Tungsten disulphide

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

No fertility, reproductive, or developmental toxicity data of sufficient quality are available for tungsten disulphide (target substance). However, fertility toxicity data are available for sodium tungstate (source substance), which will be used for reading across. Due to lower water solubility and lower toxicity for the target substance compared to the source substance, the resulting read across approach 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.

Reproductive Toxicity

 

A weight of evidence (WoE) approach was applied to fulfill Annex X, Section 8.7.3.; test method: EU B.56./OECD TG 443.

 

This approach takes several peer reviewed published rat oral studies on the read-across source substance sodium tungstate to cover the Extended One-Generation Reproductive Toxicity (EOGRT) study extent.

 

In our opinion, when all the 90-day repeated exposure, fertility, reproductive, developmental and immunotoxicity of tungstate oral results are considered collectively, no additional testing should be required as the toxicity tests already conducted offer key information on the intrinsic toxicity of tungstate to parental and F1 groups.

 

In other words, studying the toxicity of a substance in separate studies (see figure in attachment in this section) does not diminishes the scientific reliability compared to a study that uses an extended one-generation approach.

 

When comparing the EOGRT requirements against the available fertility, reproductive and developmental studies on sodium tungstate (see figure in attachment in this section) some of the testing OECD requirements are not completely cover, however the results of the individual fertility, reproductive, developmental, neuro- and immuno-toxicity cover key toxicity endpoints and additional testing is not warranted.

 

1.   Fertility/Reproductive Toxicity ‒ Weight of Evidence

 

The 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 and developmental toxicity are covered by McInturf et al (2007, 2008, and 2011) gavage study together with the US NTP perinatal drinking water study and Osterburg et al (2014) one-generation study in mice. The developmental neuro- and immuno-toxicity endpoints are covered by McInturf et al (2007, 2008, and 2011) gavage study, and the Fastje et al (2012) and Osterburg (2014). The figure in the attachment in this section depicts where each of these studies fall within the EOGRT fertility/reproductive duration and required cohorts. Below each of the studies included in the fertility/reproductive weight of evidence are discussed.

 

a.    Ballester et al (2005 & 2007) 90-day Rat Oral Fertility Studies

 

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.

 

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 ratswere 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 the male and female 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 fertilemales 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 smearswere carried out to determine ifmatinghad occurred. When vaginal smears werepositive (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 CO₂inhalation.

 

The reproductive performance of the rats was measured as the proportion of inseminated females to the total number tested (the percentage of positive vaginal smears, ie those with presence of spermatozoa, with respect to the total number of vaginal smears performed in one experimental group). This parameter was named as ‘mating index’. The number of vaginal smears varied depending on the time required by animals to show positive mating. Furthermore,the percentage of parturitions was calculated with respect to the number of positive smears. This parameter was named ‘fertility’. Finally, the mean litter size was also calculated.

 

Results showed that in healthy rats tungstate treatment caused a decrease in the body weight gain but did not modify daily food and water consumption.Tungstate treatment did not modify alanine aminotransferase (ALT) activity, progesterone, FSH or LH. In addition,tungstate treatment did not affect any reproductive parameter or affected the expression of the estrogen receptor.However, in ovaries tungstate treatment had a considerable effect on the expression ofthe progesterone receptor. In contrast, the uterine expression of the progesterone receptor was not affected by tungstate treatment.

 

b.    Osterburg et al (2014) 1-Gen Mice Study

 

A one-generation oral study in mice included in the immunotoxicity (developmental) study published by Osterburg et al (2014). Parental mice were dosed with 0, 2, 62.5, 125, or 200 mg sodiumtungstate /kg bw/d. Both P and F1 mice were maintained on these doses for the course of the study. These quantities of tungstate were selected based onprevious 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 wasexposed 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).

Results showed no statistically significant changes in body weight due to any tungstate dose levels. The 200 mg/kg bw/d 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 livebirths, litter size, or sex ratio at any dose of tungstate tested (Osterburg et al. 2014).

c.    McInturft et al (2007, 2008, & 2011) Reproductive Screening Rat Oral 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/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 P₀males 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).

