Dihydrogen hexafluorotitanate(2-)

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

Read-across from the salt K2TiF6 to the acid H2TiF6 (see read-across statement in section 13.2):

OECD 421, GLP, rat, feeding:

Under the conditions of this study and the toxicological endpoints evaluated, the no-observed-adverse-effect level (NOAEL) for Dipotassium hexafluorotitanate (IV) - 01727 was 500 ppm for male and female Sprague-Dawley rats.  The NOAEL was determined based on direct test substance-related toxicity. Stress-related, mild to moderate ovarian atrophy is considered to be secondary due to anorexia and body weight loss and not the result of direct toxicity of the test substance.  However, these secondary effects and the constellation of general toxicity microscopic and in-life findings suggest the test substance at 1000 ppm administered in the diet does have an effect on male and female rats’ ability to mate and successfully reproduce.  At levels below 1000 ppm, there is no observed adverse effect on normal adult reproduction and fertility, as well as fetal and pup development.

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Dihydrogen hexafluorotitanate is an inorganic substance which will rapidly dissociate into fluoride, potassium and titanium ions upon dissolution in the environment or the human body, and so, similar toxicological properties can be assumed. However, titanium ions do not remain in solution, only fluoride ions do. The approach follows scenario 1 of the RAAF (ECHA 2017). For details, see attached Read-Across statement in IUCLID chapter 13.2.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source: Dipotassium hexafluorotitanate (CAS 16919-27-0)
Target: Dihydrogen hexafluorotitanate (CAS 17439-11-1)

3. ANALOGUE APPROACH JUSTIFICATION
Since dihydrogen hexafluorotitanate rapidly dissociates into fluoride, protons and titanium ions upon dissolution in aqueous solutions, such as the environment or human body, and only fluoride but not titanium ions will remain in solution, it can be assumed that toxicity (if any) will be driven by the fluoride anion. The non-common dissociation products, potassium, sodium, or just hydrogen ions, are considered not to influence the (eco)toxicological profile of Ti2-F6 to a significant degree. These ions are present in the environment and in the human body in considerable amounts. Potassium and sodium levels influence multiple physiological processes. The body has mechanisms to protect from the intake of harmful concentrations of those.
This read-across is based on the hypothesis that source and target substances have similar (eco)toxicological properties, because they dissociate to common anion. The target substance dihydrogen hexafluorotitanate and the source substance dipotassium hexafluorotitanate form hexafluorotitanate species in aqueous solution. Hexafluorotitanate is considered to be (eco)toxicologically relevant, whereas potassium ions are essential elements and practically non-toxic.
For additional information, please refer to the attached read-across statement in IUCLID chapter 13.2.

4. DATA MATRIX
Please refer to the attached read-across statement in IUCLID chapter 13.2.

Reason / purpose for cross-reference:

read-across source

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Adverse test substance-related clinical signs noted over the course of the study include thin appearance and piloerection. These findings generally were consistent in severity and incidence over the course of the study and were primarily observed in Group 4 females

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Decreases in mean absolute body weight in Groups 2-4 over the course of the study were considered to be attributed to the dietary administration of the test item, with a dose-dependent trend observed. Body weights were statistically significant from control Group 1 for Groups 4 and to a lesser extent Group 3, and interpreted to be adverse in Group 4 males and females, since weight loss exceeded 15% of control levels. Mean body weight gain generally corresponded to significant changes observed in mean absolute body weight, with a lesser degree of statistical significance across dietary intervals in the low and mid dietary levels.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Mean daily food consumption decreased in a dose-dependent manner in males and in females generally across the phases of in-life from pre-mating through lactation. These changes were statistically significant in Group 4 and to a lesser extent Group 3, intermittently across dietary intervals, during female phases. Treatment-related decreases observed in food consumption is considered to be a contributing factor in the lower body weight, declining general health, leading to poor Group 4 male and female reproductive performance.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Mean daily food efficiency was significantly decreased in Group 4 males during the premating period, which consisted of all recorded dietary intervals. Female mean daily food efficiency was significantly decreased upon the initiation of dietary administration of the test item. In general, food efficiency decreased in a dose-dependent manner reaching statistical significance in Groups 3 and 4 during lactation. Overall, the dietary administration of the test item impacted the growth efficiency of male and female rats over the course of the study.

Ophthalmological findings:

not specified

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

no effects observed

Description (incidence and severity):

There were no adverse test substance-related changes in adult mean T3, T4, and TSH thyroid hormone levels. Significant changes in Group 4 male and female T3 and Group 4 female T4 were not associated with expected changes in TSH.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings were noted in the ovary of females and consisted of an increased incidence and severity of atrophy in females at ≥ 250 ppm and markedly decreased numbers of corpora lutea in two females at 1000 ppm. Mild atrophy was also present in two control animals. Sectioning may have had an effect on the amount of ovarian tissue present for evaluation, though the overall trend in incidence and severity with exposure indicates that the ovarian atrophy is test substance-related. The ovarian atrophy was best noted on subgross evaluation, as ovarian tissue appeared normal microscopically with the exception of the two females with decreased corpora lutea.

Histopathological findings: neoplastic:

not examined

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Mean estrus cycle length was comparable between treated and control groups.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

No changes were noted in the testes or epididymides of males, and normal progression of the spermatogenic cycle was present throughout the various stages of spermatogenesis.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

Male mating index was decreased in Group 4 when compare to control Group 1. Male and female fertility, and fecundity indices, were decreased in Group 4 animals, due to a lower number of pregnant females at the highest dietary level. Mating and female mean pre-coital length, were comparable for males and females administered the test item and control animals.
Test item administration resulted in significant decreases in Group 4 corpora lutea counts and implantation sites, compared to control Group 1. These changes are considered to be attributed parental weight loss and general stress and health factors, due to anorexia, and not direct test substance toxicity. There were no changes in gestational length, gestation index, resorptions, and pre-implantation loss.

Earlier stage gestational and parturition indicators that were impacted by the dietary administration of the test item include Group 4 lower litter size and decreased female offspring. Group 4 pup viability was significantly decreased and primarily attributed to the total litter loss of two dams within the first four days of lactation. Parental female weight loss and reduced food consumption was attributed to the decreased ability to nourish and care of litters. Earlier stage gestational and parturition indicators such as live birth index, fetuses dead at birth, and number of stillborn pups were comparable to control Group 1.

