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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 APR 2022 to 14 APR 2023
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to other study
Remarks:
dose range-finding study
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
14 FEB 2022 - 31 JAN 2023
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
no guideline followed
Principles of method if other than guideline:
The objective of this range-finding study was to evaluate the potential maternal toxicity and developmental effects (specifically; gestational and prenatal) of DiPotassium hexafluorotitanate (IV) – 01727 in pregnant rats likely to arise from repeated gestational exposure via dietary administration. These data will be used, along with existing data, to select dose levels for a definitive developmental toxicity study in rats. Twenty-four (24) timed-pregnant female rats were selected for the test and equally distributed into four groups (6 per group). The test substance was administered via the diet at 0, 450, 900, and 1800 ppm of DiPotassium hexafluorotitanate (IV) – 01727 for 15 consecutive days during gestation days (GD) 5-19 of a 21-day pregnancy.
GLP compliance:
no
Remarks:
This study was not performed in full compliance with GLP standards, but was conducted in a GLP-compliant facility.
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Remarks:
CRL Sprague-Dawley CD® IGS rats
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc. (CRL)
- Weight at study initiation: The weight variation did not exceed ± 20% of the mean weight for each sex
- Housing: The animals were individually housed in suspended stainless steel cages which conform to the size recommendations in the latest Guide for the Care and Use of Laboratory Animals (National Research Council, 2011). Litter paper placed beneath the cage was changed at least three times/week. Beginning no later than Day 14 of gestation (GD 14), female rats were housed in polyethylene shoebox cages (10.5”w x 19”d x 8”h) containing nesting material (Enrich-o’cobs or equivalent) with wired-mesh lids.
- Diet (e.g. ad libitum): ad libitum (OSD (Open Standard Diet) D1111225NM Rodent Diet))
- Water (e.g. ad libitum): ad libitum (Filtered tap water)
- Acclimation period: 8 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 21
- Humidity (%): 43 - 54
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: 22 FEB 22 - 11 MAR 22 (In-Life Study Initiated-Completed)
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): All diets were prepared approximately weekly or more frequently, as needed (unless stability data support an alternative schedule).
- Mixing appropriate amounts with (Type of food): The test substance was added to OSD (Open Standard Diet) D1111225NM Rodent Diet and thoroughly mixed in a high-speed mixer. Control diet was mixed under the same conditions as the diets prepared with the test substance.
- Storage temperature of food: All diets were refrigerated following preparation, unless presented to the test animals on the same day as diet preparation.
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
The test substance and selected prepared diets (at each concentration), were sampled in duplicate. Samples were frozen and/or will be discarded upon completion of the study. Dose preparations and neat test substance was not analyzed as part of this range-finding study.
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant
- Proof of pregnancy referred to as day 0 of pregnancy
Duration of treatment / exposure:
5 - 19 days pc
Frequency of treatment:
daily via food
Duration of test:
15 d (5 - 19 days pc)
Dose / conc.:
1 800 ppm (nominal)
Remarks:
86.1 mg/kg bw/d (based on mean overall (GD 5-20) daily intake)
Dose / conc.:
900 ppm (nominal)
Remarks:
49.1 mg/kg bw/d (based on mean overall (GD 5-20) daily intake)
Dose / conc.:
450 ppm (nominal)
Remarks:
30 mg/kg bw/d (based on mean overall (GD 5-20) daily intake)
Dose / conc.:
0 ppm (nominal)
No. of animals per sex per dose:
6 females
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The Sponsor, in consultation with the Study Director, selected dietary concentrations of 450, 900, and 1800 ppm (or 0.045, 0.09, and 0.18%) of DiPotassium hexafluorotitanate (IV) - 01727.
- Rationale for animal assignment (if not random): Animals were randomly assigned to test groups, stratified by body weight.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: on GD 5 and weekly thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: GD 3, 5, 8, 11, 14, 17, 20

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #20
- Organs examined: examination of the external surface of the body, all orifices, as well as the thoracic, abdominal, and cranial cavities, and their contents.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Blood sampling:
- Plasma: No
- Serum: No
Fetal examinations:
- External examinations: Yes all per litter
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No
Statistics:
Analytical methods for the compiled data in this study were based on requirements of OECD Guidelines for Testing of Chemicals, Section 4 No. 414. PSL performed statistical analysis of all quantitative data collected during the in-life phase of the study as well as organ weight and uterine data collected. Inferential comparisons, between control and test groups were made with regard to homogeneity of variance, normality, and analysis of variance (ANOVA) where appropriate. The use of the word “significant” or “significantly” indicates a statistically significant difference between control and the test groups. Means were considered to be significantly different whenever p < 0.05.
Statistical Analysis was conducted by using Provantis™ version 10, Tables and Statistics, Instem LSS, Staffordshire UK.

