Dihydrogen hexafluorotitanate(2-)

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

8 MAR 2022 to 21 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:

short-term repeated dose toxicity: oral

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 palatability and general toxicity (based on toxicological endpoints selected) of DiPotassium hexafluorotitanate (IV) - 01727 in rats following 14 days of dietary administration. These data were used, along with additional relevant data, to select dietary levels for a subsequent longer toxicity study in rats. Four groups of adult CRL: Sprague-Dawley® CD® IGS rats (5/sex/group) were maintained on diets prepared to contain 600, 1200, and 2400 ppm of DiPotassium hexafluorotitanate (IV) - 01727, for target daily intakes of 50, 100, and 200 mg/kg/day.

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

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, NC
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 6 - 7 weeks
- Weight at study initiation: the weight variation did not exceed ±20% of the mean weight for each sex. 238-261 grams (males) and 150-195 grams (females)
- 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 (Natl. Res. Council, 2011). Litter paper placed beneath the cage was changed at least three times/week.
- 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: 6 d

DETAILS OF FOOD AND WATER QUALITY: Water analysis was conducted by Precision Analytical Services, Inc., Toms River, NJ, and South Brunswick Municipal Water Supply, South Brunswick, NJ.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 21
- Humidity (%): 45 - 72
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: feed

Details on route of administration:

The test substance was administered in the diet. It was originally planned to carry out oral gavage method-based studies to comply with Decision Number CCH-D-2114471125-54-01/F at the recommendation of the Study Director and based on the method of delivery in the acute oral studies.
A pre-study done in rats by gavage administration showed that at dose levels of 40 mg/kg/day and above were considered to reflect significant irritancy in the GI tract indicating a local effect rather than systemic toxicity, the sole purpose of repeated-dose study. Animals receiving 70 or 100 mg/kg/day had to be terminated before completion of their 14-day treatment period. This local irritancy clearly limits the dose levels that can be investigated with repeated daily gavage administration and therefore impairs the assessment of systemic toxicity that can be undertaken when the test item is administered by the oral gavage route. It was considered that a more robust assessment of systemic toxicity may be possible if the test item is administered by the oral dietary route and it was therefore recommended that this route of administration should be investigated. It is anticipated that the change to the dietary route may reduce the local irritancy observed with the Test Item. This will allow higher dose levels (in terms of mg/kg/day) to be investigated thereby resulting in a more meaningful assessment of systemic toxicity and reliable NOAEC for human health risk assessment.

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): All diets were prepared 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 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:

The test substance and selected prepared diets (at each concentration), were sampled in duplicate. Samples were frozen until analyzed and/or discarded upon completion of the study. Homogeneity of the diet preparations were demonstrated in a separate analytical method validation study (PSL Study Number 58505).

Stability of Test Substance
At the initial and final diet preparations, a sample of the test substance (neat) was retained for stability and stored frozen. Analytical results of the initial and final stability samples were used to establish the stability of the test substance under normal laboratory conditions for the duration of the study.

Concentration Verification
Samples for concentration verification were collected as part of the homogeneity analysis during the first week of the study. Diet preparation calculation was verified and test diets were mixed according to PSL’s standard operating procedure. Nominal diet concentrations were used to determine the total intake of the test substance for each group.

Sample Preservation
Upon sampling, diet preparations and neat test substance were stored frozen. Samples were considered stable from the point at which they are frozen. Upon sampling, dose preparations and neat test substance were stored frozen and retained until the issuance of the final report.

Sample Analysis
The selected diet samples described above were sent to Product Safety Labs Analytical Services for analysis of diet preparation and neat test substance samples, as part of a separate method validation study (PSL Study Number 58505). Any extra sample material was retained until the issuance of a final report.

Duration of treatment / exposure:

14 days

Frequency of treatment:

daily via food

Dose / conc.:

2 400 ppm

Remarks:

The overall dietary intake could not be calculated due to the humane sacrifice of all male and females on Day 8.

Dose / conc.:

1 200 ppm

Remarks:

The mean overall (Days 0-14) daily intake of DiPotassium hexafluorotitanate (IV) – 01727 in rats fed concentrations at 1200 ppm was 72.7 mg/kg/day for the males and 78.9 mg/kg/day for the females.

Dose / conc.:

600 ppm

Remarks:

The mean overall (Days 0-14) daily intake of DiPotassium hexafluorotitanate (IV) – 01727 in rats fed concentrations at 600 ppm was 45.0 mg/kg/day for the males and 48.1 mg/kg/day for the females.

Dose / conc.:

0 ppm

No. of animals per sex per dose:

5

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The Sponsor, in consultation with the Study Director, selected dietary concentrations of 600, 1200, and 2400 ppm (or 0.06, 0.12, and 0.24%) of DiPotassium hexafluorotitanate (IV) - 01727 in the diet to target approximate exposures of 50, 100, and 200 mg/kg/day based upon a daily food consumption of 25 grams for a 300 gram rat.
- Rationale for animal assignment (if not random): randomly distributed, stratified by body weight

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: On Day 0, prior to the first treatment with the test substance, and approximately weekly thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: Day 0 (prior to study start) and on Days 3, 7, 10 and 14 or at the time of euthanasia

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- 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

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

PLASMA/SERUM HORMONES/LIPIDS: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (examination of the external surface of the body, all orifices, musculoskeletal system, and the cranial, thoracic, abdominal, and pelvic cavities, with their associated organs and tissues

HISTOPATHOLOGY: No

Statistics:

Product Safety Labs performed statistical analysis of all quantitative data collected during the in-life phase of the study as well as organ weight data. The use of the word “significant” or “significantly” indicates a statistically significant difference between the control and the experimental groups. Significance was judged at a probability value of p<0.05. Male and female rats were evaluated separately. Mean and standard deviations was calculated for all quantitative data. Male and female animals were evaluated separately. For all in-life endpoints that were identified as multiple measurements of continuous data over time (e.g., body weight parameters, food consumption, food efficiency, etc.), treatment and control groups were compared using a two-way analysis of variance (ANOVA), testing the effects of both time and treatment, with methods accounting for repeated measures in one independent variable (time; Motulsky, 2014). When a significant interaction effect is observed between treatment and time, further analysis of the p value for each individual interval may be conducted and ultimately by a post hoc multiple comparisons test (e.g., Dunnett’s test; Dunnett, 1964, 1980) of the individual treated groups to control. Statistical analysis was conducted by using the following software application: Provantis® version 10, Tables and Statistics.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Observations of hunched posture, piloerection, thin appearance with corresponding emaciation, hypoactivity, and reduced fecal volume noted in Group 4 male and/or female rats considered to be attributable to continued decreases in dietary consumption. All other observations were considered incidental and of no toxicological significance.