 

d.    US NTP Rat Perinatal Oral Study

 

The study design included drinking water doses of 0, 125, 250, 500, 1000, or 2000 mg/L (an estimated oral dose between 10-180 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 Posterwas presented detailing the following preliminary results showed:

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

·      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.

 

e.    McCain 90-day Repeated Exposure Oral Study

 

No effects on the male or female reproductive organs were observed following 90-d repeat exposure to sodium tungstate via the oral route in rats (Biotechnics, 2009).

 

Fertility/Reproductive WoE Conclusions:

 

The overall fertility reproductive parameters measured were unchanged in mice and rats after sodium tungstate exposure. No marked effects on pup survival, M: F ratio, litter size, or clinical signs were observed in the F1 rat 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 sodium tungstate, the NOAEL for reproductive toxicity was 125 mg Na₂WO₄/kg bw/d (McInturf et al, 2008 & 2011). Based on the current WoE, it is not expected that sodium tungstate is a fertility/reproductive toxicant.

Link to relevant study records

Reference

Reference 1

Endpoint:

fertility, other

Remarks:

based on test type (migrated information)

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 with acceptable restrictions

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:

REPORTING FORMAT FOR THE CATEGORY APPROACH
1. HYPOTHESIS FOR THE ANALOGUE APPROACH: The hypothesis is that properties are likely to be similar or follow a similar pattern because of the presence of a common metal ion, in this case tungstate.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES):
Source: Sodium Tungstate
Target: Tungsten disulphide
3. CATEGORY APPROACH JUSTIFICATION: See Annex 1 in CSR
4. DATA MATRIX: See Annex 1 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)

Deviations:

yes

GLP compliance:

not specified

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 48C (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 2908C 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.

Executive summary:

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

Reference 2

Endpoint:

fertility, other

Remarks:

based on test type (migrated information)

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 with acceptable restrictions

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:

REPORTING FORMAT FOR THE CATEGORY APPROACH
1. HYPOTHESIS FOR THE ANALOGUE APPROACH: The hypothesis is that properties are likely to be similar or follow a similar pattern because of the presence of a common metal ion, in this case tungstate.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES):
Source: Sodium tungstate
Target: Tungsten disulphide
3. CATEGORY APPROACH JUSTIFICATION: See Annex 1 in CSR
4. DATA MATRIX: See Annex 1 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)

Deviations:

yes

GLP compliance:

not specified

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 48C (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 290C 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 hypoglycemia, 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 reproductive toxicity data of sufficient quality are available for tungsten disulphide (target substance). However, reproductive toxicity data are available for sodium tungstate (source substance), which will be used for reading across. Due to lower water solubility and lower toxicity for the target substance compared to the source substance, the resulting read across from the source substance to the target substance is appropriate as a conservative estimate of potential toxicity for this endpoint. In addition, read across is appropriate because the classification and labelling is more protective for the source substance than the target substance, the PBT/vPvB profile is the same, and the dose descriptors are, or are expected to be, lower for the source substance. For more details, refer to the read-across category approach included in the Category section of this IUCLID submission and/or as an Annex in the CSR.

Reference 3

Endpoint:

one-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Well documented scientfically sound study similar to OECD guidelines with sufficient information provided on materials and methods to evaluate results. However as this study is used in the context of a read across, Klimisch 2 is assigne

Justification for type of information:

REPORTING FORMAT FOR THE CATEGORY APPROACH
1. HYPOTHESIS FOR THE ANALOGUE APPROACH: The hypothesis is that properties are likely to be similar or follow a similar pattern because of the presence of a common metal ion, in this case tungstate.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES):
Source: Sodium tungstate
Target: Tungsten disulphide
3. CATEGORY APPROACH JUSTIFICATION: See Annex 1 in CSR
4. DATA MATRIX: See Annex 1 in CSR

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

reference to same study

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

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):

1) 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.

2) 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.

3) 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.

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:

LOEL

Effect level:

125 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:

125 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Behaviour

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

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

Generation:

F1

Based on:

test mat.

