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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
Experiment start date (Animal arrival): 15 October 2015; Completion date of experimental phase (Last day of necropsy): 01 December 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
1. HYPOTHESIS FOR THE CATEGORY APPROACH: The hypothesis is that properties are likely to be similar or follow a similar pattern because of the presence of a common metal ion, in this case tungstate.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES):
Source: Sodium Tungstate
Target: Fused tungsten carbide
3. CATEGORY APPROACH JUSTIFICATION: See Annex 3 in CSR
4. DATA MATRIX: See Annex 3 in CSR
Cross-reference
Reason / purpose:
read-across: supporting information

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Principles of method if other than guideline:
ICH Harmonised Tripartite Guideline: Detection of Toxicity to Reproduction for Medicinal Products & Toxicity to Male Fertility S5 (R2): finalised (Step 4) November 2005
GLP compliance:
yes
Limit test:
no
Justification for study design:
Dose levels were selected in consultation with the Sponsor on the basis of results from an embryo-foetal development study performed at Sequani (Sequani Study Number: OHS0005)

Test material

Reference
Name:
Unnamed
Type:
Constituent
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
- Purity: 93.2%

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

Test animals

Species:
rat
Strain:
other: Crl:CD(SD) strain
Details on species / strain selection:
The rat is a suitable rodent species, acceptable to regulatory authorities and for which extensive background data are available. The Sprague Dawley rat is commonly used in reproduction studies because of the good fertility and fecundity of the strain.
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent, CT9 4LT, England
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: The animals were six to seven weeks of age on arrival and on examination were found to be healthy.
- Weight at study initiation: On the first day of dosing the males weighed 295 to 365 g and the females weighed 185 to 244 g.
- Housing:
- Diet (e.g. ad libitum): A pelleted rodent diet, VRF1 (manufactured by SDS) supplied by Charles River (UK) Limited, Margate, Kent, CT9 4LT, England, and mains tap water (in bottles) were freely available.
- Water (e.g. ad libitum): Tap water (in bottles) were freely available
- Acclimation period:
ENVIRONMENTAL CONDITIONS
- Temperature (°C): The target ranges for temperature were 19 °C to 23 °C.
- Humidity (%): The target ranges for humidity were 40 % to 70 %.
- Air changes (per hr): Room was air-conditioned.
- Photoperiod (hrs dark / hrs light): The study room was illuminated by fluorescent light set to give a cycle of 12 hours light and 12 hours dark.
IN-LIFE DATES: From: 15 October 2015 To: 01 December 2015

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
purified
Details on exposure:
Animals were dosed once daily, by gavage, using a rubber catheter and disposable syringe at a constant dose volume of 5 mL/kg body weight. Males were dosed for 14 days before and during pairing and until the day before necropsy. Females were dosed for 14 days before, during pairing and until Day 6 of gestation, inclusive. Individual doses were adjusted according to the most recent body weight.
Details on mating procedure:
After the pre-pairing dosing period, each female was paired with a male from the same group for up to 10 days. If a female is not mated within 10 days, the male was removed and another from the same group, that has previously mated, was substituted. This second pairing was continue for up to a further 5 days. Females with no evidence of mating after this second pairing period were sent for necropsy at the end of the pairing period. On confirmation of mating, the male was returned to the group cages and the female was caged individually. The male was allso returned to the group cage at the end of the pairing period.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis were conducted at Kymos Pharma Services using a validated method (SOP code C003-MP0023)
Duration of treatment / exposure:
The males were dosed once daily for 14 days before and during pairing until the day before necropsy. The females were dosed for 14 days before and during pairing and then until Day 6 of gestation, inclusive (Day 0 of gestation is the day of a sperm positive vaginal smear), or until the day before necropsy for unmated females.
Frequency of treatment:
Animals were dosed daily, males for a period of 14-days, and females for a period of 20 days.
Details on study schedule:
The males were dosed once daily for 14 days before and during pairing until the day before necropsy. The females were dosed for 14 days before and during pairing and then until Day 6 of gestation.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Vehicle control
Dose / conc.:
40 mg/kg bw/day (actual dose received)
Dose / conc.:
80 mg/kg bw/day (actual dose received)
Dose / conc.:
160 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
There were 40 animal per group (20 males and 20 females)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
- Rationale for animal assignment (if not random):
- Fasting period before blood sampling for clinical biochemistry:
- Other:
Positive control:
Not applicable

Examinations

Parental animals: Observations and examinations:
Animals were examined twice daily for mortality and morbidity, daily from the start of treatment for clinical signs of toxicity and changes in behaviour and were given a detailed clinical examination weekly.
Oestrous cyclicity (parental animals):
For 10 days before the start of the pairing period, vaginal smears were taken daily by lavage. During the pairing period, vaginal smears were taken daily, by lavage, until mating was confirmed by sperm being found in the smear.