Mortality:

mortality observed, treatment-related

Description (incidence):

All Group 4 males and females were humanely sacrificed on Day 8 due to observed adverse clinical signs, extreme body weight losses, and lower food consumption.

Description (incidence and severity):

Decreases in absolute body weight and body weight gain as compared to control Group 1, were observed in Groups 2-4 males and to a lesser extent in Groups 3-4 female rats. Significantly lower body weight gain in Groups 2-4 males and females were observed in the first two measured intervals, and were sporadic in statistical significance in groups thereafter. These findings considered to be attributable to the reduced dietary consumption over the course of the study.

Males
Statistically significant decreases (p<0.01-0.001) in mean body weights occurred in Group 2-3 males on Days 3-14 and on Days 3-7 for Group 4 males. Statistically significant decreases (p<0.01-0.001) in body weight gain occurred in Group 2 males on Days 0-7, Group 3 males on Days 0-7 and Days 10-14, and in Group 4 males on Days 0-7.

Females
Mean body weights for female rats in Groups 2 were comparable to control Group 1 throughout the study. Statistically significant decreases (p<0.01-0.001) in mean body weights occurred in Group 3 females on Days 7-14 and Group 4 females on Days 3-7. Statistically significant decreases (p<0.05-0.001) in body weight gain occurred in Group 2-4 females on Days 0-7.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Decreases in food consumption were observed in Groups 2-4 male and female, reaching statistical significance in all male treated groups over the course of the study. Food efficiency was significantly affected in all male treated groups and Groups 3 and 4 females, in the first two study intervals. These observations are considered to be attributed to the lack of palatability of
DiPotassium hexafluorotitanate (IV) – 01727.

Males
Statistically significant decreases (p<0.05-0.001) in food consumption occurred in Group 2 males on Days 0-7 and 10-14, Group 3 males on Days 0-14 and in Group 4 males on Days 0-7. Statistically significant decreases (p<0.01-0.001) in food efficiency occurred in Group 2-4 males
on Days 0-7.

Females
Mean daily food consumption for female rats in Groups 2-4 were lower but not significantly different from control Group 1 throughout the study. Statistically significant decreases (p<0.01-0.001) in food efficiency occurred in Group 3-4 females on Days 0-7.

Dietary Intake
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 (Days 0-14) daily intake of DiPotassium hexafluorotitanate (IV) – 01727 in rats with targeted dietary levels of 600 (Group 2) and 1200 (Group 3) ppm was 45.0 and 72.7 mg/kg/day, respectively, for the males and, 48.1 and 78.9 mg/kg/day, respectively, for the females. The overall dietary intake for Group 4 could not be calculated due to the humane sacrifice of all male and females on Day 8.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Decreases in food consumption were observed in Groups 2-4 male and female, reaching statistical significance in all male treated groups over the course of the study. Food efficiency was significantly affected in all male treated groups and Groups 3 and 4 females, in the first two study intervals. These observations are considered to be attributed to the lack of palatability of
DiPotassium hexafluorotitanate (IV) – 01727.

Males
Statistically significant decreases (p<0.05-0.001) in food consumption occurred in Group 2 males on Days 0-7 and 10-14, Group 3 males on Days 0-14 and in Group 4 males on Days 0-7. Statistically significant decreases (p<0.01-0.001) in food efficiency occurred in Group 2-4 males
on Days 0-7.

Females
Mean daily food consumption for female rats in Groups 2-4 were lower but not significantly different from control Group 1 throughout the study. Statistically significant decreases (p<0.01-0.001) in food efficiency occurred in Group 3-4 females on Days 0-7.

Dietary Intake
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 (Days 0-14) daily intake of DiPotassium hexafluorotitanate (IV) – 01727 in rats with targeted dietary levels of 600 (Group 2) and 1200 (Group 3) ppm was 45.0 and 72.7 mg/kg/day, respectively, for the males and, 48.1 and 78.9 mg/kg/day, respectively, for the females. The overall dietary intake for Group 4 could not be calculated due to the humane sacrifice of all male and females on Day 8.

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:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Conclusions:

Under the conditions of the study and based on the toxicological endpoints evaluated, there were in-life changes attributed to the dietary administration of DiPotassium hexafluorotitanate (IV) – 01727 at concentrations of 1200 and 2400 ppm. In a study of longer duration, rats are expected to tolerate dietary concentrations of up to 600 ppm, a level calculated to provide adjusted dietary intakes of 45.0 and 48.1 mg/kg/day of DiPotassium hexafluorotitanate (IV) - 01727 for males and females, respectively.