Statistical Methods (In-Life and Organ Weight Data) of the Parental (P) Generation
PSL calculated the means and standard deviations for all quantitative data collected from dams.
If warranted by sufficient group sizes, the incidence of clinical observations was evaluated through sequential application of a trend test

Statistical Methods of the Filial (F1) Generation
The litter was the experimental unit for evaluation where appropriate. Means and standard deviations were calculated independently for control and test groups. Similarly, mortality and sex were analyzed as number per litter.
Indices:
Adjusted Final Body Weight = final body weight (Gestation Day 20) – gravid uterine weight
Litter Size = number of viable fetuses + number of non-viable fetuses
Pre-Implant Loss = [(number of corpora lutea – number of implants) / number of corpora lutea] X 100
Post-Implant Loss = [(number of implants – number of viable fetuses) / number of implants] X 100
Sex Ratio (% Male Offspring) = (number of male fetuses / total number of fetuses) X 100
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical signs in general were considered to be attributed to the body weight loss observed over the duration of the study, escalating from the decreases in food consumption, in turn contributing to the increasing effects of general health.

Maternal Findings
Clinical observations included: biting objects in 1/6 Group 4 animals; slight hunched posture in 1/6 Group 4 animals; slight to moderate piloerection in 4/6 Group 3 and 6/6 Group 4 animals; slight to moderate alopecia of the abdomen, back, and left/right forelimb/forepaw in 1/6 Group 1, 1/6 Group 3, and 3/6 Group 4 animals, slight brown diarrhea in 1/6 Group 4, superficial eschar of the left/right forepaw in 1/6 Group 3 and 1/6 Group 4 animals; and reduced fecal volume in 5/6 Group 4 animals.
Detailed clinical observations included: hair loss in 1/6 Group 1, 1/6 Group 3, and 3/6 Group 4 animals; eschar in 1/6 Group 3 and 1/6 Group 4.
Mortality:
no mortality observed
Description (incidence):
All animals survived until scheduled euthanasia.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant decreases in absolute body weight and body weight gain, as compared to control Group 1, were observed in Groups 3-4 and to a lesser extent Group 2. Significantly lower body weight gain in Groups 3-4 were noted over the course of the 14-day dietary administration of the test substance during gestation. Similar trending changes in body weight gain were observed in Group 2, to a lesser extent, and were statistically significant, in a transient manner across intervals. These findings are considered to be attributable to the reduced dietary consumption over the course of the study.

Maternal Findings
Statistically significant decreases (p < 0.05-0.001) were noted in mean body weights for Group 3 on GD 11-20 and Group 4 on GD 11-20 and in mean daily body weight gain for Group 2 on GD 8-14, for Group 3 on GD 8-20, and in Group 4 on GD 5-20.
Statistically significant decreases (p < 0.05-0.001) were noted in terminal bodyweights for Groups 2-4 and adjusted bodyweights for Groups 2-4. Statistically significant decreases (p < 0.05-0.001) were noted in absolute weight gain for Group 2 on GD 8-11, GD 17-20, and for the study overall (GD 3-20) and for Groups 3 and 4 on GD 5-20 and for the study overall (GD 3-20).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Statistically significant decreases in mean daily food consumption were noted in Groups 2-4, as compared to control Group 1. Food efficiency was significantly affected in Group 4 females, and to a lesser extent Group 3. These findings are considered to be due to the lack of palatability of DiPotassium hexafluorotitanate (IV) - 01727 in the diet, being the sole food source in the study.