Dose descriptor:

NOAEL

Effect level:

500 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

other: general toxicity

Critical effects observed:

no

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Pup clinical observations included: stillborn pup(s) at an incidence of 1/162 for Group 1, and 1/158 for Group 2; found dead pup(s) at an incidence of 1/162 for Group 1, 1/152 for Group 2 and 15/42 for Group 4; pup missing/presumed dead at an incidence of 3/158 for Group 2, 4/152 for Group 3, and 21/42 for Group 4; moderate thin appearance at an incidence of 1/158 for Group 2 and 21/42 for Group 4; unthrifty at an incidence of 1/158 for Group 2 and 4/42 for Group 4; moderate to extreme emaciation at an incidence of 1/158 for Group 2 and 9/42 for Group 4; and no milk-band present for 4/42 pups in Group 4.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Group 4 pup viability was significantly decreased and primarily attributed to the total litter loss of two dams within the first four days of lactation.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean litter weight and litter weight gain were significantly decreased in all treatment groups in a dose dependent manner, with Group 4 noted as the most extreme.

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no adverse test substance-related changes in F1 generation mean T3, T4, and TSH thyroid hormone levels. Significant changes noted in Group 2 T3 and Group 3 T4 levels did not change in a dose-dependent manner and were not in a consistent trending manner across groups in LD14 samples. TSH levels in LD4 and LD14 time points were comparable to control Group 1.

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

effects observed, treatment-related

Description (incidence and severity):

Group 4 male and Group 3 and 4 female ano-genital distance was significantly reduced from control.

Nipple retention in male pups:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

In Group 4, 8 pups from 4 litters were found with no milk bands present.

Histopathological findings:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Due to lower food consumption and decreased body weight in the parental animals, there was increased difficulty in Group 4 dams to nourish and care for their litters. Clinical signs of thin appearance, unthrifty, no milk band present and moderate to extreme emaciation were primarily observed in the pups where the parental in-life endpoints were the most significant. Group 4 male and Group 3 and 4 female ano-genital distance was significantly reduced from control. Corresponding to litter observations, mean litter weight and litter weight gain were significantly decreased in all treatment groups in a dose-dependent manner, with Group 4 noted as the most extreme. There were no adverse test substance-related changes in F1 generation mean T3, T4, and TSH thyroid hormone levels. Significant changes noted in Group 2 T3 and Group 3 T4 levels did not change in a dose-dependent manner and were not in a consistent trending manner across groups in LD14 samples. TSH levels in LD4 and LD14 time points were comparable to control Group 1.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

500 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: general toxicity

Critical effects observed:

no

Reproductive effects observed:

no

Conclusions:

Under the conditions of this study and the toxicological endpoints evaluated, the no-observed-adverse-effect level (NOAEL) for Dipotassium hexafluorotitanate (IV) - 01727 was 500 ppm for male and female Sprague-Dawley rats. The NOAEL was determined based on direct test substance-related toxicity. Stress-related, mild to moderate ovarian atrophy is considered to be secondary due to anorexia and body weight loss and not the result of direct toxicity of the test substance. However, these secondary effects and the constellation of general toxicity microscopic and in-life findings suggest the test substance at 1000 ppm administered in the diet does have an effect on male and female rats’ ability to mate and successfully reproduce. At levels below 1000 ppm, there is no observed adverse effect on normal adult reproduction and fertility, as well as fetal and pup development.

Executive summary:

The objective of this study was to evaluate the potential effects on male and female reproduction and/or development (e.g. gonadal function, mating behavior, conception, development of conceptus’, parturition, and lactation), resulting from repeated exposure to DiPotassium hexafluorotitanate (IV) – 01727 in the diet. The study was performed according to OECD Guideline 421 in compliance with GLP.

96 healthy rats (48 virgin male and 48 virgin female) were selected for the test and  distributed into four groups (12 males and 12 females/dose level).  Dams were maintained on diets at concentrations of 0 (basal diet), 250 (low dose), 500 (intermediate dose), and 1000 ppm (high dose) of Dipotassium hexafluorotitanate (IV) – 01727 for Groups 1-4, respectively.

The neat test substance was determined to be stable under the conditions of storage at PSL over the course of this study.  The test substance was considered to be homogenously distributed in the dose preparations at all study concentrations.  The dietary concentrations were considered to have met the targeted levels.

Males and females received the test and control diets while housed separately for a 14-day pre-mating period, followed by a 14-day co-habitation period.  Upon determination of pregnancy or following the prescribed 14-day mating period, females were removed and placed in  a separate cage, where dosing continued through the gestation period of pregnancy until Day 13 of lactation.  Males were sacrificed following the mating phase and were evaluated for fertility, including spermatogenic stage and epididymal effects.  The males and females were evaluated histopathologically following scheduled sacrifice on Study Day 30 (males) and LD 14 (females).

Animals were observed daily for clinical signs and mortality.  The animals were observed for viability, signs of gross toxicity, and behavioral changes at least once daily during the study, and approximately weekly for a battery of detailed observations.  Individual body weights and food consumption were recorded weekly as prescribed for both sets of animals.  Body weights for females were recorded on GD 0, 7, 14, and 21, and on LD 0, 7, and 14.  Food consumption for males and females was recorded to coincide with body weight collection, with the exception of the mating period.

The mean daily intake of the test substance for male rats fed target dietary concentrations of 250, 500, and 1000 ppm of DiPotassium hexafluorotitanate (IV) – 01727 was calculated to be 0, 11.9, 19.9, and 28.3 mg/kg/day of DiPotassium hexafluorotitanate (IV) – 01727 over a period of 14 days during pre-mating.  During premating, Days 0-14, females were calculated to receive 0, 14.4, 30.3, and 46.9 mg/kg/day for Groups 1-4, respectively. During gestation, Days 0-21, females were calculated to receive 0, 14.5, 26.7, and 76.0 mg/kg/day for Groups 1-4, respectively.  During lactation, Days 0-14, females were calculated to receive 0, 27.6, 44.2, and 59.9 mg/kg/day for Groups 1-4, respectively.  These values were in generally good agreement with targeted test substance exposure concentrations in mg/kg/day.