Executive summary:

Four groups of adult CRL: Sprague-Dawley® CD® IGS rats (5/sex/group) were maintained on diets prepared to contain 600, 1200, and 2400 ppm of DiPotassium hexafluorotitanate (IV) - 01727, for target daily intakes of 50, 100, and 200 mg/kg/day. Homogeneity and stability samples were prepared in conjunction with PSL Study Number 58508 and analyzed in PSL Study Number 58505. 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 (Days 0-14) daily intake of DiPotassium hexafluorotitanate (IV) – 01727 in rats fed concentrations at 600 and 1200 ppm was 45.0 and 72.7 mg/kg/day, respectively, for the males and, 48.1 and 78.9 mg/kg/day, respectively, for the females. The overall dietary intake for Group 4 could not be calculated due to the humane sacrifice of all male and females on Day 8.
The animals were observed at least once daily for viability, signs of gross toxicity, and behavioral changes, and weekly for a battery of detailed observations. Body weights were recorded two times during the acclimation period (including prior to initial dietary administration on Day 0), on Days 3, 7, 10, and 14 of treatment, and immediately prior to sacrifice, or until death occurred. Individual food consumption was also recorded to coincide with body weight measurements. Food efficiency and dietary intake were calculated. All animals were subjected to a gross necropsy at study termination on Day 15.
There were no mortalities during the course of the 14-day study. All Group 4 males and females were humanely sacrificed on Day 8 due to observed adverse clinical signs, extreme body weight losses, and lower food consumption. Observations of hunched posture, piloerection, thin
appearance with corresponding emaciation, hypoactivity, and reduced fecal volume noted in Group 4 male and/or female rats considered to be attributable to continued decreases in dietary consumption. All other observations were considered incidental and of no toxicological significance. Decreases in absolute body weight and body weight gain as compared to control Group 1, were observed in Groups 2-4 males and to a lesser extent in Groups 3-4 female rats. Significantly lower body weight gain in Groups 2-4 males and females were observed in the first two measured intervals, and were sporadic in statistical significance in groups thereafter. These findings considered to be attributable to the reduced dietary consumption over the course of the study. Decreases in food consumption were observed in Groups 2-4 male and female, reaching statistical significance in all male treated groups over the course of the study. Food efficiency was significantly affected in all male treated groups and Groups 3 and 4 females, in the first two study intervals. These observations are considered to be attributed to the lack of palatability of DiPotassium hexafluorotitanate (IV) – 01727. There were no macroscopic, microscopic, or organ weight changes attributable to test substance administration.
Under the conditions of the study and based on the toxicological endpoints evaluated, there were in-life changes attributed to the dietary administration of DiPotassium hexafluorotitanate (IV) – 01727 at concentrations of 1200 and 2400 ppm. In a study of longer duration, rats are expected to tolerate dietary concentrations of up to 600 ppm, a level calculated to provide adjusted dietary intakes of 45.0 and 48.1 mg/kg/day of DiPotassium hexafluorotitanate (IV) - 01727 for males and females, respectively.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2023

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

2018

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: U.S. FDA Toxicological Principles for the Safety Assessment of Food Ingredients, Redbook 2000, Revised 2007, IV.C.4.a. (2003)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

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

Remarks:

CRL Sprague-Dawley CD® IGS rats

Details on species / strain selection:

The Sprague-Dawley® rat was the system of choice because, historically, it has been a preferred and commonly used species for dietary toxicity tests

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 7 - 8 weeks
- 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 caging 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 was placed beneath the cages and was changed at least three times per week.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 6 d

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 23
- Humidity (%): 32 - 70
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: feed

Details on route of administration:

The test substance was administered in the diet. It was originally planned to carry out oral gavage method-based studies to comply with Decision Number CCH-D-2114471125-54-01/F at the recommendation of the Study Director and based on the method of delivery in the acute oral studies.
A pre-study done in rats by gavage administration showed that at dose levels of 40 mg/kg/day and above were considered to reflect significant irritancy in the GI tract indicating a local effect rather than systemic toxicity, the sole purpose of repeated-dose study. Animals receiving 70 or 100 mg/kg/day had to be terminated before completion of their 14-day treatment period. This local irritancy clearly limits the dose levels that can be investigated with repeated daily gavage administration and therefore impairs the assessment of systemic toxicity that can be undertaken when the test item is administered by the oral gavage route. It was considered that a more robust assessment of systemic toxicity may be possible if the test item is administered by the oral dietary route and it was therefore recommended that this route of administration should be investigated. It is anticipated that the change to the dietary route may reduce the local irritancy observed with the Test Item. This will allow higher dose levels (in terms of mg/kg/day) to be investigated thereby resulting in a more meaningful assessment of systemic toxicity and reliable NOAEC for human health risk assessment.

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- 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.

Duration of treatment / exposure:

90 days

Frequency of treatment:

daily, 7 days each week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm

Doses / concentrations 2

Dose / conc.:

250 ppm

Remarks:

12.5 mg/kg bw/d for males and 17 mg/kg bw/d for females

Doses / concentrations 3

Dose / conc.:

500 ppm

Remarks:

25.6 mg/kg bw/d for males and 32.9 mg/kg bw/d for females

Doses / concentrations 4

Dose / conc.:

1 000 ppm

Remarks:

45.9 mg/kg bw/d for males and 61.8 mg/kg bw/d for females

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The Sponsor, in consultation with the Study Director, and based on a 14-day palatability/toxicity study, PSL 58506 (PSL, Final), selected dietary concentrations of 250, 500, and 1000 ppm to target approximate exposures of 0.025, 0.05, and 0.1% of Dipotassium hexafluorotitanate (IV) – 01727 in the diet and were expected to result in approximate dietary intakes of 19.3, 38.6, and 77.7 mg/kg/day based upon a daily food consumption of 27 grams for a 350 gram rat.
- Rationale for animal assignment (if not random): Animals were randomly assigned, stratified by body weight, to test groups.
- Fasting period before blood sampling for clinical biochemistry: overnight

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- 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

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: once during acclimation and once on Day 90 prior to test termination
- Dose groups that were examined: all dose groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of study
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked: hematocrit, platelet count, hemoglobin concentration, red blood cell count, mean corpuscular hemoglobin, red cell distribution width, mean corpuscular volume, reticulocytes count, white blood cell and differential leukocyte count. Mean corpuscular hemoglobin concentration was calculated. activated partial thromboplastin time, prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of study
- Animals fasted: Yes
- How many animals: All
- Parameters checked: albumin lipoprotein (high density), alkaline phosphatase, lipoprotein (low density), bilirubin (total), potassium, blood creatinine, serum alanine aminotransferase, calcium, serum aspartate aminotransferase, chloride, serum protein (total), cholesterol (total), sodium, fasting glucose, sorbitol dehydrogenase, globulin, triglycerides, inorganic phosphorous, urea nitrogen