Maternal Findings
Statistically significant decreases (p < 0.05-0.001) were noted in mean daily food consumption for Group 2 on GD 5-14 and GD 17-20 and for Groups 3 and 4 on GD 5-20 and in mean food efficiency for Group 3 on GD 8-14, and in Group 4 on GD 5-20.
Body weight and food consumption measurements collected throughout the study were used to calculate the mean overall daily intake of DiPotassium hexafluorotitanate (IV) – 01727. The mean overall (GD 5-20) daily intake of DiPotassium hexafluorotitanate (IV) – 01727 in rats with dietary levels of 450, 900, 1800 ppm was 30.0, 49.1, and 86.1 mg/kg/day, respectively.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant decreases in mean daily food consumption were noted in Groups 2-4, as compared to control Group 1. Food efficiency was significantly affected in Group 4 females, and to a lesser extent Group 3. These findings are considered to be due to the lack of palatability of DiPotassium hexafluorotitanate (IV) - 01727 in the diet, being the sole food source in the study.

Maternal Findings
Statistically significant decreases (p < 0.05-0.001) were noted in mean daily food consumption for Group 2 on GD 5-14 and GD 17-20 and for Groups 3 and 4 on GD 5-20 and in mean food efficiency for Group 3 on GD 8-14, and in Group 4 on GD 5-20.
Body weight and food consumption measurements collected throughout the study were used to calculate the mean overall daily intake of DiPotassium hexafluorotitanate (IV) – 01727. The mean overall (GD 5-20) daily intake of DiPotassium hexafluorotitanate (IV) – 01727 in rats with dietary levels of 450, 900, 1800 ppm was 30.0, 49.1, and 86.1 mg/kg/day, respectively.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Endocrine findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No gross necropsy observations were noted for any animal during the study.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Number of abortions:
no effects observed
Description (incidence and severity):
There were no abortions or premature deliveries throughout gestation of the pregnant females.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The rates of pre-implantation losses were 4.35, 8.79, 3.37, and 3.42% for Groups 1-4, respectively. The rates of post-implantation loss were 6.70, 3.49, 9.17, 33.33% for Groups 1-4, respectively. There were no statistical differences between the control and treated groups and the mathematical differences are considered to be random and not test substance-related.
Total litter losses by resorption:
effects observed, treatment-related
Description (incidence and severity):
Total resorptions were noted for two Group 4 females.
Early or late resorptions:
no effects observed
Description (incidence and severity):
The mean numbers of resorptions were 1.0, 0.5, 1.3, and 5.0 for Groups 1-4, respectively.
Dead fetuses:
no effects observed
Description (incidence and severity):
The numbers of live fetuses were comparable between control and treated groups. Groups 1, 2, 3, and 4 had 6, 6, 6, and 4 litters with average number of live fetuses of 13.2, 12.8, 12.2, and 9.5 per litter, respectively.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
There were no premature deliveries throughout gestation of the pregnant females.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Pregnancy rate was 100% for Groups 1-4.
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Total mean litter weights were comparable between control and treated groups with 55.490, 51.782, 45.572, and 31.455 g in Groups 1-4, respectively. Statistically significant decreases (p < 0.001) were noted in mean litter weights for Group 4.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
The numbers of live fetuses were comparable between control and treated groups. Groups 1, 2, 3, and 4 had 6, 6, 6, and 4 litters with average number of live fetuses of 13.2, 12.8, 12.2, and 9.5 per litter, respectively.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The (external) fetal sex ratios were comparable between control and treated groups with 52.88, 47.85, 42.42, and 53.85% males in Groups 1-4, respectively.
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
The numbers of live fetuses were comparable between control and treated groups. Groups 1, 2, 3, and 4 had 6, 6, 6, and 4 litters with average number of live fetuses of 13.2, 12.8, 12.2, and 9.5 per litter, respectively. Total mean litter weights were comparable between control and treated groups with 55.490, 51.782, 45.572, and 31.455 g in Groups 1-4, respectively. Statistically significant decreases (p < 0.001) were noted in mean litter weights for Group 4.
Anogenital distance of all rodent fetuses:
not examined
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
There were no gross abnormalities observed for any fetus in the study.
Skeletal malformations:
not examined
Visceral malformations:
not examined
Conclusions:
Under the conditions of this developmental range-finding study and the toxicological endpoints evaluated, dietary exposure of pregnant rats to DiPotassium hexafluorotitanate (IV) – 01727 resulted in clinical observable effects in pregnant dams, and secondarily in their conceptuses at 1800 ppm, the highest level tested. These observations were considered to be due to significant maternal body weight loss, attributable to the decrease in food consumption over the course of the study. These effects did not prevent the ability of pregnant rats in this group to produce implantation sites for evaluation. However, maternal weight loss, due to lack of dietary palatability was considered adverse, resulting in the increase of general animal health concerns. Therefore, under similar experimental conditions, pregnant rats are expected to tolerate dietary levels up to 900 ppm.
Executive summary:

A developmental range-finding/toxicity study was conducted in timed-pregnant CRL Sprague- Dawley CD® IGS rats to determine the potential of DiPotassium hexafluorotitanate (IV) – 01727 to produce pre-natal developmental toxicity, when administered via the diet throughout pregnancy, from the time of implantation to two days prior to term. Twenty-four (24) timed-pregnant female rats were selected for the test and equally distributed into four groups (6 per group). The test substance was administered via the diet at 0, 450, 900, and 1800 ppm of DiPotassium hexafluorotitanate (IV) – 01727 for 15 consecutive days during gestation days (GD) 5-19 of a 21-day pregnancy.
Homogeneity and dietary stability analyses indicate that DiPotassium hexafluorotitanate (IV) – 01727 was homogeneously distributed to within an acceptable margin of analytical variability, and stable in the dietary matrix. The dietary concentrations were considered to have met the targeted level.
Body weight and food consumption measurements collected throughout the study were used to calculate the mean overall daily intake of DiPotassium hexafluorotitanate (IV) – 01727. The mean overall (GD 5-20) daily intake of DiPotassium hexafluorotitanate (IV) – 01727 in rats with dietary levels of 450, 900, 1800 ppm was 30.0, 49.1, and 86.1 mg/kg/day, respectively.
Animals were observed daily during pregnancy for clinical signs and mortality. Individual body weights and food consumption for all pregnant females are reported on GD 3, 5, 8, 11, 14, 17, and 20. Gross necropsies and Caesarean sections (C-sections) were performed on all rats at which time the pregnancy status and uterine contents were evaluated. The conceptuses were assessed for external malformations and viability.
There were no mortalities attributable to the test substance over the course of the study. Clinical signs in general were considered to be attributed to the body weight loss observed over the duration of the study, escalating from the decreases in food consumption, in turn contributing to the increasing effects of general health. Statistically significant decreases in absolute body weight and body weight gain, as compared to control Group 1, were observed in Groups 3-4 and to a lesser extent Group 2. Significantly lower body weight gain in Groups 3-4 were noted over the course of the 14-day dietary administration of the test substance during gestation. Similar trending changes in body weight gain were observed in Group 2, to a lesser extent, and were statistically significant, in a transient manner across intervals. These findings are considered to be attributable to the reduced dietary consumption over the course of the study. Statistically significant decreases in mean daily food consumption were noted in Groups 2-4, as compared to control Group 1. Food efficiency was significantly affected in Group 4 females, and to a lesser extent Group 3. These findings are considered to be due to the lack of palatability of DiPotassium hexafluorotitanate (IV) - 01727 in the diet, being the sole food source in the study. Dose-dependent, statistically significant decreases in final body weights, average final adjusted body weight changes, and adjusted weight changes from GD 5 to terminal sacrifice were noted for Groups 2-4.
Two females from Group 4 exhibited total resorptions. All other females had viable pregnancies at scheduled sacrifice on GD 20. Reproductive and gestational parameters for Groups 2-3 were generally comparable to control Group 1. There were no test substance-related changes in pregnancy rate, maternal or placental gross abnormalities, abortions, or premature deliveries. There were no test substance-related changes in mean number of corpora lutea, implantations or resorptions, as well as pre-implantation and post-implantation loss. Statistically significant decreases (p < 0.001) were noted in gravid uterus weights and mean litter weights for Group 4, attributable to decreases in Group 4 maternal bodyweight.
A total of six litters in Group 1 (13.2 fetuses/litter), six litters in Group 2 (12.8 fetuses/litter), six litters in Group 3 (12.2 fetuses/litter), and four litters in Group 4 (9.5 fetuses/litter) were examined at C-section for external fetal malformations and developmental variations on GD 20. No external abnormalities were observed in any of the litters.
Under the conditions of this developmental range-finding study and the toxicological endpoints evaluated, dietary exposure of pregnant rats to DiPotassium hexafluorotitanate (IV) – 01727 resulted in clinical observable effects in pregnant dams, and secondarily in their conceptuses at 1800 ppm, the highest level tested . These observations were considered to be due to significant maternal body weight loss, attributable to the decrease in food consumption over the course of the study. These effects did not prevent the ability of pregnant rats in this group to produce implantation sites for evaluation. However, maternal weight loss, due to lack of dietary palatability was considered adverse, resulting in the increase of general animal health concerns. Therefore, under similar experimental conditions, pregnant rats are expected to tolerate dietary levels up to 900 ppm.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2023
Report date:
2023

