of neutrophils increased significantly, there was no associated increase in WBC counts for mice exposed to RSV-only. No group demonstrated a significant increase in WBC counts for mice exposed to RSV-only. No group demonstrated a significant increase
in WBC counts as compared to the controls or to each other. First and third quartiles for all experimental groups were within the normal range (1.8–10.7 k/ul) except for Na2WO4+ RSV whose third quartile was 12.4 k/ul. For the subgroup of Na2-WO4 + RSV mice exhibiting pathology, leukocytosis began at 2–3 months of age and continued until death/morbidity at 5–6 months of age.


Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Sexual maturation:
not specified
Anogenital distance (AGD):
not specified
Nipple retention in male pups:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Splenic to body weight ratios for mice exposed to Na2WO4-only or RSV-only did not vary significantly from the controls or from each other. Splenic ratios for Na2WO4 + RSV mice were significantly larger as compared to all other groups (p = 0.0406, 0.0184, 0.0108 for control, Na2WO4-only and RSV-only, respectively) (Fig. 2). The colors/hues of the spleens were not consistent in mice demonstrating splenomegaly and were not the same color as the control spleens.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Longitudinal controls and tungsten exposed mice did not exhibit pathological indicators.
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
Histologic slide preparation and interpretation was conducted with both spleen and bone marrow tissues from two Na2-WO4+ RSV mice exhibiting the greatest degree of splenomegaly and compared with a longitudinal control mouse. The controlexhibited a 1:1 ratio of erythropoietic and granulopoietic precursor cells with all stages represented. Both Na2WO4+ RSV mice demon strated consistent histology, a 1:20 ratio of erythropoietic to granulocytic precursors with all stages represented. Additionally, marked thrombopoiesis was reported in both spleen and bone marrow tissues.
Developmental immunotoxicity:
effects observed, treatment-related
Description (incidence and severity):
- RSV inoculation within 2 weeks of weaning was associated with a neutrophil shift in 56% of 5-month old mice. When the RSV inoculation was combined with exposure to Na2WO4 (Na2WO4+ RSV), significant splenomegaly resulted (p =0.0406, 0.0184, 0.0108 for control, Na2WO4-only and RSV-only).
- Na2WO4-only, RSV-only and Na2WO4+ RSV demonstrated significantly elevated neutrophil counts as compared to the longitudinal controls (p = 0.0162, 0.0081, 0.0059 for RSV-only, Na2WO4-only and Na2WO4 + RSV, respectively). However, the first and third quartiles for all experimental groups were within the normal range (0.1–2.4 k/ul) except for RSV-only whose third quartile was 2.76 k/ul. Additionally,
Key result
Remarks on result:
not determinable because of methodological limitations
Key result
Critical effects observed:
no
Conclusions:
Exposure to Na2WO4 and RSV resulted in hematological/immunological disease, the nature of which is currently inconclusive
Executive summary:

No fertility, reproductive, or developmental toxicity data of sufficient quality are available for 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 read-across category approach included in the Category section of this IUCLID submission on Annex 3 in the CSR.

Endpoint:
developmental immunotoxicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
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: Tungsten carbide
3. CATEGORY APPROACH JUSTIFICATION: See Annex 3 in CSR
4. DATA MATRIX: See Annex 3 in CSR
Reason / purpose for cross-reference:
read-across: supporting information
Reason / purpose for cross-reference:
assessment report
Reason / purpose for cross-reference:
assessment report
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
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]:

- Basis for dose level selection : Doses of tungstate in the two studies were selected based on previous work that used similar doses in rats (McInturf et al, 2011).
- Exclusion of extension of Cohort 1B
- Exclusion of developmental neurotoxicity Cohorts 2A and 2B
- Inclusioncof developmental immunotoxicity Cohort 3
- Route of administration : Drinking water
- 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: Sigma, St. Louis, MO
Species:
mouse
Strain:
C57BL
Remarks:
C57BL6
Details on species / strain selection:
C57BL6 Mice were obtained from Charles River Laboratories (Wilmington, MA).
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories (Wilmington, MA)
- Age at study initiation: 8–12-week-old
- Weight at study initiation: 19–22 g
- Housing: Mice were housed singly during the course of the study and pair mated for breeding. After confirmation of pregnancy, males were removed.
- Diet (eg ad libitum): All animals had ad libitum access to a low molybdenum diet
- Water (eg ad libitum): All animals had ad libitum access to filtered water
- Acclimation period: Mice were allowed to acclimatize to the animal facility for 7 days before commencement of the experimental phase
ENVIRONMENTAL CONDITIONS
- Temperature (°C): Temperature-controlled room at 22 ⁰C.
- Photoperiod (hrs dark / hrs light): During the course of the experiment, animals had a 12 h day/night cycle.
Route of administration:
oral: drinking water
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Water bottles were changed 2–3 times weekly, always using water from the source and a 1 M sodium tungstate stock supply
Details on mating procedure:
Mice were housed singly during the course of the study and pair mated for breeding. After confirmation of pregnancy, males were removed
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
The tungstate in the water bottles was administered to calculate approximate ingested doses of tungstate based on an estimated water consumption of 4.5 ml/mouse/day. Body weights were recorded weekly and quantities of tungstate adjusted appropriately in water bottles. Water consumption was determined using graduated water bottles. Measurements were made daily of water consumed by mice.
Duration of treatment / exposure:
In the one-gen exposure, mice were exposed to tungstate for 90 days prior to mating (Weeks 1–12). The next 7 weeks comprised gestation and weaning (Weeks 13–19). After pups (F1) were weaned, the parents (P) were necropsied as described, and 9 weeks after initiation of the study. The F1 generation was exposed to tungstate for a further 90 days after weaning and then necropsied. During all phases of the onegen study mice were kept on the appropriate tungstate dose.
Frequency of treatment:
Daily via drinking water.
Details on study schedule:
- Parental (P0) mice exposed for 12 weeks before mating, followed by gestation, birth, and weaning.
- F1 mice (offspring) exposed after weaning for 12 weeks.
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
2 mg/kg bw/day (nominal)
Dose / conc.:
62.5 mg/kg bw/day (nominal)
Dose / conc.:
125 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
There were eight mice per group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Selected based on previous work that used similar doses in rats (McInturf et al., 2011).

- Rationale for animal assignment (if not random):
- Fasting period before blood sampling for clinical biochemistry:
- Other:
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were weekly monitored during the course of exposure.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Measurements were made daily of water consumed by mice.
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring: number of live, births, litter size, or sex ratio.

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: The focus of these studies was to determine changes in immunological populations. Therefore, we performed complete blood counts and measured hematological parameters from the blood of animals at necropsy. With two exceptions (monocyte%
and red blood cell distribution width):
- Immune activation with Staphylococcal enterotoxin B (SEB) to induce immune response) and necropsy (spleen & blood)
- Flow cytometry: FSC vs SSC
- ELISA for or IL-6, TNFa, IL-10, and IFNg.
Statistics:
All statistical tests were performed with Systat (v11 or v13, Systat Software Inc., Chicago, IL). Statistical significance was assumed at p50.05. Comparisons between treatments (tungstate dose, immune challenge) were performed as two-way analysis of variance (ANOVA). Differences in weights were determined by repeated measures analysis of variance (RM-ANOVA). When assumptions of normality and/or equal variances were violated, non-parametric Kruskal-Wallis tests were performed. Specific post-hoc tests were performed and multiple test corrections performed when appropriate.
Clinical signs:
not specified
Mortality:
not specified
Body weight and weight changes:
no effects observed
Description (incidence and severity):
We found no statistically significant changes in body weight due to any tungstate dose levels. The 200 mg/kg/day males in the P generation show a consistent trend towards decreased weights. This observation, however, was not statistically significant.
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No significant changes in water consumption due to the quantity of tungstate present in the water
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Description (incidence and severity):
Other parameters suggest a dose-dependent trend (eg hematocrit); however, these trends were not statistically significant. There were no significant alterations in overall quantities of hematological populations.
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
not specified
Reproductive performance:
no effects observed
Description (incidence and severity):
No statistically significant changes were observed in the number of live births, litter size, or sex ratio at any dose of tungstate tested.
Key result
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
NOEL
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
reproductive performance
Key result
Dose descriptor:
NOEL
Effect level:
125 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: immunotoxicity
Key result
Critical effects observed:
no
Clinical signs:
not specified
Mortality / viability:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
The red blood cell distribution width (RDW) was higher in the P generation vs the F1 pups (p<0.004).
Developmental immunotoxicity:
effects observed, treatment-related
Description (incidence and severity):
FLOW CYTOMETRY:
With two exceptions (monocyte% and red blood cell distribution width), there were no statistical differences in the data between P and F1. There were no significant changes in any of the parameters measured in response to tungstate, with the exception of the percent monocytes (%MO). There were fewer lymphocytes in the F1 generation compared to the P generation (p<0.023), but this was not dose related. Furthermore, any statistically significant differences between the innate or immune responses of P and F1 mice were not noted.

The percentage of monocytes was dose-dependently lower at higher concentrations when compared to control (p<0.003).
Tungstate-dependent changes in the one-gen exposures 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.
The F1 controls in the SEB-treated groups were 7.20±0.76% and 2.85±0.53% for the 200 mg/kg/day tungstate groups (p<0.001). No statistically significant differences were noted in the overall quantity of CD3+ CD4+TH cells. Additionally, there were no significant effects of generation on the suppression of CD71+ TH cells in SEB-treated mice exposed to tungstate.
There were no statistically significant differences in quantities of CD3+ CD8+ cells in the one-gen study. The F1 mice CD3+ CD8+ CD71+ were 6.33±0.49% for the controls and 2.52±0.25% in the 200 mg/kg/day tungstate group (p<0.001).

CYTOKINES
Although not statistically significant, the F1 mice had an overall reduced IFN-g response, especially at the 62.5 mg/kg/day dose, compared to their parents.
Key result
Dose descriptor:
NOEL
Generation:
F1 (cohort 3)
Effect level:
125 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
developmental immunotoxicity
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Conclusions:
- Reduction in the quantity of CD71+ helper and cytotoxic T-cells present in the high-dose tungstate exposure groups (200 mg/kg/day) when challenged with SEB.
- A consistent reduced level of CD71 on SEB-treated cells in the 200 mg/kg/day tungstate group was noted. Because CD71 reductions due to tungstate were only seen in SEB-challenged mice, this would suggest that reduced CD71 levels of TH and TCTL cells in the spleens of SEB-treated mice was a result of either suppressed cell cycling or, potentially, alterations to apoptosis of these cells.
- Tungstate exposure could result in suppression of adaptive immunity. Data indicates little to no effect of tungstate at any dose on groups of mice exposed only to tungstate without being co-exposed to an immune stressor (Staphylococcal enterotoxin B, SEB).
Executive summary:

No fertility, reproductive, or developmental toxicity data of sufficient quality are available for 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 read-across category approach included in the Category section of this IUCLID submission on Annex 3 in the CSR.

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Justification for type of information:
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: Tungsten carbide
3. CATEGORY APPROACH JUSTIFICATION: See Annex 3 in CSR
4. DATA MATRIX: See Annex 3 in CSR
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
equivalent or similar to guideline
Guideline:
other: EPA Health Effects Test Guidelines OPPTS 870.3650 (which is similar to OECD TG 422)
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
- Sodium tungstate dihydrate was selected because it is the naturally occurring form of tungsten and the most water soluble
- Sodium tungstate dihydrate (purity ≥ 99%)
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Route of administration:
oral: drinking water
Vehicle:
water
Remarks:
Deionized drinking water
Details on exposure:
Drinking water was selected as the most likely route of exposure for the general population
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
Gestation Day (GD) 6 to Postnatal Day (PND) 21 (rats)
Frequency of treatment:
Daily
Dose / conc.:
0 mg/L drinking water
Dose / conc.:
250 mg/L drinking water
Remarks:
~33 mg/kg bw/day (gestational)
Dose / conc.:
500 mg/L drinking water
Remarks:
~65 mg/kg bw/day (gestational)
Dose / conc.:
1 000 mg/L drinking water
Remarks:
~143 mg/kg bw/day (gestational)
No. of animals per sex per dose:
The perinatal phase started with 8 dams/group. On PND 4, unacceptable litters (<8 pups or <2 pups of a sex) were culled and a maximum of 7 litters were standardized to 8 pups (4/sex; 2 or 3/sex was acceptable
Control animals:
yes, concurrent vehicle
Details on study design:
NTP conducted 2-year carcinogenicity studies of sodium tungstate dihydrate in rats and mice; the rat study included perinatal exposure to evaluate potential effects resulting from in utero and lactational exposure
Positive control:
Not applicable
Parental animals: Observations and examinations:
- Survival, body weights, water consumption, clinical observations
- Hematology; clinical chemistry and urinalysis in rats
- Gross pathology, histopathology, and organ weights
- Tungsten concentrations in blood and urine
- Genotoxicity (micronucleus and Comet assay)
Clinical signs:
not specified
Mortality:
not specified
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- Gestational weights were statistically decreased at gestational days (GD) 15, 18, and 21 in the 1000 mg/L group dams.
- Gestational body weights were statistically decreased at GD 6-9 in 500 and 1000 mg/L group dams
- Gestational body weights were statistically decreased at GD 12-15, 15-18, 18-21, ad 6-21 in 1000 mg/L group dams
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
effects observed, non-treatment-related
Description (incidence and severity):
At gestational days (GD) 15-18 gestational water consumption increased in the 1000 mg/l group. No other water consumption differences were noted.
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 specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed
Description (incidence and severity):
- There were no statistically treatment related effects on the litter size.
- 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
Key result
Dose descriptor:
LOAEL
Remarks:
10% body weight reduction
Effect level:
ca. 1 000 mg/L drinking water
Based on:
test mat.
Sex:
female
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 500 mg/L drinking water
Based on:
test mat.
Sex:
female
Basis for effect level:
body weight and weight gain
Critical effects observed:
no
Lowest effective dose / conc.:
1 000 mg/L drinking water
Clinical signs:
not specified
Dermal irritation (if dermal study):
not specified
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
PND 1: Preliminary data showed less pup mortality in the group exposed to 1000 mg/L than controls, 250 and 500 mg/L.
PND 4: Preliminary data showed less pup mortality in the group exposed to 1000 mg/L than controls, 250 and 500 mg/L.
PND 21: No differences in lve per litter (male + female)
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
At PND 1: No differences in pup body weights or adjusted body weights (male and female).
At PND 4: Male pups body weight in the 1000 mg/L group decreased (7%) compared to control animals
At PND 4-21: No statistically differences in body weights of male and female pups.
At lactational day (LD) 1, 17, and 21 pups body weight at the 1000 mg/L were decresed.
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No water consuption differences in lactational days (LD) 1-21.
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Histopathological findings:
not specified
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
LOAEL
Generation:
F1
Effect level:
ca. 2 000 mg/L drinking water
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
ca. 1 000 mg/L drinking water
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Conclusions:
Perinatal studies concluded that were no treatment related effects on the percentage of dams delivering, litter size, or litter weights. There is a reduced body weights greater than 10% relative to control were observed in 2000 mg/L dams, PND 4 pups, PND 21 pups, and adult rats.
Executive summary:

No fertility, reproductive, or developmental toxicity data of sufficient quality are available for tungsten 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 read-across category approach included in the Category section of this IUCLID submission on Annex 3 in the CSR.