PLASMA/SERUM HORMONES/LIPIDS: Yes, TSH, T3 and T4
- Time of blood sample collection:
- Animals fasted: Yes
- How many animals: all

URINALYSIS: Yes
- Time schedule for collection of urine: end of study (day before blood collection)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: quality, pH, ketone, color, glucose, bilirubin, clarity, specific gravity, blood, protein (total), urobilinogen, microscopic urine sediment, volume


NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, All animals in the study were subjected to a gross necropsy, which included examination of the external surface of the body, all orifices, musculoskeletal system, and the cranial, thoracic, abdominal, and pelvic cavities, with their associated organs and tissues.
The following tissues (of all animals sacrificed by design) were weighed wet as soon as possible after dissection to avoid drying: adrenals (combined), kidneys, testes (combined), brain, liver, thymus, epididymides (combined), ovaries with oviducts (combined), uterus, heart, spleen
The following tissues were weighed at least 24 hours after preservation in 10% neutral buffered formalin: prostate and seminal vesicles with coagulating gland (combined), thyroid/parathyroid, pituitary
The following organs and tissues from all surviving animals were preserved in 10% neutral buffered formalin for possible future histopathological examination:
accessory genital organs (prostate and seminal vesicles)
adrenals
all gross lesions
aorta
bone (femur)
bone marrow (from femur & sternum)
brain (sections including medulla/pons, cerebellar and cerebral cortex
cecum
cervix
colon
duodenum
esophagus
Harderian gland
heart
ileum with Peyer’s patches
jejunum
kidneys
larynx
liver
lungs
lymph node mandibular
lymph node mesenteric
mammary gland
nasal turbinates
nose
ovaries
oviducts
pancreas (with islets)
parathyroid
peripheral nerve (sciatic)
pharynx
pituitary gland
rectum
salivary glands (sublingual adrenals, submandibular, and parotid)
skeletal muscle
skin
spinal cord – 3 levels: (cervical, mid- thoracic, and lumbar)
spleen
sternum
stomach
thymus
thyroid
trachea
urinary bladder
uterus
vagina
The following organs and tissues from all animals were preserved in modified Davidson’s fixative and then stored in ethanol for possible future histopathological examination:
eyes
optic nerve
epididymides
testes

HISTOPATHOLOGY: Yes, Histological examination was performed on the preserved organs and tissues of the animals from both the control and high dose groups (Groups 1 and 4, respectively), all unscheduled death animals, all gross lesions from all animals, in addition to target organs identified during slide evaluation (kidney, parathyroid gland, adrenal gland, stomach [glandular and non-glandular], femur, sternum, cranium, tooth, prostate gland [males only], seminal vesicle/coagulating gland [males only], uterus [females only], and ovary [females only] for all Group 2 and 3 animals were processed, embedded in paraffin, sectioned and stained with hematoxylin and eosin (H&E) and examined by light microscopy. The thyroid gland was also evaluated in routine section with the parathyroid gland for the incidence of follicular cell hypertrophy, a background finding. Slide preparation, and histological assessment by a board-certified veterinary pathologist, were performed at StageBio.

Statistics:

Product Safety Labs performed statistical analysis of all data collected during the in-life phase of the study, as well as clinical pathology results and organ weight data. The use of the word “significant” or “significantly” indicates a statistically significant difference between the control and the experimental groups. Significance was judged at a probability value of p<0.05. Mean and standard deviation were calculated for all quantitative data. Male and female rats were evaluated separately.
Statistical analysis was conducted by using one or more of the following software applications: Provantis™ version 10, Tables and Statistics, Instem LSS, Staffordshire, UK.

For further details, please refer to 'Any other information on materials and methods'

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Adverse test substance-related clinical signs include noted over the course of the study include thin appearance, piloerection, irregular respiration, hypoactivity, ano-genital and other staining, ocular and nasal discharge, and urine discoloration. These findings, generally increasing in incidence and/or severity, were predominantly in Group 4 animals. Alopecia and eschar in Group 4 animals were attributable to decreasing dietary administration of the test substance over time considered to be due to searching behavior for a more palatable food source.

Mortality:

mortality observed, treatment-related

Description (incidence):

Two Group 4 female rats were humanely sacrificed at study days 78 and 85, respectively, due to degrading general health as a result of body weight loss from reduced food consumption.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean absolute body weight changes in Groups 2-4 over the course of the study were considered to be related to the dietary administration of DiPotassium hexafluorotitanate (IV)-01727, as there was a dose-dependent trend. Body weights were statistically significant from control Group 1 for Groups 3 and 4 male and female rats, and considered to be adverse in Group 3 and 4 males as well as Group 4 females, since weight loss exceeded 30% by the end of the 90-day dietary administration of the test substance. Body weight gain in Group 3 and 4 male and female rats corresponded with mean absolute body weight, with a lesser degree of statistical significance across dietary intervals.

Mean weekly body weights for the male treated rats in Groups 3-4 were statistically decreased (p<0.05-0.001) from Day 7 to Day 91 in comparison to the control Group 1 animals. Group 2 body weights were generally comparable to the control Group 1 animals during the study, with the exception of a statistical decrease (p<0.05) on Day 84.

Mean daily body weight gain for male treated rats in Group 4 was statistically decreased (p<0.01-0.001) on Days 0-63 and Days 77-84 in comparison to the control Group 1 animals. Group 3 body weight gain was statistically decreased (p<0.05-0.001) on Days 0-7 and Days 21-56. Group 2 body weight gain was statistically decreased (p<0.001) on Days 0-7 and Days 70-77. Otherwise the animals were comparable to control Group 1 animals.

Mean weekly body weights for the female treated rats in Groups 3-4 were statistically decreased (p<0.05-0.001) between Day 7 and Day 91 in comparison to the control Group 1 animals. Mean weekly body weights for Group 2 were comparable to the control Group 1 animals throughout the study.