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
EPA 712-C-98-207, August 1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted 25 June 2018
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ICH Harmonized Tripartite Guideline, Detection of Toxicity to Reproduction for Medicinal Products. Study for Effects on Embryo-Fetal Developmental (Segment II), S5 (R2), Guideline 4.1.3. Step 4
Version / remarks:
24 June 1993
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Dipotassium hexafluorotitanate
EC Number:
240-969-9
EC Name:
Dipotassium hexafluorotitanate
Cas Number:
16919-27-0
Molecular formula:
F6Ti.2K
IUPAC Name:
dipotassium hexafluorotitanate(2-)
Test material form:
solid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc.
- Age at study initiation: Animals were approximately 10-12 weeks-of-age at mating.
- Weight at study initiation: The weight variation did not exceed ± 20% of the mean weight.
- Housing: The animals were individually housed in suspended stainless steel cages which conform to the size recommendations in the most recent Guide for the Care and Use of Laboratory Animals (Natl. Res. Council, 2011). Litter paper placed beneath the cage was changed at least three times/week. Beginning no later than GD14, female rats were housed in polyethylene shoebox cages (10.5”w x 19”d x 8”h) containing nesting material (Enrich-o’cobs or equivalent) with wired-mesh lids.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 25 (Temperature was above the targeted upper limit for two days during the study.)
- Humidity (%): 27 - 58 % (Humidity was below the target lower limit for one day during the study.)
These excursions were considered minor and had no impact on this study.
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): All diets were prepared approximately weekly or more frequently, as needed.
- Mixing appropriate amounts with (Type of food): The test substance was added to OSD (Open Standard Diet) D1111225NM Rodent Diet and thoroughly mixed in a high-speed mixer. Control diet was mixed under the same conditions as the diets prepared with the test substance.
- Storage temperature of food: All diets were refrigerated following preparation, unless presented to the test animals on the same day as diet preparation.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
-Stability in the Dietary Matrix
Samples to verify the stability of the test substance in the dietary matrix, targeting the lowest and highest concentrations, were analyzed in an independent study (PSL Study Number 58505).
-Homogeneity
Samples collected from the top, middle, and bottom strata of representative dietary preparations of the lowest and highest concentrations, to demonstrate homogeneity of the test substance, as mixed in the dietary matrix, were analyzed in an independent study (PSL Study Number 58505).
-Concentration Verification
Diet preparations at each interval over the course of the study were presented to verify the nominal concentrations and mixed and presented to each treatment group.
-Sample Preservation
Upon sampling, all scheduled samples collected were stored frozen. Samples were considered stable from the point at which they are frozen.
-Sample Analysis
Frozen samples described above were sent to Product Safety Labs Analytical Services for any future possible analysis of diet preparation or neat test substance samples. Any remaining sample material was retained until issuance of the final report.

Analytical Chemistry
-Sample Storage
Upon receipt, all samples were stored and maintained frozen (approximately -20°C) prior to analysis unless analyzed at the time of collection.
-Method Validation
The method for analysis of test substance in the dietary matrix has been developed by PSL, and the suitability of the methods was demonstrated. In PSL Study 58505, method validation included, but was not limited to determination of linearity, precision, and accuracy. The details of the analysis performed were documented and reported.
-Chemical Analysis
Samples were analyzed in replicate. A detailed description of the analytical test method(s) was documented. Any remaining sample material was retained until the issuance of the final report.
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant
Duration of treatment / exposure:
GD 5 to GD 19 (5-19 days pc)
Frequency of treatment:
daily, 7 days / week
Duration of test:
GD 5 to GD 20 (5-20 days pc)
Doses / concentrationsopen allclose all
Dose / conc.:
0 ppm (nominal)
Dose / conc.:
250 ppm (nominal)
Remarks:
The mean overall (GD 3-20) daily intake of the test substance for rats fed target dietary concentration of 250 ppm of DiPotassium hexafluorotitanate (IV) – 01727 was calculated to be 15.9 mg/kg/day of DiPotassium hexafluorotitanate (IV) – 01727.
Dose / conc.:
500 ppm (nominal)
Remarks:
The mean overall (GD 3-20) daily intake of the test substance for rats fed target dietary concentration of 500 ppm of DiPotassium hexafluorotitanate (IV) – 01727 was calculated to be 32.2 mg/kg/day of DiPotassium hexafluorotitanate (IV) – 01727.
Dose / conc.:
1 000 ppm (nominal)
Remarks:
The mean overall (GD 3-20) daily intake of the test substance for rats fed target dietary concentration of 1000 ppm of DiPotassium hexafluorotitanate (IV) – 01727 was calculated to be 49.1 mg/kg/day of DiPotassium hexafluorotitanate (IV) – 01727.
No. of animals per sex per dose:
20 females
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The Sponsor, in consultation with the Study Director, and based on a previously conducted developmental toxicity study, selected target dose levels of 250, 500, and 1000 ppm (or 0.025, 0.05, and 0.1%) of the test substance. A NOAEL was expected to be achieved for this study
- Rationale for animal assignment (if not random): The animals were randomly distributed, stratified by body weight, amongst the test and control groups prior to the first administration.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: GD 3, 5, 8, 11, 14, 17, and 20