Endpoint:
developmental neurotoxicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
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: Tungsten carbide
3. CATEGORY APPROACH JUSTIFICATION: See Annex 3 in CSR
4. DATA MATRIX: See Annex 3 in CSR
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
equivalent or similar to guideline
Guideline:
other: EPA OPPTS 870.3650 "Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Study
Deviations:
yes
Remarks:
The protocol was extended beyond the minimum 54 days of treatment to accomodate evaluation of the development of the offspring through postnatal day 20 as well as their dams following the last dose on day 70.
GLP compliance:
not specified
Remarks:
Study has adequate and reliable coverage of the key parameters foreseen to be investigated in the corresponding test methods and adequate and reliable documentation of the study is provided
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories (Wilmington, MA)
- Age at study initiation: 8 weeks
- Housing: The adults (e.g., P1) were singly housed (except during mating)
- Acclimation period: 14 days
Route of administration:
oral: gavage
Vehicle:
other: deionized water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The powder readily dissolved in a deionized water (diH2O) vehicle for the concentrations used in this study at 5, 62.5 and 125 mg/mL. The solution was concentrated to administer a volume of 1 mL/kg body weight not to exceed 2 mL. Fresh solution was made daily and administered via oral gavage


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

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

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

Various organ tissues (heart, spleen, kidney, liver, lungs, brain, testes, ovaries, thymus, bone, gastrointestinal tract, etc,) were carefully removed and postfixed in 4% paraformaldehyde no longer than 24 h. After dehydration, tissues embedded in paraffin and 40 μm coronal sections were made on glass slides for histological evaluation using hematoxylin and eosin (H&E) stain.
Postmortem examinations (offspring):
Tungsten concentrations in brain tissue were measured in pups for the control and highest dose groups only. Ten male and female adults from each dose group were necropsied on the day of the last dose were necropsied on PND20 and PND70. For all necropsies, animals were anesthetized via CO2 overdose followed by exsanguination via the vena cava. Concentrations of the tungstate anion were determined using inductively coupled plasma mass spectrometry (ICP-MS).
Statistics:
All statistical testing was performed using ANOVA with repeated measures factors. Tukey'svHSD analysis was used to evaluate pair-wise comparisons.
Clinical signs:
no effects observed
Description (incidence and severity):
Animals were observed for clinical signs of toxicity throughout the exposure period, although none were noted. Additionally, no deaths were recorded for the adult females or males at the doses tested.
Mortality:
no mortality observed
Description (incidence):
No deaths were recorded for the adult females or males at the doses tested
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Sodium tungstate treatments did not have an effect on average gestational weight gain in adults
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Sodium tungstate treatments did not have an effect on average gestational weight gain in adults
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
A significant effect of tungstate exposure on spontaneous locomotor activity was detected especially with low dose treated dams. Compared to control and the high dose treated animals, the low dose treated dams spent more time on ambulatory time and distance traveled and less time in stereotypies. On the contrary, in high dose treated dams less time resting and more time in stereotypic movements than the controls or low dose group were markedly observed. Maternal retrieval test revealed that sodium tungstate exposure to dams had no effect on latency in both treatment groups. In the dams, opposing exposure effects were observed in the low and high dose groups related to locomotor activity and exploration
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathological examination showed no severe chronic injury in the organs tested. However, the heart showed histological lesions, histiocytic inflammation from minimal to mild with cardiomyocyte degeneration and necrosis in several P0 animals of 125 mg NaW dose group.
Histopathological findings: neoplastic:
not specified
Other effects:
effects observed, treatment-related
Description (incidence and severity):
- The study results indicate that a significant difference was found for one of the treatment groups on measures of reproductive toxicity. In the dams, particularly 125 mg dose group animals showed longer gestational period when compared to control, 5 and 62.5 mg groups, but did not have an effect on average gestational weight gain in adults and offspring.

- Tungstate ion concentrations in the male and female adult and pup brains after sodium tungstate exposure were significantly greater in the high dose (125 mg) treated rats than control. Similarly, in dam milk secretions tungstate ion concentrations was significantly greater in the 125 mg treatment group than in the low dose group or controls. Further, we also observed increased concentration of tungstate ion distribution in other major organs like heart, spleen, kidney, thymus, testes, lungs, liver, femur bone and gastrointestinal regions in both male and female treated adult and pups.
Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed
Description (incidence and severity):
Daily administration of sodium tungstate produced no overt evidence of toxicity and had no apparent effect on mating success
- Tungstate ion concentrations in the male and female adult rats after sodium tungstate exposure were significantly greater in the high dose (125 mg) treated rats than control
- Further, we also observed increased concentration of tungstate ion distribution in other major organs like heart, spleen, kidney, thymus, testes, lungs, liver, femur bone and gastrointestinal regions in both male and female treated adult rats.
- Histopathological examination showed no severe chronic injury in the organs tested.
Key result
Dose descriptor:
NOAEL
Effect level:
125 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
gross pathology
histopathology: non-neoplastic
reproductive performance
Key result
Dose descriptor:
LOAEL
Effect level:
125 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Dose descriptor:
LOAEL
Effect level:
5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Behaviour
Dose descriptor:
LOAEL
Effect level:
125 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Behaviour
Dose descriptor:
NOAEL
Effect level:
> 125 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Behaviour
Critical effects observed:
no
Lowest effective dose / conc.:
125 mg/kg bw/day (actual dose received)
System:
cardiovascular
Organ:
heart
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Sodium tungstate treatments did not have an effect on average gestational weight gain in adults and offspring
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
Daily administration of sodium tungstate produced no overt evidence of toxicity on offspring physical development.
Histopathological findings:
no effects observed
Description (incidence and severity):
Daily administration of sodium tungstate produced no overt evidence of toxicity on offspring physical development and no significant test article-related lesions
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Tungstate ion concentrations in the pup brains after sodium tungstate exposure were significantly greater in the high dose (125 mg) treated rats than control. Similarly, in dam milk secretions tungstate ion concentrations was significantly greater in the 125 mg treatment group than in the low dose group or controls. Further, we also observed increased concentration of tungstate ion distribution in other major organs like heart, spleen, kidney, thymus, testes, lungs, liver, femur bone and gastrointestinal regions in pups.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
For righting reflex, a significant sex effect was found on male where males were faster than females. However, there was no dose related effect on this activity (Fig. 3). Further, on ultrasonic distress vocalization test, pups showed dose-related effects during 60 s time period. The high dose treated pups exhibited more vocalization than control and 5 mg groups
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 125 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
clinical signs
mortality
body weight and weight gain
gross pathology
histopathology: non-neoplastic
Key result
Dose descriptor:
LOAEL
Generation:
F1
Effect level:
5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
developmental neurotoxicity
Key result
Dose descriptor:
LOAEL
Generation:
F1
Effect level:
125 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
developmental neurotoxicity
Reproductive effects observed:
not specified
Conclusions:
Daily administration of sodium tungstate produced no overt evidence of toxicity and had no apparent effect on mating success, and did not cause a change in gestation length and weight gain in adult females.
Executive summary:

No fertility, reproductive, or developmental toxicity data of sufficient quality are available for tungsten carbide (target substance). However, reproductive toxicity data are available for sodium tungstate (source substance), which are used for read-across. Due to lower water solubility and lower toxicity for the target substance compared to the source substance, the resulting read-across from the source substance to the target substance is appropriate as a conservative estimate of potential toxicity for this endpoint. In addition, read-across is appropriate because the classification and labelling is more protective for the source substance than the target substance, the PBT/vPvB profile is the same, and the dose descriptors are, or are expected to be, lower for the source substance. For more details, refer to the read-across category approach included in the Category section of this IUCLID submission on Annex 3 in the CSR. 

Endpoint:
developmental neurotoxicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well documented scientfically sound study similar to OECD guidelines with sufficient information provided on materials and methods to evaluate results. However as this study is used in the context of a read across, Klimisch 2 is assigned.
Justification for type of information:
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: Tungsten carbide
3. CATEGORY APPROACH JUSTIFICATION: See Annex 3 in CSR
4. DATA MATRIX: See Annex 3 in CSR
Reason / purpose for cross-reference:
read-across: supporting information
Reason / purpose for cross-reference:
assessment report
Qualifier:
equivalent or similar to guideline
Guideline:
other: EPA OPPTS 870.3650 "Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Study
Deviations:
yes
Remarks:
The protocol was extended beyond the minimum 54 days of treatment to accomodate evaluation of the development of the offspring through postnatal day 20 as well as their dams following the last dose on day 70.)
GLP compliance:
not specified
Remarks:
Study has adequate and reliable coverage of the key parameters foreseen to be investigated in the corresponding test methods and adequate and reliable documentation of the study is provided
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories (Wilmington, MA)
- Age at study initiation: 8 weeks
- Housing: The adults (e.g., P1) were singly housed (except during mating)
- Acclimation period: 14 days
Route of administration:
oral: gavage
Vehicle:
other: deionized water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The powder readily dissolved in a deionized water (diH2O) vehicle for the concentrations used in this study at 5 mg/mL and 125 mg/mL. The solution was concentrated to administer a volume of 1 mL/kg body weight not to exceed 2 mL. Fresh solution was made daily and administered via oral gavage


VEHICLE
- Justification for use and choice of vehicle (if other than water): no data
- Concentration in vehicle: no data
- Amount of vehicle (if gavage): no data
- Lot/batch no. (if required): no data
- Purity: no data
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: not specified
- Length of cohabitation: 14 days
- Verification of same strain and source of both sexes: not specified
- Proof of pregnancy: vaginal plug referred to as day 1 of pregnancy
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
70 days
Frequency of treatment:
Daily pre- and postnatal exposure
Dose / conc.:
5 mg/kg bw/day (actual dose received)
Dose / conc.:
125 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
120 males and 120 females (40 per treatment group)
Control animals:
yes, concurrent vehicle
Parental animals: Observations and examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Throughout the exposure period, dams and the males used for mating were observed for clinical signs of toxicity.

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

In the dams, reflexive motor functions were tested in acoustic startle and pre-pulse inhibition (AS/PPI) responses; emotionality was tested in maternal retrieval responding and spontaneous locomotor activity (SLA); gross motor coordination was assessed in the watermaze test and spontaneous locomotor activity (SLA); and more complex learning and memory functions were assessed in the watermaze.
Litter observations:
- Pregnant dams were weighed on gestation days (GD) 1,10,15 and 20, and gestational weight gain and gestation length were recorded. On PND1 (day of parturition=PND 0), the number of pups in each litter was recorded. Litters were culled on PND4 to eight pups, and litters were weighed. Selection of pups was pseudo-random to maintain a 4 male/4 female ratio whenever possible, and all pups remained with their biological mothers.

- For pups emotionality was assessed in maternal separation distress as measured by their emitting ultrasonic distress vocalizations, and motor coordination development was measured in the righting reflex.
Postmortem examinations (parental animals):
Tungsten concentrations in brain tissue were measured in adults for the control and highest dose groups only. Ten male and female adults from each dose group were necropsied on the day of the last dose were necropsied on PND20 and PND70. For all necropsies, animals were anesthetized via CO2 overdose followed by exsanguination via the vena cava. Concentrations of the tungstate anion were determined using inductively coupled plasma mass spectrometry (ICP-MS).
Postmortem examinations (offspring):
Tungsten concentrations in brain tissue were measured in pups for the control and highest dose groups only. Ten male and female adults from each dose group were necropsied on the day of the last dose were necropsied on PND20 and PND70. For all necropsies, animals were anesthetized via CO2 overdose followed by exsanguination via the vena cava. Concentrations of the tungstate anion were determined using inductively coupled plasma mass spectrometry (ICP-MS).
Statistics:
All measures were analyzed using ANOVA. Repeated measures ANOVA were used where appropriate. All animals in the pre-weanling litter were tested for righting reflex and separation distress so the litter was used as the unit of analysis for the ANOVA. The fiducial limit was p<0.05 and post-hoc comparisons were performed using Tukey's HSD analysis for pair-wise comparisons as appropriate.
Reproductive indices:
- Average gestation duration
- Average gestation weight
- Average number of pups in litter
Offspring viability indices:
- Weight of Litter
- Gross abnormalities
Clinical signs:
no effects observed
Description (incidence and severity):
Throughout the exposure period, dams and the males used for mating were observed for clinical signs of toxicity, although none were noted.
Mortality:
no mortality observed
Description (incidence):
No deaths were recorded during the treatment period for the adult females or males at the doses tested
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Average gestational weight gain did not differ across treatments.
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Maternal retrieval was not affected by sodium tungstate exposure and there were no apparent effects of treatment on F1 acoustic startle response or water maze navigation
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Other effects:
effects observed, treatment-related
Description (incidence and severity):
1) On PND 20, the mean tungstate ion concentrations in males and females of the 125 mg/kg/day treatment group were 0.008 ± 0.006 ppm and 0.002±0.002 ppm, respectively. No tungstate ion was detected in the males or females of the control group.
Furthermore, no tungstate ion was detected in the brains of males or females in the control or high dose group on PND 70.