Mean daily body weight gain for female treated rats in Group 4 was statistically decreased (p<0.001) on Days 0-14, Days 49-63, and Days 77-84 in comparison to the control Group 1 animals. Group 3 body weight gain was statistically decreased (p<0.05) on Days 0-7 and Days 42-49. Group 2 body weight gain was comparable to control Group 1 animals throughout the study.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Mean daily food consumption was decreased in a dose-dependent manner in Groups 2-4, and statistically significant intermittently across dietary intervals in Group 2 and 3 male and female rats. Significant decreases in Group 4 male and female food consumption over the course of the study was considered adverse, with decreases in consumption exceeding 40% of control levels by the end of the study. Decreases in food consumption is considered to be a contributing cause in body weight loss, as well as the declining health of Group 4 animals by the end of the study.

Mean daily food consumption for the male treated rats in Groups 3-4 were statistically decreased (p<0.05-0.001) between Days 0-7 to Days 84-91 in comparison to the control Group 1 animals. Group 2 mean daily food consumption were generally comparable to the control Group 1 animals during the study, with the exception of statistical decreases (p<0.05-0.01) on Days 0-7 and Days 49-70.

Mean daily food consumption for the female treated rats in Group 4 was statistically decreased (p<0.01-0.001) between Days 0-35 and Days 42-91 in comparison to the control Group 1 animals. Group 3 mean daily food consumption was generally comparable to the control Group 1 animals during the study, with the exception of statistical decreases (p<0.05) on Days 0-14. Group 2 mean daily food consumption was comparable to control Group 1 animals throughout the study.

Dietary Intake
Body weight and food consumption measurements collected throughout the study were used to calculate the mean overall daily intake of DiPotassium hexafluorotitanate (IV) – 01727, based on expected achievement of target dietary concentrations of 125, 350, and 700 mg/kg/day (Groups 2-4, respectively). Based on target nominal concentrations, intakes of 12.5, 25.6, and 45.9 mg/kg/day in male rats, and 17.0, 32.9, and 61.8 mg/kg/day in female rats were calculated for Groups 2-4, respectively.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Food efficiency for male treated rats in Group 4 was statistically decreased (p<0.05-0.001) on Days 0-28, Days 35-42, Days 49-56, and Days 70-77 in comparison to the control Group 1 animals. Group 3 food efficiency was generally comparable to the control Group 1 animals during the study, with the exception of a statistical decrease (p<0.05) on Days 0-7. Group 2 food efficiency was comparable to control Group 1 animals throughout the study.

Food efficiency for female treated rats in Group 4 was statistically decreased (p<0.05-0.001) on Days 0-14, Days 49-63, and Days 77-84 in comparison to the control Group 1 animals. Group 3 food efficiency was generally comparable to the control Group 1 animals during the study, with the exception of a statistical decrease (p<0.05) on Days 42-49. Group 2 food efficiency was comparable to control Group 1 animals throughout the study.

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

All animals included in the study were normal upon ophthalmic exam, thus the test substance is not considered an ocular irritant.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Increased WBC counts in 1000 ppm males and at ≥250 ppm in females, increased neutrophil counts in 1000 ppm males and at ≥500 ppm in females and increased lymphocytes and monocytes in 1000 ppm males and females were considered as test substance-related changes. These changes were associated with mixed cell inflammation of glandular stomach at ≥250 ppm in males and females, degenerative nephropathy (tubular neutrophils aggregates, interstitial lymphocytes infiltration) in 1000 ppm animals.
Decreased hemoglobin and hematocrit in 1000 ppm males and females, decreased MCH and MCHC in ≥ 500 ppm males and females and decreased MCV decreased in ≥ 500 ppm females and increased red cell distribution width in 1000 ppm females were considered as test substance-related secondary changes. These findings represent anaemia due to decreased erythropoietin production due to moderate to severe degenerative nephropathy.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Increased creatinine in 500 ppm females and 1000 ppm males and females, increased BUN in 1000ppm males and females, decreased total protein at ≥ 500 ppm in males and females, decreased albumin in 500 ppm females and 1000 ppm males and females, increased phosphorus and chloride in 500 ppm females and 1000 ppm males and females, increased sodium, total cholesterol, LDL and HDL in 1000 ppm females were considered as test substance related changes. All above changes were consequent to moderate to severe degenerative nephropathy observed in kidneys.
Increased ALP in 1000 ppm females was considered secondary to renal dysfunction and hyperparathyroidism. This change was microscopically correlated with fibrous osteodystrophy in bones.

Endocrine findings:

no effects observed

Description (incidence and severity):

There were no test item-substance related changes in the thyroid hormones at all dose levels tested. Increased TSH levels in 1000 ppm females was considered incidental change as it was not associated with thyroid follicular hypertrophy, decreased T3 and T4 levels.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Increased urine volume at ≥ 500 ppm males and females due to renal dysfunction was considered as test substance-related change.
Increased urine specific gravity in 1000 ppm males and females, decreased urine protein at ≥ 500 ppm males and females, decrease in urine ketones in 1000 ppm males and female were due to significant increase in urine volume due to moderate to severe degenerative nephropathy in kidneys.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

no effects in open field observations

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Administration of Dipotassium hexafluorotitanate (IV) – 01727 via diet for 90 days resulted in significantly increased adrenal gland weights in absolute, organ to body, and organ to brain weights in male rats at 1000 ppm. The increased adrenal gland weights corresponded to diffuse cortical hypertrophy microscopically.

Statistically significantly decreased prostate gland weights were noted in all three measured parameters in males at 1000 ppm, and two of the three parameters in the 250 ppm group, and corresponded microscopically to atrophy.

Females in the 1000 ppm group had statistically significantly decreased ovarian weights were present in two of three measured parameter (absolute and organ to brain weight) and corresponded microscopically to atrophy. Ovarian to body weight was also decreased in females at 1000 ppm, but without statistical significance.