There were no treatment-related mortalities over the course of this study.  There were seven Group 4 females that were not pregnant following the two-week mating period, and two with a total litter loss.  Adverse test substance-related clinical signs noted over the course of the study include thin appearance and piloerection.  These findings generally were consistent in severity and incidence over the course of the study and were primarily observed in Group 4 females.

Mean estrus cycle length was comparable between treated and control groups.  Decreases in mean absolute body weight in Groups 2-4 over the course of the study were considered to be attributed to the dietary administration of DiPotassium hexafluorotitanate (IV)-01727, with a dose-dependent trend observed.  Body weights were statistically significant from control Group 1 for Groups 4 and to a lesser extent Group 3, and interpreted to be adverse in Group 4 males and females, since weight loss exceeded 15% of control levels.  Mean body weight gain generally corresponded to significant changes observed in mean absolute body weight, with a lesser degree of statistical significance across dietary intervals in the low and mid dietary levels.   Mean daily food consumption   decreased in a dose-dependent manner in males and in females generally across the phases of in-life from pre-mating through lactation.  These changes were statistically significant in Group 4 and to a lesser extent Group 3, intermittently across dietary intervals, during female phases.  Treatment-related decreases observed in food consumption is considered to be a contributing factor in the lower body weight, declining general health, leading to poor Group 4 male and female reproductive performance.  Mean daily food efficiency was significantly decreased in Group 4 males during the premating period, which consisted of all recorded dietary intervals.  Female mean daily food efficiency was significantly decreased upon the initiation of dietary administration of DiPotassium hexafluorotitanate (IV)-01727).  In general, food efficiency decreased in a dose-dependent manner reaching statistical significance in Groups 3 and 4 during lactation.  Overall, the dietary administration of DiPotassium hexafluorotitanate (IV)-01727 impacted the growth efficiency of male and female rats over the course of the study.  There were no adverse test substance-related changes in adult mean T3, T4, and TSH thyroid hormone levels.  Significant changes in Group 4 male and female T3 and Group 4 female T₄ were not associated with expected changes in TSH.  Historically at these levels in a similar general tox dietary administration study of Dipotassium hexafluorotitanate, there were no microscopic findings of thyroid follicular hypertrophy to associate these changes with (PSL Study 58508).  There were no adverse, test substance-related macroscopic or organ weight changes in any of the animals of the parental generation.

Dietary administration of DiPotassium hexafluorotitanate (IV) – 01727 for a minimum of four weeks at exposures of 250, 500, and 1000 ppm did not result in any early deaths, organ weight differences in males or females, or microscopic findings in males. In females, microscopic findings considered related to dietary administration of DiPotassium hexafluorotitanate (IV) – 01727 were present in the ovary and consisted of an increased incidence and severity of mild to moderate atrophy in females at ≥ 250 ppm exposure. Markedly decreased corpora lutea were noted in two females at 1000 ppm.  The ovarian findings present in the current study were morphologically consistent with ovarian changes noted in the 90-day Repeat dose toxicity study with this material(PSL study 58508) and considered to be due to stress-related  factors including palatable food accessibility, and resulting in available energy, anorexia, and body weight loss, and not the result of direct toxicity of the test substance.  The reproductive stress response in female rats can result in a longer estrus cycle, atrophy of reproductive organs, and decreased corpora lutea No changes were noted in the testes or epididymides of males, and normal progression of the spermatogenic cycle was present throughout the various stages of spermatogenesis.

Male mating index was decreased in Group 4 when compared to control Group 1.  Male and female fertility, and fecundity indices, were decreased in Group 4 animals administered Dipotassium hexafluorotitanate (IV) - 01727, due to a lower number of pregnant females at the highest dietary level.  Mating and female mean pre-coital length, were comparable for males and females administered Dipotassium hexafluorotitanate (IV) - 01727 and control animals.

Dipotassium hexafluorotitanate (IV) - 01727 administration resulted in significant decreases in Group 4 corpora lutea counts and implantation sites, compared to control Group 1. These changes are considered to be attributed parental weight loss and general stress and health factors, due to anorexia, and not direct test substance toxicity.  There were no changes in gestational length, gestation index, resorptions, and pre-implantation loss.  Earlier stage gestational and parturition indicators that were impacted by the dietary administration of Dipotassium hexafluorotitanate (IV) – 01727 include Group 4 lower litter size and decreased female offspring.  Group 4 pup viability was significantly decreased and primarily attributed to the total litter loss of two dams within the first four days of lactation.  Parental female weight loss and reduced food consumption was attributed to the decreased ability to nourish and care of litters.  Earlier stage gestational and parturition indicators such as live birth index, fetuses dead at birth, and number of stillborn pups were comparable to control Group 1.

Due to lower food consumption and decreased body weight in the parental animals, there was increased difficulty in Group 4 dams to nourish and care for their litters.  Clinical signs of thin appearance, unthrifty, no milk band present, and moderate to extreme emaciation were primarily observed in the pups where the parental in-life endpoints were the most significant.  Group 4 male and Group 3 and 4 female ano-genital distance was significantly reduced from control.  Corresponding to litter observations, mean litter weight and litter weight gain were significantly decreased in all treatment groups in a dose dependent manner, with Group 4 noted as the most extreme.  There were no adverse test substance-related changes in F1 generation mean T3, T4, and TSH thyroid hormone levels.  Significant changes noted in Group 2 T3 and Group 3 T4 levels did not change in a dose-dependent manner and were not in a consistent trending manner across groups in LD14 samples.  TSH levels in LD4 and LD14 time points were comparable to control Group 1.