2) Tungstate ion concentrations in dam milk secretions measured PND10–14 were significantly greater in the 125 mg/kg/day treatment group (0.45 ± 0.007 ppm) than in the low dose group (0.021 ± 0.001 ppm) or controls (0.005±0.0 ppm). Concentrations in the low dose group did not significantly differ from controls.
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
Daily administration of sodium tungstate produced no overt evidence of toxicity and had no apparent effect on mating success.
Key result
Dose descriptor:
NOAEL
Effect level:
> 125 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Key result
Dose descriptor:
NOAEL
Effect level:
> 125 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
reproductive performance
Key result
Dose descriptor:
LOAEL
Effect level:
125 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Gestational duration
Dose descriptor:
NOAEL
Effect level:
> 125 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Behaviour
Dose descriptor:
LOAEL
Effect level:
5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Behaviour
Dose descriptor:
LOAEL
Effect level:
125 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Behaviour
Clinical signs:
not specified
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
All pups were inspected for physical birth defects, including missing digits, however, none were found in any of the treatment groups.
Histopathological findings:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
On PND 20, the mean tungstate ion concentrations in males and females of the 125 mg/kg/day treatment group were 0.008 ± 0.006 ppm and 0.002±0.002 ppm, respectively. No tungstate ion was detected in the males or females of the control group. Furthermore, no tungstate ion was detected in the brains of males or females in the control or high dose group on PND 70.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
- For righting reflex, a significant effect of sex was found (pb0.05) where males were faster (9.6±1.3 s) than females (14.5±1.6 s). No significant effects related to dose were found.
- 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 s 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)
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 125 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
gross pathology
Reproductive effects observed:
not specified
Conclusions:
The P1 dams had similar gestational weight gain, and had average litter sizes that were the same across groups. Furthermore, there were no observed differences in litter sizes or weights across treatment groups. No dose related signs of physical toxicity were apparent in either P1 adults the duration of this study.
Executive summary:

No fertility, reproductive, or developmental toxicity data of sufficient quality are available for tungsten carbide (target substance). However, reproductive toxicity data are available for sodium tungstate (source substance), which are used for read-across. Due to lower water solubility and lower toxicity for the target substance compared to the source substance, the resulting read-across from the source substance to the target substance is appropriate as a conservative estimate of potential toxicity for this endpoint. In addition, read-across is appropriate because the classification and labelling is more protective for the source substance than the target substance, the PBT/vPvB profile is the same, and the dose descriptors are, or are expected to be, lower for the source substance. For more details, refer to the read-across category approach included in the Category section of this IUCLID submission on Annex 3 in the CSR. 

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: Tungsten carbide
3. CATEGORY APPROACH JUSTIFICATION: See Annex 3 in CSR
4. DATA MATRIX: See Annex 3 in CSR
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
equivalent or similar to guideline
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 or test system 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 (eg 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 (eg 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.
Executive summary:

No fertility, reproductive, or developmental toxicity data of sufficient quality are available for tungsten carbide (target substance). However, reproductive toxicity data are available for sodium tungstate (source substance), which are used for read-across. Due to lower water solubility and lower toxicity for the target substance compared to the source substance, the resulting read-across from the source substance to the target substance is appropriate as a conservative estimate of potential toxicity for this endpoint. In addition, read-across is appropriate because the classification and labelling is more protective for the source substance than the target substance, the PBT/vPvB profile is the same, and the dose descriptors are, or are expected to be, lower for the source substance. For more details, refer to the read-across category approach included in the Category section of this IUCLID submission on Annex 3 in the CSR.

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Justification for type of information:
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: Tungsten carbide
3. CATEGORY APPROACH JUSTIFICATION: See Annex 3 in CSR
4. DATA MATRIX: See Annex 3 in CSR
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
equivalent or similar to guideline
Guideline:
other: EPA Health Effects Test Guidelines OPPTS 870.3650 (which is similar to OECD TG 422)
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
- Sodium tungstate dihydrate was selected because it is the naturally occurring form of tungsten and the most water soluble
- Sodium tungstate dihydrate (purity ≥ 99%)
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Route of administration:
oral: drinking water
Vehicle:
water
Remarks:
Deionized drinking water
Details on exposure:
Drinking water was selected as the most likely route of exposure for the general population
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
Gestation Day (GD) 6 to Postnatal Day (PND) 21 (rats)
Frequency of treatment:
Daily
Dose / conc.:
0 mg/L drinking water
Dose / conc.:
125 mg/L drinking water
Remarks:
~16 mg/kg bw/day (gestational)
Dose / conc.:
250 mg/L drinking water
Remarks:
~33 mg/kg bw/day (gestational)
Dose / conc.:
500 mg/L drinking water
Remarks:
~58 mg/kg bw/day (gestational)
Dose / conc.:
1 000 mg/L drinking water
Remarks:
~132 mg/kg bw/day (gestational)
Dose / conc.:
2 000 mg/L drinking water
Remarks:
~247 mg/kg bw/day (gestational)
No. of animals per sex per dose:
The perinatal phase started with 8 dams/group. On PND 4, unacceptable litters (<8 pups or <2 pups of a sex) were culled and a maximum of 7 litters were standardized to 8 pups (4/sex; 2 or 3/sex was acceptable
Control animals:
yes, concurrent vehicle
Details on study design:
NTP conducted 13-week studies of sodium tungstate dihydrate in rats and mice; the rat study included perinatal exposure to evaluate potential effects resulting from in utero and lactational exposure
Positive control:
Not applicable
Parental animals: Observations and examinations:
- Survival, body weights, water consumption, clinical observations
- Hematology; clinical chemistry and urinalysis in rats
- Gross pathology, histopathology, and organ weights
- Tungsten concentrations in blood and urine
- Genotoxicity (micronucleus and Comet assay)
Clinical signs:
effects observed, treatment-related
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
- One 2000 mg/L moribund dam and litter were terminated on postnatal day 6.

Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- No changes on gestional body weights and body weight gains were observed.
- Lactational body weights at 14 and 21 days were decreased in the 1000 and 2000 mg/L groups.
- Lactational body weights at 7-14 and 1-21 days were decreased in the 2000 mg/L groups.
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
- Preliminary data showed that gestational water consumption did not show statistically differences among control and exposed groups.
- Lactational water consumption decreased at 17-21 days in the 1000 mg/L group.
- Lactational water consumption decreased at 7-10, 10-14, 14-17, 17-21, 1-14 days in the 2000 mg/L group.


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 specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed
Description (incidence and severity):
- There were no statistically treatment related effects on the litter size (live and dead).
- No statistically differences in the number of male/female per exposure group or litter.
- No statistically differences in the percent male in the litter at PND 4.
- The percent of pregnant females/mated, percent of littered females/mated were reduced in the 500, 1000 and 2000 mg/L groups.
- No differences were found in the percent of littered females/pregnant and gestational lenght.
Key result
Dose descriptor:
LOAEL
Remarks:
10% body weight reduction
Effect level:
ca. 1 000 mg/L drinking water
Based on:
test mat.
Sex:
female
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 500 mg/L drinking water
Based on:
test mat.
Sex:
female
Basis for effect level:
body weight and weight gain
Critical effects observed:
no
Lowest effective dose / conc.:
1 000 mg/L drinking water
Clinical signs:
not specified
Dermal irritation (if dermal study):
not specified
Mortality / viability:
no mortality observed
Description (incidence and severity):
- At PND 1 no differences in total pup lived and dead, live males, live females, live pups (male and female), 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
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At PND 1:
- Male - Pup weights of the 2000 mg/L group were statistically reduced by 16%
- Combined pup weights (male + female) of the 2000 mg/L group were statistically reduced by 15%
- At 2000 mg/L the adjusted pup male and male+female body weights were statistically reduced by 16 and 12%, respectively.

At PND 21 male, female, and male+female pups body weights were statistically reduced at the 2000 mg/L group.
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Sexual maturation:
no effects observed
Description (incidence and severity):
No changes on testicular spermatid count, sperm motility, cauda epididymis sperm count.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- No changes on testis weights.
- Epididymitis and cauda epididymitis weights were statistically reduced by 15 & 17% respectively, in male pups at the 2000 mg/L.
Gross pathological findings:
not specified
Histopathological findings:
not specified
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
LOAEL
Generation:
F1
Effect level:
ca. 2 000 mg/L drinking water
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
ca. 1 000 mg/L drinking water
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Conclusions:
Perinatal studies concluded that were no treatment related effects on the percentage of dams delivering, litter size, or litter weights. There is a reduced body weights greater than 10% relative to control were observed in 2000 mg/L dams, PND 4 pups, PND 21 pups, and adult rats.
Executive summary:

No fertility, reproductive, or developmental toxicity data of sufficient quality are available for tungsten carbide (target substance). However, reproductive toxicity data are available for sodium tungstate (source substance), which are used for read-across. Due to lower water solubility and lower toxicity for the target substance compared to the source substance, the resulting read-across from the source substance to the target substance is appropriate as a conservative estimate of potential toxicity for this endpoint. In addition, read-across is appropriate because the classification and labelling is more protective for the source substance than the target substance, the PBT/vPvB profile is the same, and the dose descriptors are, or are expected to be, lower for the source substance. For more details, refer to the read-across category approach included in the Category section of this IUCLID submission on Annex 3 in the CSR.

Endpoint:
fertility, other
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
Study conducted only on male rats. Reproductive toxicity indicators included Leydig cell density and morphology, serum testosterone, luteinizing hormone, follicle-stimulating hormone, and percent of able males.
Justification for type of information:
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: Tungsten carbide
3. CATEGORY APPROACH JUSTIFICATION: See Annex 3 in CSR
4. DATA MATRIX: See Annex 3 in CSR
Reason / purpose for cross-reference:
reference to other study
Remarks:
Same authors published reproductive study in females
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
GLP compliance:
not specified
Remarks:
Study has adequate and reliable coverage of the key parameters foreseen to be investigated in the corresponding test methods and adequate and reliable documentation of the study is provided
Limit test:
yes
Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals or test system and environmental conditions:
Adult male Wistar rats (200 g) were kept under a constant 12-hour light-dark cycle and were allowed to eat and drink ad libitum.
Route of administration:
oral: drinking water
Vehicle:
other: 0.9% NaCl
Details on exposure:
The treatment was carried out for 3 months. During this period, glycemia and body weight were measured regularly. The time period of 3 months was chosen to allow tungstate to exert a complete effect on testicular function.
Details on mating procedure:
To evaluate reproductive performance, after 10 weeks of treatment, individual males were placed in a cage with 1 healthy adult female (body weight: 250 g). The animals were kept together overnight, and the following morning they were separated. Immediately after separation, a vaginal examination and scrape were performed to determine whether sexual intercourse had occurred. When intercourse was positive (presence of a vaginal tap with or without spermatozoa), the night routine was discontinued. When the male showed no sexual contact with the female during 9 consecutive days, this individual was not used for further mating and was termed ‘‘unable,’’ while those that showed sexual activity, as determined above, were termed ‘‘able.’’
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
3 months (time period was chosen to allow tungstate to exert complete effect on testicular function)
Frequency of treatment:
Daily via drinking water
Dose / conc.:
2 000 mg/L drinking water
Remarks:
Nominal in water
No. of animals per sex per dose:
12 male rats
Control animals:
yes, concurrent vehicle
Details on study design:
All the animals were anesthetized with diethyl ether and sacrificed by decapitation.
Parental animals: Observations and examinations:
Gycemia and body weights measured regularly
Sperm parameters (parental animals):
Time period was chosen to allow tungstate to exert complete effect on testicular function
Postmortem examinations (parental animals):
Blood was collected immediately to measure serum parameters. Testes were prepared in 3 ways: 1) Tissues were immediately fixed in 3% formaldehyde in a buffered solution containing 54 mM NaH2PO4 and 28 mM Na2HPO4 (pH 7.4) at 4°C (buffered formaldehyde). This material can be stored for up to 5 weeks. These tissues were used for optical microscope histology and immunohistochemistry; 2) Tissues were immediately fixed in 2.5% glutaraldehyde in ice-cold 0.1 M sodium cacodylate buffer (pH 7.4). These tissues were used for transmission electronic microscope histology; and 3) Tissues were immediately frozen in liquid N2 and store at -90°C until Western blot analyses were performed.
Reproductive indices:
The reproductive performance of the male rats was determined by calculating the percentage of ‘‘able’’ males out of the total number tested.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Description (incidence):
All the rats in the tungstate healthy rat group were alive
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight decease in treated animals
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Body weight decease in treated animals
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Tungstate administration to healthy rats did not modifiy the apperance or number of Leydig cells
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
To check whether tungstate has a direct effect on Leydig cells, the effects on the mLTC-1 cell line were evaluated, which is derived from Leydig cells. For this purpose, the effect of tungstate on the phosphorylation state of MAP-kinase and GSK-3, 2 were evaluated. Incubation with 1 mM tungstate induced a clear increase in the phosphorylation signal of MAP-kinase of these cells, which reached a maximum after 10 minutes of incubation. No effect of 1 mM tungstate was observed on the levels of total MAP-kinase. Incubation with 1 mM tungstate caused a significant increase in the GSK-3 phosphorylation state. No effects of tungstate were observed on the total amount of GSK-3.
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Tungstate treatment did not modify Leydig cell function in healthy rats
Reproductive performance:
no effects observed
Description (incidence and severity):
Tungstate administration did not alter the reproductive performance of healthy animals
- All the rats in the conrol and tretated tungstate group survived.
- Tungstate exposed rats have lower body weight gain than control animals.
- Tungstate administration to healthy rats did not modify the appearance or number of Leydig cells
- Administration of tungstate to healthy rats did not modify the expression of insulin receptor.
- To check whether tungstate has a direct effect on Leydig cells, the effects on the mLTC-1 cell line were evaluated, which is derived from Leydig cells. For this purpose, the effect of tungstate on the phosphorylation state of MAP-kinase and GSK-3, 2 were evaluated. Incubation with 1 mM tungstate induced a clear increase in the phosphorylation signal of MAP-kinase of these cells, which reached a maximum after 10 minutes of incubation. No effect of 1 mM tungstate was observed on the levels of total MAP-kinase. Incubation with 1 mM tungstate caused a significant increase in the GSK-3 phosphorylation state. No effects of tungstate were observed on the total amount of GSK-3.
- Tungsten treatment had no effect on serum glucose, insulin, luteinizing hormone, and follicle-stimulating hormone concentrations.
.
Key result
Dose descriptor:
NOAEL
Effect level:
> 2 000 mg/L drinking water
Based on:
test mat.
Sex:
male
Basis for effect level:
reproductive performance
Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
not examined
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Reproductive effects observed:
no
Conclusions:
Oral administration (2 mg/ml in drinking water) of sodium tungstate to adult male rats for 3 months did not show. Sodium tungstate orally exposed rats did not alter the reproductive performance of healthy males or any alteration in Leydig cell function, as shown by serum testosterone levels.
Executive summary:

No fertility, reproductive, or developmental toxicity data of sufficient quality are available for tungsten carbide (target substance). However, reproductive toxicity data are available for sodium tungstate (source substance), which are used for read-across. Due to lower water solubility and lower toxicity for the target substance compared to the source substance, the resulting read-across from the source substance to the target substance is appropriate as a conservative estimate of potential toxicity for this endpoint. In addition, read-across is appropriate because the classification and labelling is more protective for the source substance than the target substance, the PBT/vPvB profile is the same, and the dose descriptors are, or are expected to be, lower for the source substance. For more details, refer to the read-across category approach included in the Category section of this IUCLID submission on Annex 3 in the CSR. 

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: Tungsten carbide
3. CATEGORY APPROACH JUSTIFICATION: See Annex 3 in CSR
4. DATA MATRIX: See Annex 3 in CSR
Reason / purpose for cross-reference:
read-across source
Qualifier:
equivalent or similar to guideline
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: Animals were dosed once daily, by oral gavage, from Day 6 of gestation to Day 20 of lactation.
- Basis for dose level selection: Dose range finding study
- Inclusion/exclusion of extension of Cohort 1B : Cohort 1B was excluded
- Termination time for F2: F2 excluded
- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B: Inclusion of Cohorts 2A and 2B
- Inclusion/exclusion of developmental immunotoxicity Cohort 3: Cohort 1B was excluded
- Route of administration: Oral gavage
- Other considerations, e.g. on choice of species, strain, vehicle and number of animals [if applicable]: 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. Four groups of 22 time-mated female Crl: CD(SD) rats were dosed (in purified water). For the F1 generation, 20 males and 20 females per group were selected at weaning.
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 or test system 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 (eg ad libitum): A pelleted rodent diet, VRF1 (manufactured by SDS) supplied.
- Water (eg 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 / 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:
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.

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/day.
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
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):
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.

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
OESTRUS CYCLE:: 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.
OTHER: 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.
Dose descriptor:
NOAEL
Generation:
F1 (cohort 2A)
Effect level:
ca. 160 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
gross pathology
Remarks on result:
other: At necropsy there were no abnormalities noted in the reproductive organs of males.
Dose descriptor:
NOAEL
Generation:
F1 (cohort 2A)
Effect level:
ca. 160 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical signs
mortality
other: Group mean numbers of corpora lutea, implantations and number of live embryos were statistically significantly lower for females maternally given 160 mg/kg/day when compared with Controls. However, all values were within the historical Control data range
Dose descriptor:
NOEL
Generation:
F1 (cohort 2A)
Effect level:
ca. 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:
NOEL
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:
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.
Executive summary:

No fertility, reproductive, or developmental toxicity data of sufficient quality are available for 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 read-across category approach included in the Category section of this IUCLID submission in Annex 3 in the CSR.

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

No developmental toxicity data of sufficient quality are available for tungsten carbide (target substance). However, developmental (teratogenic) 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 or see Annex 3 of the CSR.

Segment II - Embryo-Foetal Development Study

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

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

 

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

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

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

·      Hanzu et al., (2010) conducted a prospective, randomized, placebo-controlled, double-blind, proof-of-concept studyin humans  (n=30). Following a 2-week lead-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.

A. First Species- Rat Embryo-Foetal Development

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

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

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

B. Second Species - Rabbit Embryo-Foetal Development

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

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

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

Conclusion: The major ECHA criticism on the previous weight of evidence submitted to fulfill the developmental toxicity endpoint was the lack of skeletal malformation information as well as a second species (rabbit) study. Under this resubmission update new ICH Segment II rat and rabbit studies

(never submitted previously) are included with the requested skeletal malformation information in two species satisfying the OECD 414 (first and second species) requirement.

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) - 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: Tungsten carbide
3. CATEGORY APPROACH JUSTIFICATION: See Annex 3 in CSR
4. DATA MATRIX: See Annex 3 in CSR
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
equivalent or similar to guideline
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. QA statement)
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 or test system 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 (eg 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 . See Tables 1a, 1b and 2 on "Any other information on results incl. tables" section below.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/day. See Tables 1a, 1b and 2 on "Any other information on results incl. tables" section below.
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
Description (incidence and severity):
See Table 3 on "Any other information on results incl. tables" section below.
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. See Tables 4a and Table 4b on "Any other information on results incl. tables" section below.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
See Table 3 on "Any other information on results incl. tables" section below.
Early or late resorptions:
no effects observed
Description (incidence and severity):
See Table 3 on "Any other information on results incl. tables" section below.
Dead fetuses:
no effects observed
Description (incidence and severity):
See Table 4b on "Any other information on results incl. tables" section below.
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. See Table 3 on "Any other information on results incl. tables" section below.
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 . See Table 5 on "Any other information on results incl. tables" section below.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
See Table 3 on "Any other information on results incl. tables" section below.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There was no effect of sodium tungstate on the sex ratio. See Table 5 on "Any other information on results incl. tables" section below.
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 . See Table 5 on "Any other information on results incl. tables" section below.
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Description (incidence and severity):
See Table 6 on "Any other information on results incl. tables" section below.
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. See Table 6 on "Any other information on results incl. tables" section below.
Visceral malformations:
no effects observed
Description (incidence and severity):
See Table 6 on "Any other information on results incl. tables" section below.
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

Table 1a. Body weight gains (g) - group mean values


 



































































































































Sex: Female



Body Weight Gain (Day of Gestation)



Group



0 to 5#



5 to 6#



6 to 7#



7 to 8#



8 to 9#



 



Group: 1


Control


0 mg/kg/day



Mean



22.4 I1



3.9 I1



2.0 R2



3.7 I1



4.1 I1



 



SD



11.0



3.8



6.6



4.8



7.7



 



N



20



20



20



20



20



 



Group: 2


Sodium Tungstate


40 mg/kg/day



Mean



22.2



4.1



1.4



4.6



4.0



 



SD



7.8



3.6



6.1



5.5



4.9



 



N



20



20



20



20



20



 



Group: 3


Sodium Tungstate


80 mg/kg/day



Mean



20.7



3.8



1.2



1.6



4.7



 



SD



9.6



4.7



6.9



4.6



4.2



 



N



19



19



19



19



19



 



Group: 4


Sodium Tungstate


160 mg/kg/day



Mean



20.4



4.8



0.3



2.7



2.8



 



SD



10.3



5.1



6.9



3.8



4.3



 



N



20



20



20



20



20



 



# [Statistically Analysed]


1 [I - Automatic Transformation: No Transformation]                                         


2 [R - Automatic Transformation: Rank]


 


Table 1b. Body weight gains (g) - group mean values


 



































































































































Sex: Female



Body Weight Gain (Day of Gestation)



Group



9 to 12#



12 to 15#



15 to 18#



6 to 18#



18 to 20#



 



Group: 1


Control


0 mg/kg/day



Mean



18.7 R1



18.4 I2



29.4 I2



76.2 I2



31.6 I2



 



SD



7.0



8.1



12.6



14.1



8.1



 



N



20



20



20



20



20



 



Group: 2


Sodium Tungstate


40 mg/kg/day



Mean



20.5



18.6



33.8



82.7



32.9



 



SD



6.0



6.3



7.0



10.2



4.7



 



N



20



20



20



20



20



 



Group: 3


Sodium Tungstate


80 mg/kg/day



Mean



18.4



18.2



32.6



76.8



36.5



 



SD



5.6



5.6



7.2



15.8



7.7



 



N



19



19



19



19



19



 



Group: 4


Sodium Tungstate


160 mg/kg/day



Mean



15.4



15.6



29.9



66.6 W3



34.8



 



SD



11.0



6.6



8.9



16.2



6.5



 



N



20



20



20



20



20



 



# [Statistically Analysed]


1 [R - Automatic Transformation: Rank]                                                           


2 [I - Automatic Transformation: No Transformation]


3 [W - Test: Williams 2 Sided p < 0.05]


 


Table 2. Terminal body weight (g) adjusted for gravid uterus weight (g) - group mean values


 












































































































Sex: Female



Dead Body Weight



Gravid Uterus Wt



BWt Adjusted for GUWt



Adjusted BWt Gain



Group



#



#



#



6 to 20#



Group: 1


Control


0 mg/kg/day



Mean



349.27 I1



71.1 R2



278.17 I1



32.42 I1



SD



30.15



23.0



17.85



10.75



N



20



20



20



20



Group: 2


Sodium Tungstate


40 mg/kg/day



Mean



359.18



73.4



285.83



39.03



SD



25.70



10.9



17.50



9.25



N



20



20



20



20



Group: 3


Sodium Tungstate


80 mg/kg/day



Mean



357.06



77.4



279.69



31.12



SD



32.53



13.9



24.31



14.85



N



19



19



19



19



Group: 4


Sodium Tungstate


160 mg/kg/day



Mean



346.06



69.3



276.81



29.71



SD



31.40



13.0



26.66



13.77



N



20



20



20



20



# [Statistically Analysed]


1 [I - Automatic Transformation: No Transformation]


2 [R - Automatic Transformation: Rank]


 


Table 3. Pregnancy data - summary


 





























































































Sex: Female



Group: 1
Control


0 mg/kg/day



Group: 2
Sodium Tungstate
40 mg/kg/day



Group: 3
Sodium Tungstate
80 mg/kg/day



Group: 4


Sodium Tungstate
160 mg/kg/day



Group Size



 



20



20



20



20



Not Pregnant



 



0



0



1



0



Not Pregnant %



 



0.0



0.0



5.0



0.0



Not Pregnant Died/Killed



Sum



0



0



0



0



Not Pregnant Schedule Kill



Sum



0



0



1



0



Pregnant



 



20



20



19



20



Pregnant %



 



100.0



100.0



95.0



100.0



Pregnant Died/Killed/Aborted



Sum



0



0



0



0



Pregnant with Total Resorption



Sum



0



0



0



0



Number with Live Foetuses



Sum



20



20



19



20



 


Table 4a. Uterine and implantation data - group mean values


 





















































































































Sex: Female



Group: 1
Control


0 mg/kg/day



Group: 2
Sodium Tungstate
40 mg/kg/day



Group: 3
Sodium Tungstate
80 mg/kg/day



Group: 4


Sodium Tungstate
160 mg/kg/day



Number with Foetuses



 



20



20



19



20



Number of Corpora Lutea#



Sum



275  R1



279



283



284



Mean



13.8  R1



14.0



14.9



14.2



SD



3.7



2.1



2.8



2.0



Number of Implantations#



Sum



259   R1



270



268



265



Mean



13.0  R1



13.5



14.1



13.3



SD



4.1



1.6



2.0



2.0



% Pre-implantation Loss #



Mean



6.1  R1



2.6



4.5



6.3



Number of Early Deaths



Sum



13



18



9



16



Mean



0.7



0.9



0.5



0.8



SD



1.0



0.7



0.7



1.1



Number of Late Deaths



Sum



0



0



0



0



Mean



0.0



0.0



0.0



0.0



SD



0.0



0.0



0.0



0.0



# [Statistically Analysed]


1 [R - Automatic Transformation: Rank]


 


Table 4b. Uterine and implantation data - group mean values (Continued)