Sex:

male/female

Basis for effect level:

developmental neurotoxicity

Remarks on result:

not determinable because of methodological limitations

Key result

Reproductive effects observed:

no

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. No significant test article related histopathological findings were observed in the F1 treatment groups. The 2011 publication mentions the investigation of neurobehavioral findings only in the control, low and high dose groups but not from the mid dose (only body weight gain, tungsten organ distribution data and histopathology) for the mid dose group animals are reported. The neurobehavioral results in the 2008 publication resemble the results reported in the 2011 publication. However, the 2011 paper does not quote the 2008 publication of the same group. This causes some doubt on the study design.

Executive summary:

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

Reference 4

Endpoint:

developmental immunotoxicity

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

Justification for type of information:

REPORTING FORMAT FOR THE CATEGORY APPROACH
1. HYPOTHESIS FOR THE ANALOGUE APPROACH: The hypothesis is that properties are likely to be similar or follow a similar pattern because of the presence of a common metal ion, in this case tungstate.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES):
Source: Sodium Tungstate
Target: Tungsten disulphide
3. CATEGORY APPROACH JUSTIFICATION: See Annex 1 in CSR
4. DATA MATRIX: See Annex 1 in CSR

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

other: USEPA Guideline OPPTS 870.3650

Principles of method if other than guideline:

This study examined the consequences of tungstate exposure on the immune response of mice after activation of the immune system with either Staphylococcal Enterotoxin B (SEB) or in a delayed-type hypersensitivity (DTH) Type IV model. The study here investigated the effects of tungstate in a 28-day and a one-gen exposure to determine acute as well as longer-term exposure toxicities. Experiments were then conducted as described in Current Protocols in Immunology (Luo & Dorf, 2001)

GLP compliance:

not specified

Limit test:

no

Justification for study design:

Support for read-across from water-soluble tungsten compounds to ammonium metatungstate (AMT) is based on consideration of structural similarities, physicochemical properties, release of ionic tungsten species, and/or toxicity data for AMT, ammonium paratungstate (APT), sodium metatungstate, and sodium tungstate. This approach is applicable for all systemic effect endpoints for all routes of exposure (more imformation is available in Annex 1 of the CSR attached in Section 13)

Species:

mouse

Strain:

C57BL

Details on species / strain selection:

c57BL6

Sex:

male/female

Details on test animals or test system and environmental conditions:

C57BL6 (28-day study ¼ 8–12-week-old, 19–22 g; one-gen study=8–12-week-old, 15–18 g) mice were obtained from Charles River Laboratories (Wilmington, MA). Animals were housed in an Association for Assessment and Accreditation of Laboratory
Animal Care (AAALAC) approved facility under animal protocols approved by the Wright-Patterson Air Force Base and/or University of Cincinnati Institutional Animal Care and Use Committee (IACUC). Mice were allowed to acclimatize to the animal facility for 7 days before commencement of the experimental phase. During the course of the experiment, animals had a 12 h day/night cycle in a temperature-controlled room (22 C). All animals had ad libitum access to filtered water and a low molybdenum diet; CA.170481 AIN-76 A Purified Diet

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

Individual mice were identified by ear punch. 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. 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. In the one-gen study parental mice were dosed with 0, 2, 62.5, 125, or 200 mg/kg/day of tungstate.

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

Duration of treatment / exposure:

Both P and F1 mice were maintained on these doses for the course of the study. Water bottles were changed 2–3 times weekly, always using water from the source and a 1 M sodium tungstate stock supply. 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, &19 weeks after initiation of the study. The F1 generation was exposed to tungstate for a further 90 days after weaning and then necropsied.

Dose / conc.:

0.2 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 (nominal)

Control animals:

yes, concurrent vehicle

Parental animals: Observations and examinations:

After euthanasia, splenocytes and blood were collected and stained with lymphocyte and/or myeloid immunophenotyping panels and analyzed by flow cytometry. I

Statistics:

All statistical tests were performed with Systat (v11 or v13, Systat Software Inc., Chicago, IL). Statistical significance was assumedat p<0.05. 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.

Reproductive indices:

Litter size, and sex ratio

Offspring viability indices:

Live births

Clinical signs:

not specified

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No statistically significant changes in body weight due to any tungstate dose levels in one-gen study. 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):

There were no significant changes in water consumption due to the quantity of tungstate present in the water.

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

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. 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.