Under the conditions of this study and the toxicological endpoints evaluated, the no-observed-adverse-effect level (NOAEL) for Dipotassium hexafluorotitanate (IV) - 01727 was 500 ppm for male and female Sprague-Dawley rats.  The NOAEL was determined based on direct test substance-related toxicity. Stress-related, mild to moderate ovarian atrophy is considered to be secondary due to anorexia and body weight loss and not the result of direct toxicity of the test substance.  However, these secondary effects and the constellation of general toxicity microscopic and in-life findings suggest the test substance at 1000 ppm administered in the diet does have an effect on male and female rats’ ability to mate and successfully reproduce.  At levels below 1000 ppm, there is no observed adverse effect on normal adult reproduction and fertility, as well as fetal and pup development.

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 MAY 2022 to 21 APR 2023

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

Adopted 29 July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3550: Reproduction/Developmental Toxicity Screening Test.

Version / remarks:

EPA 712-C-00-367, July 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The Sprague Dawley® rat is the system of choice because this strain of rat has been widely accepted and used for developmental and reproductive toxicity studies throughout industry.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc.
- Age at study initiation: (P) 11-12 wks
- Weight at study initiation: (P) Males: 462.0-463.1 g; Females: 237.6-239.7 g
- Housing: Animals were individually housed, except during cohabitation and lactation periods. For males throughout in-life and females during pre-mating and mating periods, animals were housed in 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, except during the mating/cohabitation period, when paperboard was placed beneath the cage and replaced daily. During cohabitation, each pair of rats was housed in the male’s cage. Once considered to be successfully mated, female rats were individually housed in polycarbonate shoebox cages (10.5”w x 19”d x 8”h) containing nesting material (Enrich-o’cobs®) with wire mesh lids. Each dam and delivered litter were housed in a common nesting box during the lactation/postnatal period. This nesting material was changed as often as necessary to keep the animals dry and clean.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 22 d
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-23
- Humidity (%): 51-81
- Photoperiod (hrs dark / hrs light): 12 / 12

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.
- 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.

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: 14 d
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After 7 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): Individually in polycarbonate shoebox cages (10.5”w x 19”d x 8”h) containing nesting material (Enrich-o’cobs®) with wire mesh lids.

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:

Males: During the two week pre-mating period, mating, and post-mating periods, at least until the minimum total administration period of 28 days.
Females: During the two week pre-mating period, mating period, as well as the gestation period, and through lactation to LD13, the day prior to terminal sacrifice.

Frequency of treatment:

daily, 7 days/week

Dose / conc.:

0 ppm (nominal)

Dose / conc.:

250 ppm (nominal)

Dose / conc.:

500 ppm (nominal)

Dose / conc.:

1 000 ppm (nominal)

Remarks:

The mean daily intake of the test substance for male rats fed target dietary concentrations of 250, 500, and 1000 ppm of DiPotassium hexafluorotitanate (IV) – 01727 was calculated to be 0, 11.9, 19.9, and 28.3 mg/kg/day of DiPotassium hexafluorotitanate (IV) – 01727 over a period of 14 days during pre-mating. During premating, Days 0-14, females were calculated to receive 0, 14.4, 30.3, and 46.9 mg/kg/day for Groups 1-4, respectively. During gestation, Days 0-21, females were calculated to receive 0, 14.5, 26.7, and 76.0 mg/kg/day for Groups 1-4, respectively. During lactation, Days 0-14, females were calculated to receive 0, 27.6, 44.2, and 59.9 mg/kg/day for Groups 1-4, respectively.

No. of animals per sex per dose:

12

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, PSL Study No. 58507 selected target dietary concentrations of 250, 500, and 1000 ppm (0.025, 0.05, and 0.1%). A no-observed-adverse-effect-level (NOAEL) was expected to be achieved for this study.
- Rationale for animal assignment (if not random): Selected rats were distributed by randomization so that there was no statistically significant difference among group body weight means and standard deviations within a sex.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: All male rats were weighed weekly during the pre-mating, mating, and post-mating periods, as well as prior to terminal sacrifice. All female rats were weighed approximately weekly (during pre-mating and mating and gestation periods), within 24 hours of parturition (LD 0 or LD 1) and approximately weekly thereafter, as well as at terminal sacrifice (LD 14). Females showing no evidence of mating at the end of the cohabitation period were assigned GD 0 and body weights were measured approximately 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

Oestrous cyclicity (parental animals):

Estrus cycles were monitored as a baseline assessment for two weeks prior to the first day of dosing, daily during the pre-mating (beginning on Day 1) and mating periods, until evidence of mating, by vaginal smears to evaluate for regular cyclicity. A vaginal smear was also collected at termination to determine the stage of estrus at sacrifice.

Sperm parameters (parental animals):

Parameters examined in all male parental generations: testis weight, epididymis weight, histological examination was performed on the epididymides, and testes (with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure) of animals from both the control and high dose groups (Groups 1 and 4, respectively).

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes, maximum of 10 pups/litter (5/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals were sacrificed following a minimum treatment of four weeks.
- Maternal animals: All surviving animals were sacrificed on LD 14.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
Histological examination was performed on the ovaries, epididymides, and testes (with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure) of animals from both the control and high dose groups (Groups 1 and 4, respectively). The fixed tissues were trimmed, processed, embedded in paraffin, sectioned with a microtome, placed on glass microscope slides, stained with hematoxylin and eosin and examined by light microscopy. Slide preparation and histological assessment was performed at StageBio.
The following tissues (of all animals sacrificed by design) were weighed wet as soon as possible after dissection to avoid drying:
epididymides, testes, ovaries with oviducts, Cowper’s glands, glans penis, thyroid (post-fixed), Levitor ani plus bulbocavernosus muscle complex

Postmortem examinations (offspring):

SACRIFICE
- The offspring were sacrificed at 14 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: All pups were examined externally for gross abnormalities. The thorax, abdomen, and pelvis of each pup were examined internally.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

Statistics:

-In-Life Data
For all in-life endpoints that are identified as multiple measurements of continuous data over time (e.g. body weight parameters, food consumption, and food efficiency), treatment and control groups were compared using a repeated measures 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). Significant interactions observed between treatment and time as well as main effects may be further analyzed by a post hoc multiple comparisons test (e.g., Dunnett’s test; Dunnett, 1964 and 1980) of the individual treated groups to control.
-Organ Weight Data
If warranted by sufficient group sizes, all endpoints with single measurements of continuous data within groups (e.g. organ weight and relative organ weight) will be evaluated for homogeneity of variances (Bartlett, 1937) and normality. Where homogeneous variances and normal distribution is observed, treatment and control groups will be compared using a one-way analysis of variance (ANOVA). When one-way analysis of variance is significant, a comparison of the treated groups to control will be performed with a multiple comparisons test (e.g., Dunnett’s test; Dunnett, 1964 and 1980). Where variances are considered significantly different, groups will be compared using a non-parametric method (e.g. Kruskal-Wallis non-parametric analysis of variance; Kruskal-Wallis, 1952). When non-parametric analysis of variance is significant, a comparison of treated groups to control will be performed (e.g., Dunn’s test; Dunn, 1964).
-F1 Data
The litter was the experimental unit for evaluation where appropriate. Mean and standard deviations were calculated independently for all test groups. Similarly, mortality and sex was analyzed as number per litter. Inferential comparisons, for quantitative data, were performed on litter size, viability, litter weight, ano-genital distance, number of nipples/areolae

Reproductive indices:

Mating
Male Mating Index = (number of confirmed males mating/number of males cohabitated) x 100
Male Fertility Index = (number of males impregnating females/number of males cohabitated) x 100
Male Fecundity Index = (number of males siring litters/number of males impregnating females) x 100
Female Mating Index = (number of confirmed females mating/number of cohabitated females) x 100
Female Fertility Index = (number of pregnant females/number of cohabitated females) x 100
Female Fecundity = (number of pregnant females/number of females with confirmed mating) x 100

Pregnancy
Implantation Index = (number of implants/number of corpora lutea) x 100
Pre-implant Loss = [(number of corpora lutea – number of implants)/number of corpora lutea)]
Post-implant Loss = [(number of implants – number of viable fetuses)/number of implants] x 100
Gestation Index = (number of females with live born/number of females with evidence of pregnancy) x 100
Live Birth Index = (number of pups born alive/total number of pups born) x 100
Summation Per Group = (∑ pre(post)-implantation loss/litter (%))/number of litters in group
Post Implant Loss/Litter = [(number of dead fetuses + total resorptions/litter)/(number of corpora lutea/litter)] x 100

Offspring viability indices:

Post-Natal
Resorptions = Implant Sites – (Viable + Non-viable Pups)
Stillborn Index = (number of stillborn fetuses/number of pups) x 100
Survival Index = (total number of live pups (at designated time point)/number of pups) x 100
% Male Offspring = (number of male offspring/total number of offspring) x 100
% Female Offspring = (number of female offspring/total number of offspring) x 100

Malformations/Findings
Viable Fetuses Affected/Litter (%)= [(number of viable fetuses affected/litter)/(number of viable fetuses/litter)] x 100
Summation Per Group (%) = (∑ viable fetuses affected/litter (%))/number of litters/group
Neonatal/Postnatal Survival LD 1 and 4 (%/Litter)=[(∑ viable pups/litter on LD (X))/(number of pups born/litter)/number of litters per group] x 100
Postnatal Survival (all other intervals) = [(∑ viable pups /litter at the end of interval N)/(viable pups/litter at the start of interval)/number of litters per group] x 100
Viability Index = (number of viable fetuses LD (X)/No. Live fetuses LD (X)) x 100
Prenatal Mortality / Unaccounted for Sites = (number of former implant sites – number of pups born)/number of former implant sites x 100
Litter Size (Postnatal Day 0) = total number of pups delivered (live and stillborn)/number of dams that delivered
Live Litter Size = total viable pups on LD0/number of litters with viable pups on LD0

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Adverse test substance-related clinical signs noted over the course of the study include thin appearance and piloerection. These findings generally were consistent in severity and incidence over the course of the study and were primarily observed in Group 4 females

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Decreases in mean absolute body weight in Groups 2-4 over the course of the study were considered to be attributed to the dietary administration of the test item, with a dose-dependent trend observed. Body weights were statistically significant from control Group 1 for Groups 4 and to a lesser extent Group 3, and interpreted to be adverse in Group 4 males and females, since weight loss exceeded 15% of control levels. Mean body weight gain generally corresponded to significant changes observed in mean absolute body weight, with a lesser degree of statistical significance across dietary intervals in the low and mid dietary levels.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Mean daily food consumption decreased in a dose-dependent manner in males and in females generally across the phases of in-life from pre-mating through lactation. These changes were statistically significant in Group 4 and to a lesser extent Group 3, intermittently across dietary intervals, during female phases. Treatment-related decreases observed in food consumption is considered to be a contributing factor in the lower body weight, declining general health, leading to poor Group 4 male and female reproductive performance.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Mean daily food efficiency was significantly decreased in Group 4 males during the premating period, which consisted of all recorded dietary intervals. Female mean daily food efficiency was significantly decreased upon the initiation of dietary administration of the test item. In general, food efficiency decreased in a dose-dependent manner reaching statistical significance in Groups 3 and 4 during lactation. Overall, the dietary administration of the test item impacted the growth efficiency of male and female rats over the course of the study.

Ophthalmological findings:

not specified

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

no effects observed

Description (incidence and severity):

There were no adverse test substance-related changes in adult mean T3, T4, and TSH thyroid hormone levels. Significant changes in Group 4 male and female T3 and Group 4 female T4 were not associated with expected changes in TSH.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings were noted in the ovary of females and consisted of an increased incidence and severity of atrophy in females at ≥ 250 ppm and markedly decreased numbers of corpora lutea in two females at 1000 ppm. Mild atrophy was also present in two control animals. Sectioning may have had an effect on the amount of ovarian tissue present for evaluation, though the overall trend in incidence and severity with exposure indicates that the ovarian atrophy is test substance-related. The ovarian atrophy was best noted on subgross evaluation, as ovarian tissue appeared normal microscopically with the exception of the two females with decreased corpora lutea.