 









































































Sex: Female



Group: 1
Control


0 mg/kg/day



Group: 2
Sodium Tungstate
40 mg/kg/day



Group: 3
Sodium Tungstate
80 mg/kg/day



Group: 4


Sodium Tungstate
160 mg/kg/day



Number of Dead Foetuses



Sum



0



0



0



0



Mean



0.0



0.0



0.0



0.0



SD



0.0



0.0



0.0



0.0



Number of Live Foetuses#



Sum



246   R1



252



259



249



Mean



12.3  R1



12.6



13.6



12.5



SD



4.0



1.8



2.3



2.2



% Post-implantation Loss #



Mean



4.6  R1



6.8



3.6



6.1



Mean % of Implantations



Mean



95.4



93.2



96.4



93.9



# [Statistically Analysed]


1 [R - Automatic Transformation: Rank]


 


Table 5. Litter weights (g) / foetal data - group mean values


 





























































































Sex: Female



Group: 1
Control


0 mg/kg/day



Group: 2
Sodium Tungstate
40 mg/kg/day



Group: 3
Sodium Tungstate
80 mg/kg/day



Group: 4


Sodium Tungstate
160 mg/kg/day



Number with Live Foetuses



 



20



20



19



20



No of Live Foetuses



Sum



246



252



259



249



No of Male Foetuses



Sum



133



145



141



114



No of Female Foetuses



Sum



113



107



118



135



% of Male Foetuses#



Mean



56.3 I1



57.3



54.8



44.3 W2



Litter Weight (g)#



Mean



46.66 R3     



47.85



51.40



46.15



Foetal Weight (M+F) (g)#



Mean



3.83 I1



3.80



3.77



3.70



Foetal Weight (M) (g)



Mean



3.93



3.88



3.88



3.82



Foetal Weight (F) (g)



Mean



3.65



3.68



3.67



3.60



Placental Weight (g)#



Mean



0.58 R3       



0.55



0.52



0.52



# [Statistically Analysed]


1 [I - Automatic Transformation: No Transformation]                                                


2 [W - Test: Williams 2 Sided p < 0.05]
3 [R - Automatic Transformation: Rank]


 


Table 6. Examination of foetuses - summary of group mean values



























































































Combined examination


(external/visceral/skeletal)



Group: 1 Control


0 mg/kg/day



Group: 2
Sodium Tungstate
40 mg/kg/day



Group: 3
Sodium Tungstate
80 mg/kg/day



Group: 4


Sodium Tungstate
160 mg/kg/day



 



Total number of litters examined



20



20



19



20



Total number of foetuses examined



246



252



259



249



Number with major abnormalities



1



1



1



0



Mean % of foetuses examined



0.4



0.5



0.5



0.5


 



Number of litters affected#



1



1



1



0



Number with minor abnormalities



56



74



75



81



Mean % of foetuses examined



23.2



30.1



28.4



33.1



Number of litters affected



19



20



19



20J



Number with variations



136



129



136



150



Mean % of foetuses examine



58.1



51.5



52.4



60.2



Number of litters affected#



20



20



19



20


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.
Executive summary:

No fertility, reproductive, or developmental toxicity data of sufficient quality are available for tungsten carbide (target substance). However, developmental toxicity data are available for sodium tungstate (source substance), which are used for read-across. Due to lower water solubility and lower toxicity for the target substance compared to the source substance, the resulting read-across from the source substance to the target substance is appropriate as a conservative estimate of potential toxicity for this endpoint. In addition, read-across is appropriate because the classification and labelling is more protective for the source substance than the target substance, the PBT/vPvB profile is the same, and the dose descriptors are, or are expected to be, lower for the source substance. For more details, refer to the read-across category approach in the Category section of this IUCLID submission or Annex 3 in the CSR.

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): 11 June 2015 - Completion date of experimental phase (Foetal pathology) - 08 July 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: Tungsten carbide
3. CATEGORY APPROACH JUSTIFICATION: See Annex 3 in CSR
4. DATA MATRIX: See Annex 3 in CSR
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental 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
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%
- Storage condition of test material: The test item was stored at room temperature (15 °C to 25 °C), protected from light.
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Covance Research Products Inc, 310 Swampbridge Road, Denver, PA 17517, USA
- Age at study initiation: The animals were approximately five months of age on arrival
- Weight at study initiation: On the first day of dosing the females weighed 2.72 kg to 3.93 kg
- Housing: The animals were housed individually in perforated-floor cages suspended over paper-lined trays.
- Diet (e.g. ad libitum): A pelleted diet, STANRAB (P) SQC supplied by Special Diet Services (SDS) freely available.
- Water (e.g. ad libitum): Tap water were freely available.
- Acclimation period: After five days acclimatisation they were re-examined and confirmed to be suitable for use.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): The target range for temperature was 16 °C to 20 °C and recorded values were within these limits.
- Humidity (%): Humidity was not controlled but recorded
- 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 and was air-conditioned

IN-LIFE DATES: From: 07 August 2015 To: 04 November 2015
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
purified
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Formulations for use on the first and last day of dosing were analysed to determine their achieved concentrations. Vehicle (for Controls) from the first preparation and that for use on the last day of dosing were analysed to confirm the absence of the test item. Analysis were conducted at Kymos Pharma Services using method code C003-MP0023
Details on mating procedure:
The females were obtained from the supplier timed-mated. For mating, each female will have been paired with a sexually mature male of the same strain. The day on which mating is detected is designated Day 0 of gestation. The uterus of any apparently non-pregnant female was stained with ammonium sulphide to confirm pregnancy status.
Duration of treatment / exposure:
Animals will be dosed once daily, from Day 6 of gestation to Day 18 of gestation inclusive. Control animals will receive the vehicle only, following the same regimen as the other groups
Frequency of treatment:
Animals will be dosed once daily
Duration of test:
From Day 6 of gestation to Day 18 of gestation inclusive.
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Twenty-two females per group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected on the basis of results from a preliminary dose range-finding study performed at Sequani (Sequani Study Number: OHS0002. In that study, a dose level of 150 mg/kg/day sodium tungstate resulted in body weight losses, however, at 100 mg/kg/day there was only reduced body weight gain. A dose level of 100 mg/kg/day therefore, was considered to be a suitable high dose level for the current study

- Rationale for animal assignment (if not random): Allocation to groups was performed using a stratified randomisation procedure based on individual body weights recorded on Day 0 of gestation at the suppliers.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes
- Animals were examined twice daily for mortality and morbidity and were given a detailed clinical examination daily.

BODY WEIGHT: Yes
- Body weights were recorded on Day 0 of gestation by the supplier. At Sequani, body weights were recorded daily from Day 4 to 19 of gestation inclusive, and then on Days 22, 25 and 28 of gestation.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
The amount of food consumed by each animal was recorded daily over Days 5 to 6 of gestation and every two days thereafter during gestation

POST-MORTEM EXAMINATIONS: Yes
The females were killed on Day 28 of gestation. 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:
The ovaries and uterine content was examined after termination: Yes
For pregnant females the following observations were made:
1. Number of corpora lutea
2. Number and distribution of implantations in uterine horns, classified as early intrauterine deaths, late intrauterine deaths, dead foetuses or live foetuses
3. Gravid uterus weight (Day 28 of gestation only)
4. Placental weight (Day 28 of gestation only)
The implantations are numbered separately for the right and left horns. Numbering is sequential, commencing at the ovarian end through to the cervix.
Fetal examinations:
- External examinations: Yes
- Soft tissue examinations: Yes
- Skeletal examinations: Yes
- Head examinations: Yes

The following observations were made for live foetuses killed on Day 28 of gestation: 1. Foetal weights 2. Foetal sexes (internal) 3. Foetal abnormalities (to include external, fresh visceral, fixed heads and/or skeletal examinations). Approximately 50 % of the foetuses in each litter were decapitated and the heads fixed in Bouins' fluid for subsequent serial sectioning to permit examination of the eyes, brain, nasal passages and tongue.

The head along the frontal parietal suture and the brain was examined. The carcasses were subsequently cleared in potassium hydroxide, stained with Alizarin
red S to visualise the ossified skeleton and examined for skeletal variants and abnormalities. Structural congenital abnormalities that impair or potentially impair the survival, development or function of the foetus are classified as major abnormalities. Other defects were classified as minor abnormalities. Commonly observed variations are recorded as variants.
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. 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 were applied.
Indices:
- 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 foetuses)/no. of implantation sites ]x 100

Mean foetal body weights were calculated separately by sex for each litter; group mean body weights were calculated (separately by sex) from the litter means. Mean pre- and post-implantation losses were calculated on a proportional litter basis. The percentage of foetuses in each litter exhibiting each classification of abnormality was calculated; group mean percentages were calculated from the litter percentages. The percentage of male foetuses, out of the total number of foetuses, was calculated for each litter.
Historical control data:
Background data on the rate of spontaneous malformations have been accumulated.
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 three early decedent females, all given 10 mg/kg/day Sodium Tungstate. Female 428 died on Day 16 of gestation; necropsy findings were consistent with a dosing trauma. Female 434 showed signs of abortion on Day 26 of gestation and Female 423 aborted on Day 28 of gestation; as there were no abortions at higher doses of Sodium Tungstate these two events were considered not to be related to test item administration. There were no clinical signs associated with sodium tungstate.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In the group given 100 mg/kg/day, body weight gain was significantly lower than Controls during the dosing period (p<0.01) but thereafter, weight gain was similar to Controls. Body weights and body weight gains were similar to Controls for animals given 10 or 30 mg/kg/day Sodium Tungstate. See Tables 1a, 1b and 2 on "Any other information on results incl. tables" section below.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There was no adverse effect of sodium tungstate on food intake; group mean intake was similar in all groups throughout the study.
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 maternal macroscopic abnormalities at necropsy that were considered to be related to sodium tungstate administration.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Number of abortions:
effects observed, non-treatment-related
Description (incidence and severity):
Female 434 showed signs of abortion on Day 26 of gestation and Female 423 aborted on Day 28 of gestation; as there were no abortions at higher doses of sodium tungstate these two events were considered not to be related to test item administration. See Table 3 on "Any other information on results incl. tables" section below.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There were no effects of sodium tungstate on the mean numbers of implantations, the incidences of pre- and post-implantation loss or on the mean number of live foetuses. See Table 4b on "Any other information on results incl. tables" section below.
Total litter losses by resorption:
effects observed, non-treatment-related
Description (incidence and severity):
One pregnant rabbit with total resorption at the 30 mg/kg/day group. See Table 3 on "Any other information on results incl. tables" section below.
Early or late resorptions:
effects observed, non-treatment-related
Description (incidence and severity):
See Table 4a and 5a on "Any other information on results incl. tables" section below.
Dead fetuses:
no effects observed
Description (incidence and severity):
Female 450 given 30 mg/kg/day had total early intrauterine deaths and Females 469 and 486 given 100 mg/kg/day were not pregnant. There were no effects of sodium tungstate on the mean number of live foetuses. See Table 4b and 5b on "Any other information on results incl. tables" section below.
Changes in pregnancy duration:
not specified
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
See Table 3 on "Any other information on results incl. tables" section below.
Key result
Dose descriptor:
NOEL
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
NOAEL
Effect level:
100 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):
There was no adverse effect of sodium tungstate on the mean foetal or placental weights. See Table 6 on "Any other information on results incl. tables" section below.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
There were 22, 19, 21 and 20 females with live foetuses on Day 28 of gestation in the groups given 0, 10, 30 or 100 mg/kg/day, respectively. See Tables 4b and 5b on "Any other information on results incl. tables" section below.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There was no adverse effect of sodium tungstate on the mean sex ratio. See Table 6 on "Any other information on results incl. tables" section below.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
See Table 6 on "Any other information on results incl. tables" section below.
Changes in postnatal survival:
not specified
External malformations:
no effects observed
Description (incidence and severity):
Major foetal abnormalities were noted in three, seven, four and four foetuses from three, five, three and two litters given 0, 10, 30 or 100 mg/kg/day, respectively. The nature, incidence and intergroup distribution of these abnormalities do not indicate an adverse effect of the test item. The incidences of minor and variant foetal abnormalities were similar in all groups.
Skeletal malformations:
no effects observed
Description (incidence and severity):
Major foetal abnormalities were noted in three, seven, four and four foetuses from three, five, three and two litters given 0, 10, 30 or 100 mg/kg/day, respectively. The nature, incidence and intergroup distribution of these abnormalities do not indicate an adverse effect of the test item. The incidences of minor and variant foetal abnormalities were similar in all groups.
Visceral malformations:
no effects observed
Description (incidence and severity):
Major foetal abnormalities were noted in three, seven, four and four foetuses from three, five, three and two litters given 0, 10, 30 or 100 mg/kg/day, respectively. The nature, incidence and intergroup distribution of these abnormalities do not indicate an adverse effect of the test item. The incidences of minor and variant foetal abnormalities were similar in all groups.
Key result
Dose descriptor:
NOEL
Effect level:
> 100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
external malformations
skeletal malformations
visceral malformations
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Table 1a. Body weight gains (kg) - group mean values.






















































































































Sex: Female



Body Weight Gain (Day of Gestation)