Statistically significantly decreased uterine weights were present in all three measured parameters in 1000 ppm females and corresponded to the microscopic change of atrophy.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Dipotassium hexafluorotitanate (IV) – 01727-related macroscopic findings were noted in the kidneys of six males (Animal 7061, 7062, 7063, 7064, 7069, and 7070) and all eight surviving females (Animals 7071, 7072, 7073, 7074, 7075, 7077, 7079, and 7080) at 1000 ppm. All kidney observations were recorded as ‘pale’ and occasionally with other modifiers such as ‘enlarged’ or ‘irregular surface’. In all cases the macroscopic kidney findings correlated with moderate to severe degenerative nephropathy in males and marked to severe degenerative nephropathy in females.

Other macroscopic observations noted at necropsy included ‘uterus: fluid filled’ in three Group 1 females (Animal 7012, 7013, and 7018), one Group 250 ppm female (Animal 7038), and two 500 ppm females (Animal 7051 and 7057). In all cases, no microscopic correlate was present for the finding, as these changes were considered part of the normal physiologic variation related to estrous cyclicity.

In a single Group 4 female (Animal 7077) macroscopic findings were reported in the small intestines as ‘duodenum: distended, jejunum: distended, and ileum with Peyer’s patches: distended.’ In this same female the stomach was reported as grossly ‘distended.’ No microscopic correlates were present for any of the findings noted in the stomach or small intestine in Animal 7077. The thymus was also reported as ‘small’ in Group 4 female, Animal 7077 and corresponded to moderately decreased lymphocyte cellularity of the thymic cortex.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Dipotassium hexafluorotitanate (IV) – 01727-related microscopic findings were noted in the kidney, parathyroid gland, adrenal gland, stomach (glandular and non-glandular), bones (femur, sternum, and cranium), tooth, prostate gland, seminal vesicles, ovary, and uterus.

Moderate to severe degenerative nephropathy was noted in nine of ten males at 1000 ppm and in all surviving females at 1000 ppm with marked to severe severity and is considered a primary toxic effect of test article administration. The most severe cases of degenerative nephropathy were characterized by a range of changes involving both tubules and glomeruli. The tubules within the cortex and medulla had one or more of the following changes: enlarged epithelial cells or cells with vacuolated cytoplasm (degeneration), tubular dilation with tubules lined by attenuated or even absent epithelium, basophilic cytoplasm with a few mitotic figures (regeneration). Some ectatic tubules contained aggregates of neutrophils (tubulitis), eosinophilic hyaline casts, or karyorrhectic debris. The renal interstitium contained increased acellular pale hyalinized eosinophilic matrix that separated or engulfed tubules (fibrosis) and contained low numbers of scattered lymphocytes and plasma cells. In mild cases, degenerative tubular changes were present in conjunction with the more typical chronic progressive nephropathy changes characterized by small clusters of basophilic tubules with hyalinized basement membranes. In these cases, both diagnoses were recorded.

The kidney changes present resulted in a cascade of secondary findings due to loss of renal function. Diffuse hyperplasia of the parathyroid gland occurred secondarily to the loss of renal function and was present in all ten males and six females at 1000 ppm and ranged from mild to marked. Secondary renal hyperparathyroidism arises from decreased kidney function resulting in elevated phosphorous levels in the blood. The parathyroid gland responds to elevated phosphorous by secreting excess parathyroid hormone to raise calcium levels in the blood to bind phosphate. The result of increased parathyroid hormone is release of calcium from bones. Minimal to moderately decreased bone was noted in the femur of five male rats at 1000 ppm and in seven female rats at 1000 ppm with mild to marked severity. Decreased bone was also noted in the sternum with four males and seven females at 1000 ppm affected. The severity of the finding in the sternum ranged from mild to moderate in males and minimal to moderate in females. Fibrous osteodystrophy was present in the femur and cranium (maxilla) in males and females at 1000 ppm. Specifically, this change occurred in the femur of three males and four females and in the cranium of seven males and eight females. In the femur, fibrous osteodystrophy ranged from minimal to mild in males and from minimal to marked in females. In the cranium, fibrous osteodystrophy ranged in severity from minimal to moderate in males and from minimal to marked in females.

Dipotassium hexafluorotitanate (IV) – 01727-related findings were present in the teeth which were evaluated in nasal sections 1 and 2. Ameloblast degeneration occurred in two, seven, and nine males at ≥ 250 ppm, respectively, and in nine and eight females at 500 and 1000 ppm, respectively. Minimal to marked ameloblast necrosis occurred in four males at 1000 ppm and occurred with minimal to mild severity in five females at 1000 ppm. The finding in ameloblasts was determined to be a primary toxic insult from administration of Dipotassium hexafluorotitanate (IV) – 01727, as these findings have not been reported as a result of secondary hyperparathyroidism and are consistent with other toxic insults to enamel formation in the teeth.
Matrix alteration of the dentin was also present of six males in the 250 ppm, six 500 ppm males, and in nine 1000 ppm group males. The finding was characterized by expansion of the dentin layer which appears pale and disorganized and contains multiple cellular inclusions. In females the change occurred in seven females at 250 ppm, ten females at 500 ppm and in eight females at 1000 ppm. The change in the dentin matrix ranged from minimal to severe in males and females with severity increasing with exposure concentration.

The reproductive organs of both males and females appeared atrophied on both subgross examination and organ weight evaluation. Microscopic examination revealed a normal morphologic appearance; however, the tissue was deemed to be smaller than comparative control tissue. The diagnosis of atrophy is best made subgrossly and with the aid of organ weight data which was statistically significantly decreased in this study for the organs discussed (prostate glands, seminal vesicles, ovary, and uterus).
In males, atrophy of the prostate was diagnosed in two, four, and five males at ≥ 250 ppm, respectively, and ranged from minimal to moderate severity which increased with exposure concentration. The seminal vesicle was also atrophied in two, four, and five males at ≥ 250 ppm, respectively with severity ranging from minimal to moderate and increasing with exposure.
In females, the uterus was noted as atrophied in five, six, and seven females at doses ≥ 250 ppm, respectively; with severity increasing from minimal to marked with increasing exposure. The ovary was atrophied in six, six, and eight females at doses ≥ 250 ppm, respectively, and the severity ranged from minimal to marked with the highest severities noted in the 1000 ppm group.