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #20
- Organs examined: All pregnant females in the study were subjected to a necropsy and C-section), which included examinations of the external surface of the body, all orifices, as well as the thoracic, abdominal, and cranial cavities, and their contents. Internal examination included but was not limited to, the examination of mammary tissue. Upon examination of the abdominal and pelvic cavities, the uterus was excised and a gravid uterine weight was recorded. Beginning at the distal end of the right uterine horn, extending caudally across the cervix to the left uterine horn, the location of viable and nonviable fetuses, early and late resorptions were recorded for each uterine horn, their position on the uterus, and total number of implantations.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Blood sampling:
- Plasma: Yes
- Serum: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: all per litter
Statistics:
Analytical methods for this study were based on the compiled data collected and requirements of the regulatory guidelines. Statistical analyses of all quantitative data collected during the in-life and post-mortem phases of the study were performed. Statistical analyses were conducted using the following software application: Provantis® version 10, Tables and Statistics, Instem LSS, Staffordshire UK, and Prism Biostatistics, Graph Pad Software, San Diego, CA.

Statistical Methods of the Parental (P) Generation
Means and standard deviations were calculated for all quantitative data collected from dams. When warranted by sufficient group sizes, data within groups were evaluated for homogeneity of variances and normality by Bartlett’s test (Bartlett, 1937). Where Bartlett’s test indicated normal data distributions, treated and control groups were compared using parametric repeated measures one-way analysis of variance (RM-ANOVA). When a parametric ANOVA was significant, pairwise comparisons of the treated groups to control were performed using Dunnett’s test (Dunnett, 1964, 1980). Where the data distributions were not normal as determined by a significant Bartlett’s test, a non-parametric method was used (Kruskal-Wallis non-parametric ANOVA; Kruskal and Wallis, 1952). When Kruskal-Wallis ANOVA was significant, pairwise comparisons between the control and test groups were performed using Dunn’s test (Dunn, 1964).

Statistical Methods of the Filial (F1) Generation
The litter was the experimental unit for evaluation where appropriate. Mean and standard deviations were calculated independently for control and each treatment group. Similarly, mortality, sex, skeletal/visceral changes were analyzed as number of occurrences per litter. Inferential comparisons between control and the test groups were made with regards to homogeneity of variance, normality, and ANOVA, where appropriate as described for the P generation.
Indices:
Litter Size = number of viable fetuses + number of non-viable fetuses
Pre-Implant Loss = [(number of corpora lutea – number of implants)/number of corpora lutea] x 100
Post-Implant Loss = [(number of implants – number of viable fetuses)/number of implants] x 100
Sex Ratio (% Male Offspring) = (number of male fetuses/total number of fetuses) x 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related, adverse cage-side and detailed clinical observations include piloerection, soft feces, and reduced fecal volume were noted predominantly in Group 4 animals. These observations correlate to decreased weight parameters in Group 4, resulting in a degradation of general health assessment.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Decreased absolute body weights were observed in a dose dependent manner in Groups 2-4, and statistically significant in Group 4, as compared to control Group 1. Lower body weight gain in general corresponded to changes observed in absolute body weights, with or without a significant statistical difference, when compared within weekly intervals. These Group 4 adverse test substance-related decreases in body weight correspond to significantly reduced food consumption and to a lesser body weight impact in Groups 2 and 3.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Statistically significant decreases (p < 0.05-0.001) in mean daily food consumption were noted for Group 2 on GD 5-8, for Group 3 on GD 5-20, and for Group 4 on GD 5-20.
The mean overall (GD 3-20) daily intake of the test substance for rats fed target dietary concentrations of 250, 500, and 1000 ppm of DiPotassium hexafluorotitanate (IV) – 01727 was calculated to be 15.9, 32.2, and 49.1 mg/kg/day of DiPotassium hexafluorotitanate (IV) – 01727. These values were in generally good agreement with targeted test substance exposure concentrations in mg/kg/day.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant decreases (p < 0.05-0.001) in mean food efficiency were noted for Group 2 on GD 5-8, for Group 3 on GD 5-8, and for Group 4 on GD 5-14.
Ophthalmological findings:
not specified
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Endocrine findings:
no effects observed
Description (incidence and severity):
Dietary exposure of the timed-pregnant rats to the test substance resulted in statistically significant decreases of T4 and T3 in animals from Groups 4 and Groups 3 and 4, respectively. However, the mean levels are within the historical control limit ranges for female rats.

