Tungsten oxide

Toxicity to reproduction

Administrative data

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 oxide
3. CATEGORY APPROACH JUSTIFICATION: See Annex 3 in CSR
4. DATA MATRIX: See Annex 3 in CSR

Cross-reference

Reason / purpose for cross-reference:

read-across source

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

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.

Test material

Test material information

Constituent 1

Reference substance name:

Disodium wolframate

EC Number:

236-743-4

EC Name:

Disodium wolframate

Cas Number:

13472-45-2

Molecular formula:

Na2O4W

IUPAC Name:

Disodium dioxido(dioxo)tungsten

Test material form:

solid: crystalline

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).

Test animals

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.

Administration / exposure

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.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

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).

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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.

Details on results (P0)

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

Effect levels (P0)

1

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

2

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

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

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

Developmental neurotoxicity (F1)

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 (F1)

Developmental immunotoxicity:

not specified

Details on results (F1)

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.

Effect levels (F1)

1

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.

2

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

3

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

4

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

5

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 (functional observations)

6

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

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

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 oxide (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 description in the Category section of this IUCLID submission or Annex 3 of the CSR.