Other microscopic findings were considered secondary and associated with stress, poor body condition, and periods of anorexia. These findings occurred in the adrenal gland and stomach. In the glandular stomach, minimal to moderate eosinophilic globules were present in three, three, and eight males at ≥ 250 ppm, respectively. This change also occurred in females with one affected at 250 ppm, four at 500 ppm and one at 1000 ppm. Severities for this change were minimal in females at 250 and 500 ppm, and moderate in the single female affected at 1000 ppm. Eosinophilic globules are often present in conjunction with inflammation and mixed cell inflammation of the glandular stomach was noted in three, four, and eight males at ≥ 250 ppm, respectively, and ranged from minimal to marked. Mixed cell inflammation of the glandular stomach was also noted in four, six, and five females at ≥ 250 ppm, respectively, and ranged from minimal to moderate severity. In the non-glandular stomach hyperkeratosis was noted in one, three, and six males and in one, three, and three females at ≥ 250 ppm, respectively. Severities for both sexes ranged from minimal to mild with severity increasing with exposure concentration. The change in the non-glandular stomach were attributed to anorexia.
Diffuse adrenal cortical hypertrophy occurred in all test article treated males, with four affected at 250 ppm, five at 500 ppm and all ten males affected at 1000 ppm. In females this finding was present in one 250 ppm, eight 500 ppm, and eight at 1000 ppm rats. This change occurred with minimal to marked severity in both males and females, with the highest severity occurring in the 1000 ppm group in both sexes. Diffuse adrenal hypertrophy was considered secondary to stress.

Histopathological findings: neoplastic:

no effects observed

Effect levels

1

Dose descriptor:

LOAEL

Effect level:

17 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

gross pathology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

serum/plasma biochemistry

2

Dose descriptor:

LOAEL

Effect level:

12.5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

gross pathology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

serum/plasma biochemistry

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

Hematology

The hematology parameters changes associated with administration of test substance when compared to vehicle control are tabulated below:

Sex	Males			Females
Group	G2	G3	G4	G2	G3	G4
Dietary concentration (ppm)	250	500	1000	250	500	1000
No. of rats	10	10	10	10	10	6
Total Leucocyte count	─	─	↑77%^*	↑34%^*	↑42%^*	↑233%^*
Neutrophil count	─	─	↑141%^*	─	↑83%^*	↑458%^*
Lymphocyte count	─	─	↑61%^*	─	─	↑178%^*
Monocyte count	─	─	↑54%^*	─	─	↑192%^*
Hemoglobin	─	─	↓8%	─	─	↓16%^*
Hematocrit	─	─	↓7%	─	─	↓10%
MCV	─	─	─	─	↓3%^*	↓7%^*
MCH	─	↓7%^*	↓4%	─	↓9%^*	↓15%^*
MCHC	─	↓4%^*	↓4%^*	─	↓5%^*	↓7%^*
RDW	─	─	─	─	─	↑9%

   ↑: Increase     ↓: Decrease   ^*: Statistically significant

   ─:  indicates that values are not considered meaningfully different from controls.

 

Clinical Chemistry

The clinical chemistry parameters changes associated with administration of test substance when compared to vehicle control are tabulated below: 

Sex	Males			Females
Group	G2	G3	G4	G2	G3	G4
Dose (ppm)	250	500	1000	250	500	1000
No. of rats	10	10	10	10	10	6
Creatinine	─	─	↑263%	─	↑17%^*	↑537%^*
Blood urea nitrogen	─	─	↑194%^*	─	─	↑355%^*
Total protein	─	↓8%^*	↓16%^*	─	↓15%^*	↓23%^*
Albumin	─	─	↓7%	─	↓14%^*	↓28%^*
Inorganic Phosphorus	─	─	↑21%^*	─	↑15%^*	↑59%^*
Sodium	─	─	─	─	─	↑3%^*
Chloride	─	─	↑3%^*	─	─	↑5%^*
Total Cholesterol	─	─	─	─	─	↑94%^*
Low Density Lipoprotein	─	─	─	─	─	↑292%^*
High Density Lipoprotein	─	─	─	─	─	↑70%^*
Alkaline Phosphatase	─	─	─	─	─	↑78%^*

↑: Increase     ↓: Decrease   ^*: Statistically significant

   ─:  indicates that values are not considered meaningfully different from controls.

 

Urinalysis

The urinalysis parameters changes associated with administration of test substance when compared to vehicle control are tabulated below: 

Sex	Males			Females
Group	G2	G3	G4	G2	G3	G4
Dose (ppm)	250	500	1000	250	500	1000
No. of rats	10	10	10	10	10	6
Urine volume	─	↑145%^*	↑646%^*	─	↑88%	↑416%^*

↑: Increase     ↓: Decrease   ^*: Statistically significant

   ─:  indicates that values are not considered meaningfully different from controls.

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study and based on the toxicological endpoints evaluated, a no-observed-adverse-effect level (NOAEL) was not reached in males or females for DiPotassium hexafluorotitanate (IV) – 01727. Microscopic findings that were considered adverse, consisting of degenerative nephropathy, ameloblast degeneration and necrosis, dentin alteration, parathyroid gland hyperplasia, decreased bone, fibrous osteodystrophy, and stress-response reproductive organ atrophy were present in both sexes at the lowest dietary level, 250 ppm.

Executive summary:

The objective of this study was to evaluate the potential subchronic toxicity of DiPotassium hexafluorotitanate (IV) - 01727 in male and female rats continuously exposed to the test substance in the diet for at least 90 days (94 days for males and 95 days for females). The study was performed according to OECD Guideline 408 in compliance with GLP.

Four groups of adult CRL: Sprague-Dawley CD^® IGS rats (10/sex/group) were maintained on diets prepared to contain 0, 250, 500, or 1000 ppm of DiPotassium hexafluorotitanate (IV) – 01727.

The neat test substance was considered to be stable under the conditions of storage at PSL over the course of the study.  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 levels.