TSH
TSH values for Groups 2-4 were comparable to control Group 1.

T3
T3 values for Groups 2-3 were comparable to control Group 1. Statistically significant decreases (p < 0.05) in T3 were noted for Group 3 and 4 animals. However, the observed values are within the historical control limit range (0.672 - 5.618 ng/mL).

T4
T4 values for Group 2 were comparable to control Group 1. Statistically significant decreases (p < 0.001) in T4 were noted for Group 4 animals. However, the observed values are within the historical control limit range (26.459-56.404 ng/mL).
Urinalysis findings:
not examined
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No gross necropsy observations were noted for any animal during the study.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
There were no abortions or premature deliveries throughout gestation of the pregnant females.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Pre-implantation loss in Groups 2-4 was comparable to control Group 1 with mean values of 8.98, 2.85, 3.48, and 7.77% for Groups 1-4, respectively.
Post-implantation loss was 2.49, 4.56, 2.98, and 6.41% for Groups 1-4, respectively
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Description (incidence and severity):
Total number of resorptions in Groups 2-4 was comparable to control Group 1 with mean percentages of 0.3, 0.7, 0.4, and 0.7% for Groups 1-4, respectively.
Dead fetuses:
no effects observed
Description (incidence and severity):
The number of live fetuses was comparable for all test animals. The average numbers of live fetuses per litter were 12.8 (20 litters), 13.6 (20 litters), 14.2 (20 litters), and 12.7 (19 litters), respectively for Groups 1-4.
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
The pregnancy rate was 100.0, 100.0, 100.0 and 95.0% for female rats in
Groups 1-4, respectively.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
ca. 32.2 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
food efficiency
Dose descriptor:
NOAEL
Effect level:
500 ppm (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
food efficiency

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Male and female mean fetal weights for Groups 2-3 were comparable between control and treated groups. Statistically significant (p < 0.001) decreases in mean fetal weight for males and females and total mean fetal weight were noted for Group 4 animals.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The (external) fetal male sex ratio was 49.61, 52.34, 48.03, and 58.14% for Groups 1-4, respectively, and was comparable across treatment groups.
Changes in litter size and weights:
no effects observed
Anogenital distance of all rodent fetuses:
not examined
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
Approximately half of each litter, 17 fetuses from Group 1 (3 litters), 21 fetuses from Group 2 (3 litters), 29 fetuses from Group 3 (4 litters), and 20 fetuses from Group 4 (3 litters), were evaluated for skeletal malformations and variations.
The following malformations were observed:
• Wavy 6th right thoracic rib in 1 Group 4 fetus.
• Wavy 7th right thoracic rib in 1 Group 4 fetus.
• Wavy 8th right thoracic rib in 1 Group 4 fetus.
• Wavy 9th right thoracic rib in 1 Group 4 fetus.
• Wavy 10th right thoracic rib in 1 Group 4 fetus.
• Bilateral wavy 11th thoracic ribs in 1 Group 4 fetus.
• Wavy 11th right thoracic rib in 1 Group 4 fetus.
• Wavy 12th right thoracic rib in 1 Group 4 fetus.
• Malpositioned 4th sternebrae in 1 Group 3 fetus.
Due to the nature and incidence, these malformations are considered to have limited biological or toxicological impact on the developing fetuses and are thus interpreted to be not adverse.
Visceral malformations:
no effects observed
Details on embryotoxic / teratogenic effects:
An increased incidence and severity in ossification delays was observed in multiple skeletal bones in Group 3 and/or Group 4 fetuses, when compared to study control and historical control values, correlating with lower litter and fetal weights, as well as lower maternal body weights. In addition, increased Group 4 ossification delays could potentially correlate to observations including decreases in bone and/or the fibrous osteodystrophy noted in a cascade of secondary renal effects in general toxicity assessments.