The smear was examined under light microscopy and the stage of the oestrous cycle was determined by the type of cell present. The number of copulation plugs was also recorded to give an assessment of the mating activity of the animals.
Sperm parameters (parental animals):
When conducted, seminal fluid from the epididymis of all males was collected at scheduled necropsy and assessed for sperm concentration and motility using the Hamilton Thorne IVOS CASA system.
Postmortem examinations (parental animals):
- Females were killed on Day 13 of gestation. The males were killed approximately two weeks after completion of the mating period and a necropsy was performed.. The thoracic and abdominal cavities were opened by a ventral mid-line incision and the major organs were examined. Organs or tissues showing macroscopic abnormalities were removed and retained in fixative.
- The ovaries, testes, epididymides, prostate and seminal vesicles (including coagulating gland) were removed and the ovaries and testes were weighed. - - Pregnancy status was checked and the uterus of any apparently non-pregnant female was stained with ammonium sulphide to confirm pregnancy status.
Postmortem examinations (offspring):
For all pregnant females, the number of corpora lutea and the number and distribution of implantations in each uterine horn were recorded. Implantations were classified as early intrauterine deaths, dead embryos or live embryos. The implantations were numbered separately for the right and left horns. Numbering was sequential, commencing at the ovarian end through to the cervix.
Statistics:
Comparisons: Group 1 against Groups 2, 3 and 4
Statistical tests and parameters: Data was processed to give group mean values and standard deviations, where appropriate. Where the data allow, the following methods were used for statistical analysis. Depending on the nature of the data set to be analysed, appropriate tests were applied as indicated in the table below. Where parametric tests may be appropriate they were preceded by a check for homogeneity of variance using the Levene test and, where available, the Shapiro-Wilks test for normality. If either of these two assumptions fails a log transformation was applied before retesting. If the transformation fails, appropriate non-parametric tests was applied.
Probability values of less than 5 % were regarded as providing sufficient evidence to reject the null hypothesis and therefore statistical significance was identified at the p<0.05 level. For illustrative purposes, significance levels of p<0.01 and p<0.001 were also noted.
Reproductive indices:
1) Copulation index (%) = (no. of animals mated/no. of animals paired) x 100
2) Female Fertility index (%) = (no. of pregnant females/no. of females paired) x 100
3) Male Fertility index (%) = (no. of males siring one or more pregnancies/ no. of males paired) x 100
4) Fecundity index (%) female = no. of pregnant females no. of females mated x 100
5) Pre-implantation loss (%) = [(no. of corpora lutea –no. of implantation sites)/no. of corpora lutea] x 100
6) Post implantation loss (%) = [(no. of implantation sites –no. of live embryos)/no. of implantation sites] x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Description (incidence):
There was no mortality or clinical signs associated with sodium tungstate. There was only one death: Control Male 10 was euthanised on Day 17 of the study following a hind limb injury which was unrelated to sodium tungstate.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In males given 160 mg/kg/day, mean body weight gain was lower than Controls throughout the study, with some sporadic individual body weight losses and consequently, mean absolute body weight at the end of the study was statistically significantly lower than Controls (- 8.2 %; p<0.01). In females given 160 mg/kg/day, mean body weight gains were also lower than Controls during the dosing period but, this was less apparent than effects in males and after the cessation of dosing, compensatory body weight gains resulted in mean absolute body weight being comparable with Controls on Day 13 of gestation. Body weight gains in males and females given 40 or 80 mg/kg/day were similar to Controls.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Male and female food intake was unaffected by sodium tungstate.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
There was no effect of sodium tungstate on the number of oestrous cycles recorded during the pre-pairing period, or on the average cycle length.
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed
Description (incidence and severity):
There was no effect of sodium tungstate on fertility or mating; all animals mated and only one Control female (Animal 86) was not pregnant.

Details on results (P0)

Pregnancy data were similar in all groups, with no adverse effect on the mean numbers of corpora lutea, implantations, the incidence of pre- or post-implantation loss or on the number of live embryos.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
> 160 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive function (oestrous cycle)
reproductive performance

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

Effect levels (F1)

Key result
Remarks on result:
not measured/tested

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
Administration of sodium tungstate to the Crl:CD(SD) rat for 14 days before pairing until Day 6 of gestation in females, or necropsy in males (five weeks), was generally well tolerated, with only non-adverse body weight changes seen in both sexes. On this basis, the No Observed Adverse Effect Level (NOAEL) for female fertility and early embryonic development, or male fertility, was considered to be 160 mg/kg/day.