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

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):

No observed statistically significant changes in the number of live births, litter size, or sex ratio at any dose of tungstate tested.

Key result

Dose descriptor:

NOEL

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/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: Immunological: 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

Key result

Dose descriptor:

LOEL

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: immunological: 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 (co-exposure) with SEB, and this effect cannot be attributed to tungstate only.

Clinical signs:

not specified

Mortality / viability:

no mortality observed

Description (incidence and severity):

No observed statistically significant changes in the number of live births

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No statistically significant changes in body weight due to any tungstate dose levels in one-gen study. 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):

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

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. 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.

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Histopathological findings:

not specified

Other effects:

no effects observed

Description (incidence and severity):

Not observed statistically significant changes in the number of llitter size, or sex ratio at any dose of tungstate tested.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

effects observed, treatment-related

Description (incidence and severity):

- The one-gen exposures resulted in reduced quantities of CD71+ TH cells in the parental mice, 6.21 0.39% for the controls and 2.28+/-0.41% for the 200 mg/kg/day dose group. 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.
- The cytotoxic CD3+ CD8+ CD71+ cells in the parental mice were 7.98+/-0.49% in the 0 mg/kg/day SEB-challenged groups and 1.58+/-0.23% in the corresponding 200 mg/kg/day group. The F1 mice were 6.33+/-0.49% for the controls and 2.52+/-0.25% in the 200 mg/kg/day tungstate group (p<0.001).
- Interferon gamma in the P mice of the one-gen exposure had a reduction from the control value of 7.11 1.7 to 3.74 2.9 pg/ml (p<0.001). Although not statistically significant, the F1 mice had an overall reduced IFNg response, especially at the 62.5 mg/kg/day dose, compared to their parents

Key result

Dose descriptor:

NOEL

Generation:

F1

Effect level:

125 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: 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 (co-exposure) with SEB, and this effect cannot be attributed to tungstate only.

Key result

Dose descriptor:

LOEL

Generation:

F1

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

developmental immunotoxicity

Key result

Reproductive effects observed:

no

Conclusions:

No observed statistically significant changes in the number of live births, litter size, or sex ratio at any dose of tungstate tested.

Executive summary:

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

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

125 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Well documented scientfically sound study similar to OECD guidelines with sufficient information provided on materials and methods to evaluate results. However as this study is used in the context of a read across, Klimisch 2 is assigned.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

No developmental toxicity data of sufficient quality is available for tungsten disulphide (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.

The US Navy has sponsored a study conducted following US EPA Guideline OPPTS 870.3650 (equivalent to OECD TG 422) – Combined Repeated Dose Toxicity Study with the Reproduction-Developmental Toxicity Study. The study 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). Preliminary results 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 (Johnson, EW Boeckman, HJ Arfsten et al. 2006). However, for unknown the results of this dose group were never peer reviewed and published.

The objective of the study was to evaluate 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 two separate peer review publications (McInturf et al 2008 & 2011) and on an unpublished summary report (McInturf et al., 2007). Unfortunately, a review of the laboratory final study report has not been conducted as is not publicly available and all the information described here are coming from the fore mentioned documents.

Daily administration of sodium tungstate produced no overt evidence of toxicity and had no apparent effect on mating success or offspring physical development. At the 125 mg/kg bw/d no effects on pup survival, M/F ratio, liter size, clinical signs, or physical birth defects were observed in the F1 litters. 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, EW Boeckman, HJ Arfsten et al. 2006), but as it was mentioned previously the results of the 250 mg/kg/d dose group were never peer reviewed and published.

The US EPA Guideline OPPTS 870.3650 calls for a gross necropsy of the offspring which includes visceral malformations (but not skeletal ones), and on this subject, “all pups were inspected for physical birth defects, including missing digits, however, none were found”. Based on the lack of F1 significant effects, the NOAEL for developmental toxicity is 125 mg/kg/d. (McInturf et al., 2008 & 2011).

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

125 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

No reproductive/developmental studies are available for tungsten disulphide. 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 tungsten disulphide is a reproductive/developmental (teratogenic) toxicant. Therefore, based on the lack of reproductive and/or developmental effects observed, no classification is warranted for tungsten disulphide.