Histopathological findings: neoplastic:

not examined

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Mean estrus cycle length was comparable between treated and control groups.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

No changes were noted in the testes or epididymides of males, and normal progression of the spermatogenic cycle was present throughout the various stages of spermatogenesis.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

Male mating index was decreased in Group 4 when compare to control Group 1. Male and female fertility, and fecundity indices, were decreased in Group 4 animals, due to a lower number of pregnant females at the highest dietary level. Mating and female mean pre-coital length, were comparable for males and females administered the test item and control animals.
Test item administration resulted in significant decreases in Group 4 corpora lutea counts and implantation sites, compared to control Group 1. These changes are considered to be attributed parental weight loss and general stress and health factors, due to anorexia, and not direct test substance toxicity. There were no changes in gestational length, gestation index, resorptions, and pre-implantation loss.

Earlier stage gestational and parturition indicators that were impacted by the dietary administration of the test item include Group 4 lower litter size and decreased female offspring. Group 4 pup viability was significantly decreased and primarily attributed to the total litter loss of two dams within the first four days of lactation. Parental female weight loss and reduced food consumption was attributed to the decreased ability to nourish and care of litters. Earlier stage gestational and parturition indicators such as live birth index, fetuses dead at birth, and number of stillborn pups were comparable to control Group 1.

Dose descriptor:

NOAEL

Effect level:

500 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

other: general toxicity

Critical effects observed:

no

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Pup clinical observations included: stillborn pup(s) at an incidence of 1/162 for Group 1, and 1/158 for Group 2; found dead pup(s) at an incidence of 1/162 for Group 1, 1/152 for Group 2 and 15/42 for Group 4; pup missing/presumed dead at an incidence of 3/158 for Group 2, 4/152 for Group 3, and 21/42 for Group 4; moderate thin appearance at an incidence of 1/158 for Group 2 and 21/42 for Group 4; unthrifty at an incidence of 1/158 for Group 2 and 4/42 for Group 4; moderate to extreme emaciation at an incidence of 1/158 for Group 2 and 9/42 for Group 4; and no milk-band present for 4/42 pups in Group 4.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Group 4 pup viability was significantly decreased and primarily attributed to the total litter loss of two dams within the first four days of lactation.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean litter weight and litter weight gain were significantly decreased in all treatment groups in a dose dependent manner, with Group 4 noted as the most extreme.

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no adverse test substance-related changes in F1 generation mean T3, T4, and TSH thyroid hormone levels. Significant changes noted in Group 2 T3 and Group 3 T4 levels did not change in a dose-dependent manner and were not in a consistent trending manner across groups in LD14 samples. TSH levels in LD4 and LD14 time points were comparable to control Group 1.

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

effects observed, treatment-related

Description (incidence and severity):

Group 4 male and Group 3 and 4 female ano-genital distance was significantly reduced from control.

Nipple retention in male pups:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

In Group 4, 8 pups from 4 litters were found with no milk bands present.

Histopathological findings:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Due to lower food consumption and decreased body weight in the parental animals, there was increased difficulty in Group 4 dams to nourish and care for their litters. Clinical signs of thin appearance, unthrifty, no milk band present and moderate to extreme emaciation were primarily observed in the pups where the parental in-life endpoints were the most significant. Group 4 male and Group 3 and 4 female ano-genital distance was significantly reduced from control. Corresponding to litter observations, mean litter weight and litter weight gain were significantly decreased in all treatment groups in a dose-dependent manner, with Group 4 noted as the most extreme. There were no adverse test substance-related changes in F1 generation mean T3, T4, and TSH thyroid hormone levels. Significant changes noted in Group 2 T3 and Group 3 T4 levels did not change in a dose-dependent manner and were not in a consistent trending manner across groups in LD14 samples. TSH levels in LD4 and LD14 time points were comparable to control Group 1.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

500 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: general toxicity

Critical effects observed:

no

Reproductive effects observed:

no

Conclusions:

Under the conditions of this study and the toxicological endpoints evaluated, the no-observed-adverse-effect level (NOAEL) for Dipotassium hexafluorotitanate (IV) - 01727 was 500 ppm for male and female Sprague-Dawley rats. The NOAEL was determined based on direct test substance-related toxicity. Stress-related, mild to moderate ovarian atrophy is considered to be secondary due to anorexia and body weight loss and not the result of direct toxicity of the test substance. However, these secondary effects and the constellation of general toxicity microscopic and in-life findings suggest the test substance at 1000 ppm administered in the diet does have an effect on male and female rats’ ability to mate and successfully reproduce. At levels below 1000 ppm, there is no observed adverse effect on normal adult reproduction and fertility, as well as fetal and pup development.

Executive summary:

The objective of this study was to evaluate the potential effects on male and female reproduction and/or development (e.g. gonadal function, mating behavior, conception, development of conceptus’, parturition, and lactation), resulting from repeated exposure to DiPotassium hexafluorotitanate (IV) – 01727 in the diet. The study was performed according to OECD Guideline 421 in compliance with GLP.

96 healthy rats (48 virgin male and 48 virgin female) were selected for the test and  distributed into four groups (12 males and 12 females/dose level).  Dams were maintained on diets at concentrations of 0 (basal diet), 250 (low dose), 500 (intermediate dose), and 1000 ppm (high dose) of Dipotassium hexafluorotitanate (IV) – 01727 for Groups 1-4, respectively.

The neat test substance was determined to be stable under the conditions of storage at PSL over the course of this study.  The test substance was considered to be homogenously distributed in the dose preparations at all study concentrations.  The dietary concentrations were considered to have met the targeted levels.

Males and females received the test and control diets while housed separately for a 14-day pre-mating period, followed by a 14-day co-habitation period.  Upon determination of pregnancy or following the prescribed 14-day mating period, females were removed and placed in  a separate cage, where dosing continued through the gestation period of pregnancy until Day 13 of lactation.  Males were sacrificed following the mating phase and were evaluated for fertility, including spermatogenic stage and epididymal effects.  The males and females were evaluated histopathologically following scheduled sacrifice on Study Day 30 (males) and LD 14 (females).

Animals were observed daily for clinical signs and mortality.  The animals were observed for viability, signs of gross toxicity, and behavioral changes at least once daily during the study, and approximately weekly for a battery of detailed observations.  Individual body weights and food consumption were recorded weekly as prescribed for both sets of animals.  Body weights for females were recorded on GD 0, 7, 14, and 21, and on LD 0, 7, and 14.  Food consumption for males and females was recorded to coincide with body weight collection, with the exception of the mating period.