Group



0 to 6#



6 to 7#



7 to 8#



8 to 9#



9 to 12#



Group: 1


Control


0 mg/kg/day



Mean



-0.033 R1



0.030 I2



0.004 R1



0.011 R1



0.066 I2



SD



0.303



0.061



0.046



0.036



0.040



N



22



22



22



22



22



Group: 2


Sodium Tungstate


10 mg/kg/day



Mean



0.007



0.005



0.007



0.005



0.073



SD



0.100



0.052



0.046



0.036



0.052



N



20



20



20



20



20



Group: 3


Sodium Tungstate


30 mg/kg/day



Mean



-0.035



0.005



0.004



0.007



0.072



SD



0.141



0.039



0.068



0.062



0.060



N



21



21



21



21



21



Group: 4


Sodium Tungstate


100 mg/kg/day



Mean



0.033



-0.012 W3



0.003



0.008



0.062



SD



0.075



0.049



0.045



0.030



0.054



N



20



20



20



20



20



# [Statistically Analysed]


1 [R - Automatic Transformation: Rank]


2 [I - Automatic Transformation: No Transformation]
3 [W - Test: Williams 2 Sided p < 0.05]


 


Table 1b. Body weight gains (kg) – group mean values (Continued)






















































































































Sex: Female



Body Weight Gain (Day of Gestation)



Group



12 to 15#



15 to 19#



6 to 19#



19 to 28#



 



Group: 1


Control


0 mg/kg/day



Mean



0.092 I1



0.072 R2



0.275 R2



0.149 R2



 



SD



0.055



0.049



0.108



0.075



 



N



22



22



22



22



 



Group: 2


Sodium Tungstate


10 mg/kg/day



Mean



0.078



0.065



0.233



0.138



 



SD



0.054



0.066



0.104



0.157



 



N



20



20



20



20



 



Group: 3


Sodium Tungstate


30 mg/kg/day



Mean



0.107



0.076



0.271



0.191



 



SD



0.041



0.062



0.152



0.066



 



N



21



21



21



21



 



Group: 4


Sodium Tungstate


100 mg/kg/day



Mean



0.096



0.024 S3



0.180 SS4



0.191



 



SD



0.044



0.077



0.105



0.072



 



N



20



20



20



20



 



# [Statistically Analysed]


1 [I - Automatic Transformation: No Transformation]


2 [R - Automatic Transformation: Rank]


3 [S - Test: Shirley 2 Sided p < 0.05]


4 [SS - Test: Shirley 2 Sided p < 0.01]


 


 Table 2. Terminal body weight (kg) adjusted for gravid uterus weight (g) - group mean values.


 





































































































Sex: Female  (Group)



Dead Body Weight (g)#



Gravid Uterus  Wt#



BWt Adjusted for GUWt#



Adjusted BWt Gain 6 to 28#



Group: 1


Control


0 mg/kg/day



Mean



3.55 R1



443.2 I2



3.11 I2



-0.05 R1



SD



0.30



73.5



0.25



0.11



N



22



22



22



22



Group: 2


Sodium Tungstate


10 mg/kg/day



Mean



3.60



421.5



3.24



-0.02



SD



0.34



92.7



0.19



0.13



N



20



19



19



19



Group: 3


Sodium Tungstate


30 mg/kg/day



Mean



3.65



447.9



3.20



-0.01



SD



0.22



75.5



0.20



0.16



N



21



21



21



21



Group: 4


Sodium Tungstate


100 mg/kg/day



Mean



3.56



412.2



3.14



-0.06



SD



0.21



71.9



0.22



0.10



N



20



20



20



20



# [Statistically Analysed]


1 [R - Automatic Transformation: Rank]                 


2 [I - Automatic Transformation: No Transformation]


 


Table 3. Pregnancy data – summary





























































































Sex: Female



Group: 1
Control


0 mg/kg/day



Group: 2
Sodium Tungstate
10 mg/kg/day



Group: 3
Sodium Tungstate
30 mg/kg/day



Group: 4


Sodium Tungstate
100 mg/kg/day



Group Size



 



22



22



22



22



Not Pregnant



 



0



0



0



2



Not Pregnant %



 



0.0



0.0



0.0



9.1



Not Pregnant Died/Killed



Sum



0



0



0



0



Not Pregnant Schedule Kill



Sum



0



0



0



2



Pregnant



 



22



22



22



20



Pregnant %



 



100.0



100.0



100.0



90.9



Pregnant Died/Killed/Aborted



Sum



0



3



0



0



Pregnant with Total Resorption



Sum



0



0



1



0



Number with Live Foetuses



Sum



22



19



21



20



 


Table 4a. Uterine and implantation data - group mean values - by implant





















































































































Sex: Female



Group: 1
Control


0 mg/kg/day



Group: 2
Sodium Tungstate
10 mg/kg/day



Group: 3
Sodium Tungstate
30 mg/kg/day



Group: 4


Sodium Tungstate
100 mg/kg/day



Number with Implantations



 



22



20



22



20



Number of Corpora Lutea#



Sum



217R1



198



221



200



Mean



9.9R1



9.9



10.0



10.0



SD



1.1



1.2



2.1



1.5



Number of Implantations#



Sum



205R1



174



203



181



Mean



9.3R1



8.7



9.2



9.1



SD



1.5



2.3



2.0



1.5



% Pre-implantation Loss #



Mean



5.4R1



12.3



8.1



9.2



Number of Early Deaths#



Sum



7 R1



3



7



12



Mean



0.3R1



0.2



0.3



0.6



SD



0.6



0.4



0.8



1.3



Number of Late Deaths#



Sum



1 R1



0



0



0



Mean



0.0R1



0.0



0.0



0.0



SD



0.2



0.0



0.0



0.0



# [Statistically Analysed]


1 [R - Automatic Transformation: Rank]


 


Table 4b. Uterine and implantation data - group mean values - by implant (Continued)









































































Sex: Female



Group: 1
Control


0 mg/kg/day



Group: 2
Sodium Tungstate
10 mg/kg/day



Group: 3
Sodium Tungstate
30 mg/kg/day



Group: 4


Sodium Tungstate
100 mg/kg/day



Number of Dead Foetuses



Sum



0



0



0



0



Mean



0.0



0.0



0.0



0.0



SD



0.0



0.0



0.0



0.0



Number of Live Foetuses#



Sum



197 R1



162



196



169



Mean



9.0 R1



8.1



8.9



8.5



SD



1.6



2.9



2.5



1.7



% Post-implantation Loss #



Mean



3.8 R1



6.5



6.6



6.5



Mean % of Implantations



Mean



96.2



93.5



93.4



93.5



# [Statistically Analysed]


1 [R - Automatic Transformation: Rank]


 


Table 5a. Uterine and implantation data - group mean values - by foetus





















































































































Sex: Female



Group: 1
Control


0 mg/kg/day



Group: 2
Sodium Tungstate
10 mg/kg/day



Group: 3
Sodium Tungstate
30 mg/kg/day



Group: 4


Sodium Tungstate
100 mg/kg/day



Number with Foetuses



 



22



20



21



20



Number of Corpora Lutea#



Sum



217 L1



198



215



200



Mean



9.9 L1



9.9



10.2



10.0



SD



1.1



1.2



2.0



1.5



Number of Implantations#



Sum



205 I2



174



200



181



Mean



9.3 I2



8.7



9.5



9.1



SD



1.5



2.3



1.4



1.5



% Pre-implantation Loss #



Mean



5.4 R3



12.3



6.1



9.2



Number of Early Deaths#



Sum



7R3



3



4



12



Mean



0.3 R3



0.2



0.2



0.6



SD



0.6



0.4



0.5



1.3



Number of Late Deaths#



Sum



1 R3



0



0



0



Mean



0.0 R3



0.0



0.0



0.0



SD



0.2



0.0



0.0



0.0



 


# [Statistically Analysed]


1 [L - Automatic Transformation: Log]                                                       


2 [I - Automatic Transformation: No Transformation]


3 [R - Automatic Transformation: Rank]


 


Table 5b. Uterine and implantation data - group mean values - by foetus (Continued)









































































Sex: Female



Group: 1
Control


0 mg/kg/day



Group: 2
Sodium Tungstate
10 mg/kg/day



Group: 3
Sodium Tungstate
30 mg/kg/day



Group: 4


Sodium Tungstate
100 mg/kg/day



Number of Dead Foetuses



Sum



0



0



0



0



Mean



0.0



0.0



0.0



0.0



SD



0.0



0.0



0.0



0.0



Number of Live Foetuses#



Sum



197 R1



162



196



169



Mean



9.0 R1



8.1



9.3



8.5



SD



1.6



2.9



1.6



1.7



% Post-implantation Loss #



Mean



3.8 R1



6.5



2.2



6.5



Mean % of Implantations



Mean



96.2



93.5



97.8



93.5



# [Statistically Analysed]


1 [R - Automatic Transformation: Rank]


 


Table 6. Litter weights (g) / foetal data - group mean values





























































































Sex: Female



Group: 1
Control


0 mg/kg/day



Group: 2
Sodium Tungstate
10 mg/kg/day



Group: 3
Sodium Tungstate
30 mg/kg/day



Group: 4       Sodium Tungstate
100 mg/kg/day



Number with Live Foetuses



 



22



19



21



20



No of Live Foetuses



 



197



162



196



169



No of Male Foetuses



 



97



80



86



78



No of Female Foetuses



 



100



82



110



91



% of Male Foetuses#



Mean



48.7 I1



49.7



43.7



45.9



Litter Weight (g)#



Mean



298.56 R2



272.37



304.30



281.52



Foetal Weight (M+F) (g)#



Mean



33.57 I1



34.35



32.73



33.65



Foetal Weight (M) (g)



Mean



34.27



34.32



33.41



34.16



Foetal Weight (F) (g)



Mean



32.98



34.20



32.19



33.63



Placental Weight (g)#



Mean



3.45 I1



3.55



3.33



3.38



# [Statistically Analysed]


1 [I - Automatic Transformation: No Transformation]                                         


2 [R - Automatic Transformation: Rank]


 


Table 7. Examination of foetuses - summary of group mean values


























































































Combined examination


(external/visceral/skeletal)



Group: 1 Control


0 mg/kg/day



Group: 2
Sodium Tungstate
10 mg/kg/day



Group: 3
Sodium Tungstate
30 mg/kg/day



Group: 4


Sodium Tungstate
100 mg/kg/day



Total number of litters examined



22



19



21



20



Total number of foetuses examined



197



162



196



169



Number with major abnormalities



3



7



4



4



Mean % of foetuses examined



1.5



5.3



2.2



3.7



Number of litters affected#



3



5



3



2



Number with minor abnormalities



76



64



74



73



Mean % of foetuses examined



38.9



41.0



36.9



41.9



Number of litters affected#



22



18



20



18



Number with variations



197



162



196



169



Mean % of foetuses examine



100



100



100



100



Number of litters affected#



22



19



21



20



# = statistically analysed

Conclusions:
Administration of sodium tungstate once daily by oral gavage from Day 6 of gestation until Day 18 of gestation to New Zealand White rabbits at dose levels of 10, 30 or 100 mg/kg/day was well tolerated with reductions in body weight gain at 100 mg/kg/day only. There was no effect on the pregnancy data and no foetal abnormalities considered to be related to administration of sodium tungstate. Based on the above findings the No Observed Effect Level (NOEL) for maternal toxicity was considered to be 30 mg/kg/day and the No Observed Adverse Effect Level (NOAEL) was considered to be 100 mg/kg/day. The NOEL and the NOAEL for embryo-foetal development were considered to be 100 mg/kg/day. There were no deaths or clinical signs considered to be related to sodium tungstate administration.   In the group given 100 mg/kg/day, mean body weight gain was significantly lower than that of the Controls during the dosing period.  Body weights and body weight gains were similar to Controls for animals given 10 or 30 mg/kg/day sodium tungstate.  Group mean food intake was similar in all groups throughout the study. There were 22, 19, 21 and 20 females with live foetuses on Day 28 of gestation in the groups given 0, 10, 30 or 100 mg/kg/day, respectively.  There was no effect of sodium tungstate on the uterine/implantation or foetal data. There were no test item-related major foetal abnormalities and the incidences of minor and variant foetal abnormalities were similar in all groups.
Executive summary:

No fertility, reproductive, or developmental toxicity data of sufficient quality are available for tungsten carbide (target substance). However, reproductive toxicity data are available for sodium tungstate (source substance), which are used for read-across. Due to lower water solubility and lower toxicity for the target substance compared to the source substance, the resulting read-across from the source substance to the target substance is appropriate as a conservative estimate of potential toxicity for this endpoint. In addition, read-across is appropriate because the classification and labelling is more protective for the source substance than the target substance, the PBT/vPvB profile is the same, and the dose descriptors are, or are expected to be, lower for the source substance. For more details, refer to the read-across category approach in the Category section of this IUCLID submission or Annex 3 in the CSR. 