Effect levels (fetuses)

open allclose all
Dose descriptor:
NOAEL
Effect level:
500 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
skeletal malformations
Dose descriptor:
NOAEL
Effect level:
ca. 32.2 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
skeletal malformations

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
Under the conditions of the study and based on the maternal and fetal developmental toxicity endpoints evaluated, the no-observed-adverse-effect-level (NOAEL) for maternal and developmental toxicity for the dietary administration of DiPotassium hexafluorotitanate (IV)-01727 were both determined to be 500 ppm, with a calculated 32.2 mg/kg/day. Developmental effects observed were considered to be consequentially attributable to the maternal toxicity findings noted in this study.
Executive summary:

A prenatal developmental toxicity study was conducted in timed-pregnant CRL Sprague-Dawley CD® IGS rats to determine the potential of the test item to produce pre-natal developmental toxicity, when administered in the diet throughout pregnancy, from gestational day (GD) 5 - 19. The study was performed according to OECD Guideline 414 under GLP-compliance. Eighty (80) timed-pregnant female rats were selected for the test and equally distributed into four groups (twenty females per group). Dams were maintained on diets calculated to provide target dietary intakes of 0 (basal diet), 250 (low dose), 500 (intermediate dose), and 1000 ppm (high dose) for Groups 1-4, respectively.


The neat test substance was considered to be stable under the conditions of storage over the course of the study. Homogeneity and dietary stability analyses indicate that the test item was homogeneously distributed within an acceptable margin of analytical variability, and stable in the dietary matrix.  The dietary concentrations were considered to have met the targeted levels.


Animals were observed daily during pregnancy for clinical signs and mortality.  Individual body weights and food consumption for all pregnant females were recorded on gestation days (GD) 3, 5, 8, 11, 14, 17, and 20.  Gross necropsies and Caesarean sections (C-sections) were performed on all rats, where the pregnancy status and uterine contents were evaluated.  The fetuses were assessed for viability, external malformations, and then for visceral or skeletal variations and malformations.


Test substance-related, adverse cage-side and detailed clinical observations include piloerection, soft feces, and reduced fecal volume were noted predominantly in Group 4 animals.  These observations correlate to decreased weight parameters in Group 4, resulting in a degradation of general health assessment. 


Decreased absolute body weights were observed in a dose dependent manner in Groups 2-4, and statistically significant in Group 4, as compared to control Group 1. Lower body weight gain in general corresponded to changes observed in absolute body weights, with or without a significant statistical difference, when compared within weekly intervals. The Group 4 adverse test substance-related decrease in body weight corresponds to significantly reduced food consumption and to a lesser body weight impact in Groups 2 and 3. Dietary exposure of the timed-pregnant rats to the test substance resulted in statistically significant decreases to T4 and T3 in animals from Groups 4 and Groups 3 and 4, respectively. However, the average levels are within the historical control limit ranges for female rats. Moreover, TSH levels in group 2-3 were comparable to control group.


Statistically significant decreases in total mean fetal weight parameters were observed in Group 4. These changes were considered a secondary developmental effect attributed to test substance-related decrease maternal body weight parameters over the course of the study.


There were no malformations or variations of toxicological significance observed for any fetus during the study. An increased incidence and severity in ossification delays was observed in multiple skeletal bones in Group 3 and/or Group 4 fetuses, when compared to study and historical control values, correlating with lower litter and fetal weights, as well as lower maternal body weights, with a potential correlation to decreases in bone observed in general toxicity assessments.


Under the conditions of the study and based on the maternal and fetal developmental toxicity endpoints evaluated, the no-observed-adverse-effect-level (NOAEL) for maternal and developmental toxicity for the dietary administration of the test item were both determined to be 500 ppm, with a calculated 32.2 mg/kg/day. Developmental effects observed were considered to be consequentially attributable to the maternal toxicity findings noted in this study.