The mean daily intake of the test substance for male rats fed target dietary concentrations of 250, 500, and 1000 ppm of DiPotassium hexafluorotitanate (IV) – 01727 was calculated to be 0, 11.9, 19.9, and 28.3 mg/kg/day of DiPotassium hexafluorotitanate (IV) – 01727 over a period of 14 days during pre-mating.  During premating, Days 0-14, females were calculated to receive 0, 14.4, 30.3, and 46.9 mg/kg/day for Groups 1-4, respectively. During gestation, Days 0-21, females were calculated to receive 0, 14.5, 26.7, and 76.0 mg/kg/day for Groups 1-4, respectively.  During lactation, Days 0-14, females were calculated to receive 0, 27.6, 44.2, and 59.9 mg/kg/day for Groups 1-4, respectively.  These values were in generally good agreement with targeted test substance exposure concentrations in mg/kg/day.

There were no treatment-related mortalities over the course of this study.  There were seven Group 4 females that were not pregnant following the two-week mating period, and two with a total litter loss.  Adverse test substance-related clinical signs noted over the course of the study include thin appearance and piloerection.  These findings generally were consistent in severity and incidence over the course of the study and were primarily observed in Group 4 females.

Mean estrus cycle length was comparable between treated and control groups.  Decreases in mean absolute body weight in Groups 2-4 over the course of the study were considered to be attributed to the dietary administration of DiPotassium hexafluorotitanate (IV)-01727, with a dose-dependent trend observed.  Body weights were statistically significant from control Group 1 for Groups 4 and to a lesser extent Group 3, and interpreted to be adverse in Group 4 males and females, since weight loss exceeded 15% of control levels.  Mean body weight gain generally corresponded to significant changes observed in mean absolute body weight, with a lesser degree of statistical significance across dietary intervals in the low and mid dietary levels.   Mean daily food consumption   decreased in a dose-dependent manner in males and in females generally across the phases of in-life from pre-mating through lactation.  These changes were statistically significant in Group 4 and to a lesser extent Group 3, intermittently across dietary intervals, during female phases.  Treatment-related decreases observed in food consumption is considered to be a contributing factor in the lower body weight, declining general health, leading to poor Group 4 male and female reproductive performance.  Mean daily food efficiency was significantly decreased in Group 4 males during the premating period, which consisted of all recorded dietary intervals.  Female mean daily food efficiency was significantly decreased upon the initiation of dietary administration of DiPotassium hexafluorotitanate (IV)-01727).  In general, food efficiency decreased in a dose-dependent manner reaching statistical significance in Groups 3 and 4 during lactation.  Overall, the dietary administration of DiPotassium hexafluorotitanate (IV)-01727 impacted the growth efficiency of male and female rats over the course of the study.  There were no adverse test substance-related changes in adult mean T3, T4, and TSH thyroid hormone levels.  Significant changes in Group 4 male and female T3 and Group 4 female T₄ were not associated with expected changes in TSH.  Historically at these levels in a similar general tox dietary administration study of Dipotassium hexafluorotitanate, there were no microscopic findings of thyroid follicular hypertrophy to associate these changes with (PSL Study 58508).  There were no adverse, test substance-related macroscopic or organ weight changes in any of the animals of the parental generation.

Dietary administration of DiPotassium hexafluorotitanate (IV) – 01727 for a minimum of four weeks at exposures of 250, 500, and 1000 ppm did not result in any early deaths, organ weight differences in males or females, or microscopic findings in males. In females, microscopic findings considered related to dietary administration of DiPotassium hexafluorotitanate (IV) – 01727 were present in the ovary and consisted of an increased incidence and severity of mild to moderate atrophy in females at ≥ 250 ppm exposure. Markedly decreased corpora lutea were noted in two females at 1000 ppm.  The ovarian findings present in the current study were morphologically consistent with ovarian changes noted in the 90-day Repeat dose toxicity study with this material(PSL study 58508) and considered to be due to stress-related  factors including palatable food accessibility, and resulting in available energy, anorexia, and body weight loss, and not the result of direct toxicity of the test substance.  The reproductive stress response in female rats can result in a longer estrus cycle, atrophy of reproductive organs, and decreased corpora lutea No changes were noted in the testes or epididymides of males, and normal progression of the spermatogenic cycle was present throughout the various stages of spermatogenesis.

Male mating index was decreased in Group 4 when compared to control Group 1.  Male and female fertility, and fecundity indices, were decreased in Group 4 animals administered Dipotassium hexafluorotitanate (IV) - 01727, due to a lower number of pregnant females at the highest dietary level.  Mating and female mean pre-coital length, were comparable for males and females administered Dipotassium hexafluorotitanate (IV) - 01727 and control animals.

Dipotassium hexafluorotitanate (IV) - 01727 administration resulted in significant decreases in Group 4 corpora lutea counts and implantation sites, compared to control Group 1. These changes are considered to be attributed parental weight loss and general stress and health factors, due to anorexia, and not direct test substance toxicity.  There were no changes in gestational length, gestation index, resorptions, and pre-implantation loss.  Earlier stage gestational and parturition indicators that were impacted by the dietary administration of Dipotassium hexafluorotitanate (IV) – 01727 include Group 4 lower litter size and decreased female offspring.  Group 4 pup viability was significantly decreased and primarily attributed to the total litter loss of two dams within the first four days of lactation.  Parental female weight loss and reduced food consumption was attributed to the decreased ability to nourish and care of litters.  Earlier stage gestational and parturition indicators such as live birth index, fetuses dead at birth, and number of stillborn pups were comparable to control Group 1.

Due to lower food consumption and decreased body weight in the parental animals, there was increased difficulty in Group 4 dams to nourish and care for their litters.  Clinical signs of thin appearance, unthrifty, no milk band present, and moderate to extreme emaciation were primarily observed in the pups where the parental in-life endpoints were the most significant.  Group 4 male and Group 3 and 4 female ano-genital distance was significantly reduced from control.  Corresponding to litter observations, mean litter weight and litter weight gain were significantly decreased in all treatment groups in a dose dependent manner, with Group 4 noted as the most extreme.  There were no adverse test substance-related changes in F1 generation mean T3, T4, and TSH thyroid hormone levels.  Significant changes noted in Group 2 T3 and Group 3 T4 levels did not change in a dose-dependent manner and were not in a consistent trending manner across groups in LD14 samples.  TSH levels in LD4 and LD14 time points were comparable to control Group 1.