Endpoint:
developmental toxicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting 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: Tungsten carbide
3. CATEGORY APPROACH JUSTIFICATION: See Annex 3 in CSR
4. DATA MATRIX: See Annex 3 in CSR
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
equivalent or similar to guideline
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. QA statement)
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 or test system 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 (eg 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. See Tables 1, 2 and 3 on "Any other information on results incl. tables" section below.
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
Description (incidence and severity):
See Table 4 on "Any other information on results incl. tables" section below.
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. See Tables 5 on "Any other information on results incl. tables" section below.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
See Tables 4 on "Any other information on results incl. tables" section below.
Early or late resorptions:
no effects observed
Description (incidence and severity):
See Tables 5 on "Any other information on results incl. tables" section below.
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. See Table 5 on "Any other information on results incl. tables" section below.
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. See Table 4 on "Any other information on results incl. tables" section below.
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 . See Table 6 on "Any other information on results incl. tables" section below.
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. See Table 4 on "Any other information on results incl. tables" section below.
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. See Table 6 on "Any other information on results incl. tables" section below.
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. See Table 6 on "Any other information on results incl. tables" section below.
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. See Table 7 on "Any other information on results incl. tables" section below.
Skeletal malformations:
no effects observed
Description (incidence and severity):
See Table 7 on "Any other information on results incl. tables" section below.
Visceral malformations:
no effects observed
Description (incidence and severity):
See Table 7 on "Any other information on results incl. tables" section below.
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

Table 1. Body weights (g) - group mean values


 







































































































Sex: Female



Body Weight
(Day of Gestation)



Group



0



6



7



8



9



10



Group: 1


Control


0 mg/kg/day



Mean



240.1



253.7



248.7



250.1



257.3



263.3



SD



23.7



8.9



12.2



12.2



13.7



13.1



N



7



7



7



7



7



7



Group: 2


Sodium Tungstate


50 mg/kg/day



Mean



241.4



252.7



245.4



251.0



254.3



262.6



SD



23.2



18.0



17.1



15.3



15.4



16.9



N



7



7



7



7



7



7



Group: 3


Sodium Tungstate


100 mg/kg/day



Mean



241.4



251.0



247.9



256.0



259.3



268.6



SD



22.5



20.7



21.1



22.2



22.5



23.5



N



7



7



7



7



7



7



 










































































Sex: Female



Body Weight
(Day of Gestation)



Group



17



18



20



Group: 1


Control


0 mg/kg/day



Mean



328.3



333.1



367.0



SD



10.2



10.2



13.3



N



7



7



7



Group: 2


Sodium Tungstate


50 mg/kg/day



Mean



318.6



327.1



358.7



SD



19.5



22.8



25.0



N



7



7



7



Group: 3


Sodium Tungstate


100 mg/kg/day



Mean



321.0



326.6



358.7



SD



24.4



26.7



29.8



N



7



7



7



  


Table 2. Body weight gains (g) - group mean values


 







































































































Sex: Female



Body Weight


Gain (Day of Gestation)



Group



0 to 6



6 to 7



7 to 8



8 to 9



9 to 12



12 to 15



Group: 1


Control


0 mg/kg/day



Mean



13.6



-5.0



1.4



7.1



25.9



21.6



SD



24.8



6.1



2.2



4.0



8.8



5.5



N



7



7



7



7



7



7



Group: 2


Sodium Tungstate


50 mg/kg/day



Mean



11.3



-7.3



5.6



3.3



22.7



19.4



SD



11.0



4.0



3.2



4.0



5.7



5.5



N



7



7



7



7



7



7



Group: 3


Sodium Tungstate


100 mg/kg/day



Mean



9.6



-3.1



8.1



3.3



21.4



19.3



SD



4.9



1.2



4.0



5.6



1.5



3.9



N



7



7



7



7



7



7



 









































































Sex: Female



Body Weight


Gain (Day of Gestation)



Group



15 to 18



6 to 18



18 to 20



Group: 1


Control


0 mg/kg/day



Mean



28.4



79.4



33.9



SD



5.7



6.4



5.7



N



7



7



7



Group: 2


Sodium Tungstate


50 mg/kg/day



Mean



30.7



74.4



31.6



SD



4.9



9.6



3.6



N



7



7



7



Group: 3


Sodium Tungstate


100 mg/kg/day



Mean



26.6



75.6



32.1



SD



5.7



9.2



4.2



N



7



7



7



 


Table 3. Terminal body weight (g) adjusted for gravid uterus weight (g) - group mean values


 



















































































Sex: Female



Dead
Body Weight



Gravid
Uterus Wt



BWt Adjusted
for GUWt



Adjusted
BWt Gain



Group



6 to 20



Group: 1


Control


0 mg/kg/day



Mean



362.33



78.9



283.47



29.76



SD



14.10



3.9



13.94



8.88



N



7



7



7



7



Group: 2


Sodium Tungstate


50 mg/kg/day



Mean



355.54



75.3



280.26



27.54



SD



25.09



7.7



19.68



10.06



N



7



7



7



7



Group: 3


Sodium Tungstate


100 mg/kg/day



Mean



354.83



70.3



284.54



33.54



SD



29.14



10.9



23.78



7.32



N



7



7



7



7



 


Table 4. Pregnancy data - summary


 





























































































Sex: Female



Group: 1
Control


0 mg/kg/day



Group: 2
Sodium Tungstate
50 mg/kg/day



Group: 3
Sodium Tungstate
100 mg/kg/day



Group: 4


Sodium Tungstate
250 mg/kg/day



Group Size



 



7



7



7



7



Not Pregnant



 



0



0



0



1



Not Pregnant %



 



0.0



0.0



0.0



14.3



Not Pregnant Died/Killed



Sum



0



0



0



1



Not Pregnant Schedule Kill



Sum



0



0



0



0



Pregnant



 



7



7



7



6



Pregnant %



 



100.0



100.0



100.0



85.7



Pregnant Died/Killed/Aborted



Sum



0



0



0



6



Pregnant with Total Resorption



Sum



0



0



0



0



Number with Live Foetuses



Sum



7



7



7



0



 


Table 5. Uterine and implantation data - group mean values


 



















































































Sex: Female



Group: 1
Control


0 mg/kg/day



Group: 2
Sodium Tungstate
50 mg/kg/day



Group: 3


Sodium Tungstate
100 mg/kg/day



Number with Foetuses



 



7



7



7



Number of Corpora Lutea



Sum



108



103



93



Mean



15.4



14.7



13.3



SD



1.6



1.4



1.0



Number of Implantations



Sum



107



98



91



Mean



15.3



14.0



13.0



SD



1.6



1.4



1.3



% Pre-implantation Loss



Mean



0.9



4.6



2.2



Number of Early Deaths



Sum



4



2



6



Mean



0.6



0.3



0.9



SD



1.1



0.5



0.7



 


 


Table 6. Litter weights (g) / foetal data - group mean values


 


















































































Sex: Female



Group: 1
Control


0 mg/kg/day



Group: 2
Sodium Tungstate
50 mg/kg/day



Group: 3


Sodium Tungstate
100 mg/kg/day



Number with Live Foetuses



 



7



7



7



No of Live Foetuses



Sum



102



96



85



No of Male Foetuses



Sum



49



45



44



No of Female Foetuses



Sum



53



51



41



% of Male Foetuses



Mean



48.2



46.8



51.9



Litter Weight (g)



Mean



51.35



49.65



46.52



Foetal Weight (M+F) (g)



Mean



3.53



3.62



3.82



Foetal Weight (M) (g)



Mean



3.59



3.72



3.91



Foetal Weight (F) (g)



Mean



3.47



3.55



3.72



Placental Weight (g)



Mean



0.52



0.53



0.56



 


 


Table 7. Examination of foetuses - summary of group mean values


 














































































Combined examination


(external/visceral/skeletal)



Group: 1 Control


0 mg/kg/day



Group: 2
Sodium Tungstate


50 mg/kg/day



Group: 3


Sodium Tungstate
100 mg/kg/day



Total number of litters examined



7



7



7



Total number of foetuses examined



102



96



85



Number with major abnormalities



0



0



0



Mean % of foetuses examined



0



0



0



Number of litters affected



0



0



0



Number with minor abnormalities



0



0



1



Mean % of foetuses examined



0



0



1.3



Number of litters affected



0



0



1



Number with variations



0



0



0



Mean % of foetuses examine



0



0



0



Number of litters affected



0



0



0


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.
Executive summary:

No fertility, reproductive, or developmental toxicity data of sufficient quality are available for tungsten carbide (target substance). However, developmental toxicity data are available for sodium tungstate (source substance), which are used for read-across. Due to lower water solubility and lower toxicity for the target substance compared to the source substance, the resulting read-across from the source substance to the target substance is appropriate as a conservative estimate of potential toxicity for this endpoint. In addition, read-across is appropriate because the classification and labelling is more protective for the source substance than the target substance, the PBT/vPvB profile is the same, and the dose descriptors are, or are expected to be, lower for the source substance. For more details, refer to the read-across category approach in the Category section of this IUCLID submission or Annex 3 in the CSR.

Endpoint:
developmental toxicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Study period:
Experiment start date (Animal arrival): 11 June 2015 - Completion date of experimental phase (Foetal pathology) - 08 July 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: Tungsten carbide
3. CATEGORY APPROACH JUSTIFICATION: See Annex 3 in CSR
4. DATA MATRIX: See Annex 3 in CSR
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Principles of method if other than guideline:
The objective of this study was to investigate the effects of the test item, sodium tungstate, on the embryonic and foetal development of the female New Zealand White rabbit when administered during the period of organogenesis, in order to select dose levels for a subsequent embryo-foetal development study.
GLP compliance:
yes
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: 85.4%
- Storage condition of test material: The test item was stored at room temperature (15 °C to 25 °C), protected from light.
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Covance Research Products Inc, 310 Swampbridge Road, Denver, PA 17517, USA
- Age at study initiation: The animals were approximately five months of age on arrival
- Weight at study initiation: On the first day of dosing the females weighed 2.72 kg to 3.93 kg
- Housing: The animals were housed individually in perforated-floor cages suspended over paper-lined trays.
- Diet (e.g. ad libitum): A pelleted diet, STANRAB (P) SQC supplied by Special Diet Services (SDS) freely available.
- Water (e.g. ad libitum): Tap water were freely available.
- Acclimation period: After five days acclimatisation they were re-examined and confirmed to be suitable for use.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): The target range for temperature was 16 °C to 20 °C and recorded values were within these limits.
- Humidity (%): Humidity was not controlled but recorded
- 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 and was air-conditioned

IN-LIFE DATES: From: 11 June 2015 To: 08 July 2015
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
purified
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Formulations for use on the first and last day of dosing were analysed to determine their achieved concentrations. Vehicle (for Controls) from the first preparation and that for use on the last day of dosing were analysed to confirm the absence of the test item. Analysis were conducted at Kymos Pharma Services using method code C003-MP0023
Details on mating procedure:
The females were obtained from the supplier timed-mated. For mating, each female will have been paired with a sexually mature male of the same strain. The day on which mating is detected is designated Day 0 of gestation. The uterus of any apparently non-pregnant female was stained with ammonium sulphide to confirm pregnancy status.
Duration of treatment / exposure:
Animals will be dosed once daily, from Day 6 of gestation to Day 18 of gestation inclusive. Control animals will receive the vehicle only, following the same regimen as the other groups
Frequency of treatment:
Animals will be dosed once daily
Duration of test:
From Day 6 of gestation to Day 18 of gestation inclusive.
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Seven females per group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected in consultation with the Sponsor after examining existing toxicity data and the data generated at Sequani from an MTD study (1). In that study a dose level of 200 mg/kg/day was associated with progressive body weight losses, however, there were only minimal effects on body weight at a dose of 100 mg/kg/day, therefore 150 m/gk/day has been selected as a high dose level for the current study.

- Rationale for animal assignment (if not random):
- Other:
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes
- Animals were examined twice daily for mortality and morbidity and were given a detailed clinical examination daily from the start of dosing. Animals were observed before, shortly after and about one hour after dosing. On weekdays a final check was made around four hours after dosing.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded on Day 0 of gestation by the supplier. At Sequani, body weights were recorded daily from Day 4 to 19 of gestation, inclusive, then again on Days 20, 22, 25 and 28 of gestation.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
Individual food intake was recorded daily over Days 5 to 6 of gestation and every two days thereafter.

POST-MORTEM EXAMINATIONS: Yes
Females were killed on Day 28 of gestation. Animals were weighed, the thoracic and abdominal cavities opened and the major organs examined. Organs or tissues showing any macroscopic abnormalities were removed and retained. For pregnant females, gravid uterus weight and placental weights were recorded. The uterus of any apparently non-pregnant female was stained with ammonium sulphide to confirm pregnancy status.


Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes / No / No data
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: For pregnant females, the number of corpora lutea and the number and distribution of implantations in uterine horns were recorded. Implantations were classified as early intrauterine deaths, late intrauterine deaths, dead foetuses or live foetuses. The implantations were numbered separately for the right and left horns. Numbering was sequential, commencing at the ovarian end through to the cervix. The live foetuses and their placentae were removed and the uterus and ovaries retained.
Fetal examinations:
- External examinations: Yes
- Soft tissue examinations: Yes
- Skeletal examinations: Yes
- Head examinations: Yes

Foetuses on Day 28 of gestation were killed, weighed and examined externally for abnormalities. Within 24 hours the abdomen was incised and the sex assessed from examination of the internal genitalia. Structural congenital abnormalities that impair or potentially impair the survival or constitution of the foetus were classified as major abnormalities. Other external defects were classified as minor abnormalities.
Indices:
- 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 foetuses)/no. of implantation sites ]x 100