Body weight and food consumption measurements collected throughout the study were used to calculate the mean overall daily intake of DiPotassium hexafluorotitanate (IV) – 01727, based on expected achievement of target dietary concentrations of 250, 500, and 1000 ppm(Groups 2-4, respectively). Based on target nominal concentrations, intakes of 12.5, 25.6, and 45.9 mg/kg/day in male rats, and 17.0, 32.9, and 61.8 mg/kg/day in female rats were calculated for Groups 2-4, respectively.

Both eyes of all animals on study were examined by focal illumination, slit lamp biomicroscopy, and indirect ophthalmoscopy prior to study initiation, and for all animals on Day 92. All animals were observed for viability, signs of gross toxicity, and behavior changes at least once daily during the study or until death occurred, and weekly for a battery of detailed clinical observations. Body weights were recorded twice during acclimation, including prior to test initiation on Day 0, weekly thereafter until Day 91, and prior to terminal sacrifice. Food consumption measurements were taken to coincide with body weight measurements.  Food efficiency and dietary intake were calculated and reported. Urine samples and clinical pathology samples were collected from all animals at the time of humane sacrifice or prior to terminal sacrifice on Day 95 for males and Day 96 for females.  At terminal sacrifice, a vaginal smear was collected from all females. Gross necropsies were performed on all animals and histological evaluation of selected organs and tissues were performed.

Four Group 4 female rats were humanely sacrificed (between Days 78 and 85) due to degrading general health as a result of body weight loss from reduced food consumption. Adverse test substance-related clinical signs noted over the course of the study include thin appearance, piloerection, irregular respiration, hypoactivity, ano-genital and other staining, ocular and nasal discharge, and urine discoloration. These findings, generally increasing in incidence and/or severity, were predominantly in Group 4 animals.  Alopecia and eschar in Group 4 animals were attributable to decreasing dietary intake of the test substance over time considered to be due to searching behavior for a more palatable food source.

Mean absolute body weight changes in Groups 2-4 over the course of the study were considered to be related to the dietary administration of DiPotassium hexafluorotitanate (IV)-01727, as there was a dose-dependent trend. Body weights were significantly decreased from control Group 1 for Groups 3 and 4 male and female rats, and considered to be adverse in Group 3 and 4 males as well as Group 4 females, since weight loss exceeded 30% by the end of the 90-day dietary administration of the test substance. Body weight gain in Group 3 and 4 male and female rats corresponded with mean absolute body weight, with a lesser degree of statistical significance across dietary intervals.

Mean daily food consumption was decreased in a dose-dependent manner in Groups 2-4, and with statistical significance intermittently across dietary intervals in Group 2 and 3 male and female rats.  Significant decreases in Group 4 male and female food consumption over the course of the study was considered adverse, with decreases in consumption exceeding 40% of control levels by the end of the study. Decreases in food consumption is considered to be a contributing cause in body weight loss, as well as the declining health of Group 4 animals by the end of the study.

Administration of test substance, Dipotassium hexafluorotitanate (IV) – 01727 via diet for at least 90 days at dietary levels of 250, 500 and 1000 ppm to rats resulted in changes in hematology, clinical chemistry and urinalysis parameters. Most significant clinical pathology alterations in the study were attributable to renal dysfunction. In females and males at ≥ 500 ppm MCHC was decreased significantly.  Hemoglobin was significantly decreased in 1000 ppm females, MCV was decreased in all treated groups of females, and MCH was decreased in females at ≥ 500 ppm and males at 500 ppm only.  Hematocrit was also low in males and females but without statistical significance in males and females at 1000 ppm.  These findings represent anemia due to decreased erythropoietin production by the kidney.  Secondary to anemia, reticulocytes were increased in females at ≥ 500 ppm and likely represent a response to decreased erythrocyte production. Likewise, red cell distribution width was increased in 1000 ppm group females due to release of immature red cells from the bone marrow. Other markers of renal dysfunction were increased creatinine in females at ≥ 500 ppm and increased BUN and IPHS in 1000 ppm males and females.  Decreased total protein was present in males and females at ≥ 500 ppm and significantly increased sodium was present in 1000 ppm females. All of the previous findings are indicative of renal dysfunction.  Increased chloride was noted in both males and females at 1000 ppm. All of the findings listed are associated with functional abnormalities of the nephron.  As a result of renal impairment, urine volume was significantly increased in males at ≥ 500 ppm and in 1000 ppm females with specific gravity decreased in males and females at 1000 ppm.

Dipotassium hexafluorotitanate (IV) – 01727-related microscopic findings were present in the kidney, parathyroid gland, bones (femur, sternum, cranium), tooth, prostate gland, seminal vesicles, uterus, ovary, adrenal gland, and stomach (glandular and non-glandular). Of these findings the kidney changes (degenerative nephropathy) and tooth changes (ameloblast degeneration and necrosis) were considered to be direct toxic effects of compound exposure. Other findings in the study were deemed secondary to renal dysfunction (parathyroid gland hyperplasia, decreased bone, and fibrous osteodystrophy) or due to stress, anorexia, and poor body condition (prostate gland and seminal vesicle atrophy, uterine and ovarian atrophy, adrenal gland hypertrophy, glandular stomach eosinophilic globules and inflammation, and hyperkeratosis of the non-glandular stomach). The matrix alteration of dentin noted in the teeth was considered secondary to enamel loss.

Findings considered adverse consisted of degenerative nephropathy, ameloblast degeneration and necrosis, dentin alteration, parathyroid gland hyperplasia, decreased bone, fibrous osteodystrophy, and stress-related reproductive organ atrophy in both males and females. Non-adverse findings were confined to the adrenal gland and stomach.

Under the conditions of this study and based on the toxicological endpoints evaluated, a no-observed-adverse-effect level (NOAEL) was not reached in males or females for DiPotassium hexafluorotitanate (IV) – 01727. Microscopic findings that were considered adverse, consisting of degenerative nephropathy, ameloblast degeneration, and necrosis, dentin alteration, parathyroid gland hyperplasia, decreased bone, fibrous osteodystrophy, and stress-response reproductive organ atrophy were present in both sexes at the lowest dietary level, 250 ppm.