Mean foetal body weights were calculated separately by sex for each litter; group mean body weights were calculated (separately by sex) from the litter means. Mean pre- and post-implantation losses were calculated on a proportional litter basis. The percentage of foetuses in each litter exhibiting each classification of abnormality was calculated; group mean percentages were calculated from the litter percentages. The percentage of male foetuses, out of the total number of foetuses, was calculated for each litter.
Historical control data:
Background data on the rate of spontaneous malformations have been accumulated.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Description (incidence):
There were no deaths or adverse clinical observations related to Sodium Tungstate administration
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Animals given 150 mg/kg/day sodium tungstate lost weight, between 0.06 to 0.41 kg, during the dosing period (Days 6 to 18 of gestation). At 30 or 100 mg/kg/day, group mean body weight gains over the dosing period were lower than that of the Control group (10 % and 32 %, respectively); however, at 100 mg/kg/day, this was largely due to one animal that ate a minimal amount from Day 14 of gestation. See Tables 1a, 1b and 2 on "Any other information on results incl. tables" section below.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 150 mg/kg/day, group mean food intake was markedly lower than that of the Controls (48 %) over the dosing period (Days 6 to 18 of gestation). These animals ate more after dose administration stopped. There was no effect of sodium tungstate on group mean food intake at 30 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 specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no maternal macroscopic abnormalities at necropsy that were considered to be related to sodium tungstate administration.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Number of abortions:
no effects observed
Description (incidence and severity):
See Table 3 on "Any other information on results incl. tables" section below.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There were no effects of sodium tungstate on the mean numbers of implantations, the incidences of pre- and post-implantation loss. See Table 4 on "Any other information on results incl. tables" section below.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
See Table 3 on "Any other information on results incl. tables" section below.
Early or late resorptions:
no effects observed
Description (incidence and severity):
See Table 3 on "Any other information on results incl. tables" section below.
Dead fetuses:
no effects observed
Description (incidence and severity):
See Table 4 on "Any other information on results incl. tables" section below.
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
There were 6, 5, 7 and 6 pregnant females in the groups given 0, 30, 100 or 150 mg/kg/day, respectively. See Table 3 on "Any other information on results incl. tables" section below.
Key result
Dose descriptor:
LOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
Fetal body weight changes:
no effects observed
Description (incidence and severity):
There was no effect of sodium tungstate on the mean foetal or placental weights. See Table 5 on "Any other information on results incl. tables" section below.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
There were no effects of sodium tungstate on the mean number of live foetuses. See Table 4 on "Any other information on results incl. tables" section below.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There was no effect of sodium tungstate on the mean sex ratio. See Table 5 on "Any other information on results incl. tables" section below.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
See Table 5 on "Any other information on results incl. tables" section below.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
There were no external foetal abnormalities. See Table 6 on "Any other information on results incl. tables" section below.
Skeletal malformations:
no effects observed
Description (incidence and severity):
See Table 6 on "Any other information on results incl. tables" section below.
Visceral malformations:
no effects observed
Description (incidence and severity):
See Table 6 on "Any other information on results incl. tables" section below.
Key result
Dose descriptor:
NOAEL
Effect level:
> 150 mg/kg bw/day (actual dose received)
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

Table 1a. Body weight gains (kg) - group mean values


 






















































































































Sex: Female



Body Weight


Gain (Day of Gestation)



Group



0 to 6



6 to 7



7 to 8



8 to 9



9 to 12



Group: 1


Control


0 mg/kg/day



Mean



0.033



-0.035



-0.005



0.048



0.072



SD



0.099



0.071



0.043



0.043



0.044



N



6



6



6



6



6



Group: 2


Sodium Tungstate


30 mg/kg/day



Mean



0.036



-0.064



0.042



-0.012



0.104



SD



0.081



0.038



0.043



0.019



0.067



N



5



5



5



5



5



Group: 3


Sodium Tungstate


100 mg/kg/day



Mean



0.027



-0.049



0.021



0.001



0.084



SD



0.095



0.077



0.042



0.024



0.019



N



7



7



7



7



7



Group: 4


Sodium Tungstate


150 mg/kg/day



Mean



0.133



-0.078



-0.013



0.005



0.003



SD



0.155



0.054



0.033



0.008



0.082



N



6



6



6



6



6



 


Table 1b. Body weight gains (kg) - group mean values (Continued)









































































































Sex: Female



Body Weight


Gain (Day of Gestation)



Group



12 to 15



15 to 19



6 to 19



19 to 28



Group: 1


Control


0 mg/kg/day



Mean



0.087



0.105



0.272



0.173



SD



0.043



0.044



0.077



0.086



N



6



6



6



6



Group: 2


Sodium Tungstate


30 mg/kg/day



Mean



0.094



0.082



0.246



0.166



SD



0.036



0.041



0.137



0.059



N



5



5



5



5



Group: 3


Sodium Tungstate


100 mg/kg/day



Mean



0.057



0.069



0.184



0.194



SD



0.100



0.066



0.219



0.053



N



7



7



7



7



Group: 4


Sodium Tungstate


150 mg/kg/day



Mean



-0.032



-0.068



-0.183



0.352



SD



0.059



0.081



0.110



0.071



N



6



6



6



6



 


Table 2. Terminal body weight (kg) adjusted for gravid uterus weight (g) - group mean values


 









































































































Sex: Female



Dead
Body Weight



Gravid
Uterus Wt



BWt Adjusted
for GUWt



Adjusted
BWt Gain



Group



6 to 28



Group: 1


Control


0 mg/kg/day



Mean



3.60



441.8



3.16



-0.06



SD



0.38



27.3



0.37



0.10



N



6



6



6



6



Group: 2


Sodium Tungstate


30 mg/kg/day



Mean



3.44



414.4



3.03



-0.05



SD



0.31



46.0



0.27



0.14



N



5



5



5



5



Group: 3


Sodium Tungstate


100 mg/kg/day



Mean



3.47



438.0



3.03



-0.14



SD



0.28



87.6



0.26



0.31



N



7



7



7



7



Group: 4


Sodium Tungstate


150 mg/kg/day



Mean



3.37



424.0



2.94



-0.30



SD



0.30



107.6



0.20



0.12



N



6



6



6



6



 


Table 3. Pregnancy data - summary


 





























































































Sex: Female



Group: 1
Control


0 mg/kg/day



Group: 2
Sodium Tungstate
30 mg/kg/day



Group: 3
Sodium Tungstate
100 mg/kg/day



Group: 4


Sodium Tungstate
150 mg/kg/day



Group Size



 



7



7



7



7



Not Pregnant



 



1



2



0



1



Not Pregnant %



 



14.3



28.6



0.0



14.3



Not Pregnant Died/Killed



Sum



0



0



0



0



Not Pregnant Schedule Kill



Sum



1



2



0



1



Pregnant



 



6



5



7



6



Pregnant %



 



85.7



71.4



100.0



85.7



Pregnant Died/Killed/Aborted



Sum



0



0



0



0



Pregnant with Total Resorption



Sum



0



0



0



0



Number with Live Foetuses



Sum



6



5



7



6



 


Table 4. Uterine and implantation data - group mean values by implant


 





















































































































Sex: Female



Group: 1
Control


0 mg/kg/day



Group: 2
Sodium Tungstate
30 mg/kg/day



Group: 3
Sodium Tungstate
100 mg/kg/day



Group: 4


Sodium Tungstate
150 mg/kg/day



Number with Implantations



 



6



5



7



6



Number of Corpora Lutea



Sum



61



48



64



63



Mean



10.2



9.6



9.1



10.5



SD



1.5



1.1



1.3



2.7



Number of Implantations



Sum



56



42



58



56



Mean



9.3



8.4



8.3



9.3



SD



1.2



1.5



1.9



2.9



% Pre-implantation Loss



Mean



7.8



12.2



9.7



12.5



Number of Early Deaths



Sum



1



0



1



1



Mean



0.2



0.0



0.1



0.2



SD



0.4



0.0



0.4



0.4



Number of Late Deaths



Sum



0



0



1



0



Mean



0.0



0.0



0.1



0.0



SD



0.0



0.0



0.4



0.0



 


Table 4. Uterine and implantation data - group mean values by implant (Continued)


 









































































Sex: Female



Group: 1
Control


0 mg/kg/day



Group: 2
Sodium Tungstate
30 mg/kg/day



Group: 3
Sodium Tungstate
100 mg/kg/day



Group: 4


Sodium Tungstate
150 mg/kg/day



Number of Dead Foetuses



Sum



0



0



0



0



Mean



0.0



0.0



0.0



0.0



SD



0.0



0.0



0.0



0.0



Number of Live Foetuses



Sum



55



42



56



55



Mean



9.2



8.4



8.0



9.2



SD



1.2



1.5



1.7



2.9



% Post-implantation Loss



Mean



1.7



0.0



3.1



1.5



Mean % of Implantations



Mean



98.3



100.0



96.9



98.5



 


Table 5. Litter weights (g) / foetal data - group mean values


 





























































































Sex: Female



Group: 1
Control


0 mg/kg/day



Group: 2
Sodium Tungstate
30 mg/kg/day



Group: 3
Sodium Tungstate
100 mg/kg/day



Group: 4


Sodium Tungstate
150 mg/kg/day



Number with Live Foetuses



 



6



5



7



6



No of Live Foetuses



 



55



42



56



55



No of Male Foetuses



 



20



17



36



26



No of Female Foetuses



 



35



25



20



29



% of Male Foetuses



Mean



36.2



39.4



66.1



47.0



Litter Weight (g)



Mean



303.70



287.13



292.86



288.64



Foetal Weight (M+F) (g)



Mean



33.36



34.46



37.31



32.83



Foetal Weight (M) (g)



Mean



33.56



34.16



37.31



34.53



Foetal Weight (F) (g)



Mean



33.65



34.47



36.63



32.18



Placental Weight (g)



Mean



3.12



3.14



3.69



3.27



 


Table 6. Examination of foetuses - summary of group mean values


 


























































































Combined examination


(external/visceral/skeletal)



Group: 1
Control


0 mg/kg/day



Group: 2
Sodium Tungstate
30 mg/kg/day



Group: 3
Sodium Tungstate
100 mg/kg/day



Group: 4


Sodium Tungstate
150 mg/kg/day



Total number of litters examined



6



5



7



6



Total number of foetuses examined



55



42



56



55



Number with major abnormalities



0



0



0



0



Mean % of foetuses examined



0



0



0



0



Number of litters affected



0



0



0



0



Number with minor abnormalities



0



0



0



0



Mean % of foetuses examined



0



0



0



0



Number of litters affected



0



0



0



0



Number with variations



0



0



0



0



Mean % of foetuses examine



0



0



0



0



Number of litters affected



0



0



0



0


Conclusions:
Administration of 150 mg/kg/day sodium tungstate, once daily, by oral gavage to the rabbit, from Day 6 to Day 18 of gestation, elicited maternal toxicity in terms of body weight loss over the dosing period. At 30 or 100 mg/kg/day there were slight reductions in body weight gain only. There was no embryo-foetal toxicity. A dose level of 100 mg/kg/day is considered to be a suitable high dose level for the subsequent pivotal embryo-foetal development study.
Executive summary:

No fertility, reproductive, or developmental toxicity data of sufficient quality are available for tungsten carbide (target substance). However, developmental toxicity data are available for sodium tungstate (source substance), which are used for read-across. Due to lower water solubility and lower toxicity for the target substance compared to the source substance, the resulting read-across from the source substance to the target substance is appropriate as a conservative estimate of potential toxicity for this endpoint. In addition, read-across is appropriate because the classification and labelling is more protective for the source substance than the target substance, the PBT/vPvB profile is the same, and the dose descriptors are, or are expected to be, lower for the source substance. For more details, refer to the read-across category approach in the Category section of this IUCLID submission or Annex 3 in the CSR.

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

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


The neurotoxicity of sodium tungstate is reported three publications by McInturf et al. (2007, 2008, 2011). McInturf et al. (2007) represents a study report, and the 2008 publication is based on exactly these data. According to the results presented, the 2011 publication uses the same data already published in 2008, but with the extension of one additional dose group and more reproductive parameters. The 2008 paper is not referenced in the 2011 one.


A study conducted following EPA OPPTS 870.3650 evaluated the reproductive and developmental (teratogenic) effects of sodium tungstate in rats following 70 days of daily pre-and postnatal exposure via oral gavage to 5, 62.5 and 125 mg/kg/ day through mating, gestation and weaning (Postnatal day, PND 0–20). In this study, a range of neurobehavioral capacities in sodium tungstate exposed dams and their offspring were assessed. The tests evaluated reflexive responding, emotionality and spatial learning and memory in the low and high dose groups, but not in the mid dose group. The following neurobehavioral test batteries were performed on pups and adult females after exposure to sodium tungstate. The righting reflex and separation distress were done on PD4 and PND7, respectively. The adult females were tested for maternal retrieval latency when pups were age PND2, and spontaneous locomotor activity (SLA) on post-dosing day 7, acoustic Startle/Pre-Pulse Inhibition (AS/PPI) on post-dosing day 8, and water maze navigation on post-dosing days 15–18 (McInturf et al., 2007, 2008 & 2011).


Results from one of the two tests in the pups, separation distress, suggest neurobehavioral perturbations because of exposure to sodium tungstate (McInturf et al., 2007, 2008 and 2011). The high dose group was reported to have a greater number of ultrasonic distress vocalizations when separated from the dam and littermates. However, in the absence of single animal data from the study and historical control data, this effect cannot be evaluated. The other pup assessment, righting reflex latency, showed sex differences where males demonstrated faster righting than females, however, the effects were not dose-dependent. In the absence of single animal and historical control data the relevance of this finding cannot be evaluated. In 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.


 


 References: 


 


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


 


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


 


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


 


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


 


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


 


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


 


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


 


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


 


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


 


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


 


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

Justification for classification or non-classification

No reproductive/developmental studies are available for tungsten carbide. However, data were available on sodium tungstate, which were used for read-across. Based on the weight-of-evidence from fertility and one-generation reproductive/developmental studies in which no significant effects were observed on reproductive parameters, a lack of significant developmental effects was reported, as well as a lack of reproductive organ effects following 90-d of oral exposure to sodium tungstate in a repeat dose study, it is not expected that sodium tungstate is a reproductive/developmental (teratogenic) toxicant. Furthermore, based on bioaccessibility studies of tungsten carbide (read across source substance)  that reported a 0.04% bioelution of tungsten after 5-hrs. incubation in gastric fluid, the equivalent amount of tungsten carbide to reach NOAEL doses is substantially higher than 1,000 mg/kg/day (limit dose). Therefore, based on the lack of reproductive and/or developmental effects observed, no classification is warranted for tungsten carbide.

Additional information