Under the conditions of this study and the toxicological endpoints evaluated, the no-observed-adverse-effect level (NOAEL) for Dipotassium hexafluorotitanate (IV) - 01727 was 500 ppm for male and female Sprague-Dawley rats.  The NOAEL was determined based on direct test substance-related toxicity. Stress-related, mild to moderate ovarian atrophy is considered to be secondary due to anorexia and body weight loss and not the result of direct toxicity of the test substance.  However, these secondary effects and the constellation of general toxicity microscopic and in-life findings suggest the test substance at 1000 ppm administered in the diet does have an effect on male and female rats’ ability to mate and successfully reproduce.  At levels below 1000 ppm, there is no observed adverse effect on normal adult reproduction and fertility, as well as fetal and pup development.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

19.9 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

Read-across from the salt K2TiF6 to the acid H2TiF6 (see read-across statement in section 13.2):
Key, OECD 414, GLP, rat, oral via diet, NOAEL = 32.2 mg/kg bw/d

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Dihydrogen hexafluorotitanate is an inorganic substance which will rapidly dissociate into fluoride, potassium and titanium ions upon dissolution in the environment or the human body, and so, similar toxicological properties can be assumed. However, titanium ions do not remain in solution, only fluoride ions do. The approach follows scenario 1 of the RAAF (ECHA 2017). For details, see attached Read-Across statement in IUCLID chapter 13.2.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source: Dipotassium hexafluorotitanate (CAS 16919-27-0)
Target: Dihydrogen hexafluorotitanate (CAS 17439-11-1)

3. ANALOGUE APPROACH JUSTIFICATION
Since dihydrogen hexafluorotitanate rapidly dissociates into fluoride, protons and titanium ions upon dissolution in aqueous solutions, such as the environment or human body, and only fluoride but not titanium ions will remain in solution, it can be assumed that toxicity (if any) will be driven by the fluoride anion. The non-common dissociation products, potassium, sodium, or just hydrogen ions, are considered not to influence the (eco)toxicological profile of Ti2-F6 to a significant degree. These ions are present in the environment and in the human body in considerable amounts. Potassium and sodium levels influence multiple physiological processes. The body has mechanisms to protect from the intake of harmful concentrations of those.
This read-across is based on the hypothesis that source and target substances have similar (eco)toxicological properties, because they dissociate to common anion. The target substance dihydrogen hexafluorotitanate and the source substance dipotassium hexafluorotitanate form hexafluorotitanate species in aqueous solution. Hexafluorotitanate is considered to be (eco)toxicologically relevant, whereas potassium ions are essential elements and practically non-toxic.
For additional information, please refer to the attached read-across statement in IUCLID chapter 13.2.

4. DATA MATRIX
Please refer to the attached read-across statement in IUCLID chapter 13.2.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

reference to other study

Remarks:

dose range-finding study

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

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.

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

Abnormalities:

no effects observed

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.

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

Abnormalities:

no effects observed

Developmental effects observed:

no

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.

Reference 2

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

Reason / purpose for cross-reference:

reference to other study

Remarks:

dose range-finding study

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

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

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)

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.

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

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

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.

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

Abnormalities:

no effects observed

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.

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

Abnormalities:

no effects observed

Developmental effects observed:

no

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.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

32.2 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Read-across is applied from the salt K2TiF6 to the acid H2TiF6. This read-across is justified based on the chemical similarity of these two substances. The salt K2TiF6 is soluble in water and dissociates into inorganic ions. H2TiF6 is an aqueous solution, also containing the respective ions. Under physiological conditions, both substances would yield the same ionic species, which would be responsible for any toxicological or physiological effect (see IUCLID section 13.2 for further information).

OECD 421, GLP

The objective of this study was to evaluate the potential effects on male and female reproduction and/or development (e.g. gonadal function, mating behavior, conception, development of conceptus’, parturition, and lactation), resulting from repeated exposure to DiPotassium hexafluorotitanate (IV) – 01727 in the diet. The study was performed according to OECD Guideline 421 in compliance with GLP.

96 healthy rats (48 virgin male and 48 virgin female) were selected for the test and distributed into four groups (12 males and 12 females/dose level).  Dams were maintained on diets at concentrations of 0 (basal diet), 250 (low dose), 500 (intermediate dose), and 1000 ppm (high dose) of Dipotassium hexafluorotitanate (IV) – 01727 for Groups 1-4, respectively.

The neat test substance was determined to be stable under the conditions of storage at PSL over the course of this study.  The test substance was considered to be homogenously distributed in the dose preparations at all study concentrations. The dietary concentrations were considered to have met the targeted levels.

Males and females received the test and control diets while housed separately for a 14-day pre-mating period, followed by a 14-day co-habitation period. Upon determination of pregnancy or following the prescribed 14-day mating period, females were removed and placed in  a separate cage, where dosing continued through the gestation period of pregnancy until Day 13 of lactation. Males were sacrificed following the mating phase and were evaluated for fertility, including spermatogenic stage and epididymal effects. The males and females were evaluated histopathologically following scheduled sacrifice on Study Day 30 (males) and LD 14 (females).

Animals were observed daily for clinical signs and mortality. The animals were observed for viability, signs of gross toxicity, and behavioral changes at least once daily during the study, and approximately weekly for a battery of detailed observations. Individual body weights and food consumption were recorded weekly as prescribed for both sets of animals. Body weights for females were recorded on GD 0, 7, 14, and 21, and on LD 0, 7, and 14. Food consumption for males and females was recorded to coincide with body weight collection, with the exception of the mating period.

The mean daily intake of the test substance for male rats fed target dietary concentrations of 250, 500, and 1000 ppm of DiPotassium hexafluorotitanate (IV) – 01727 was calculated to be 0, 11.9, 19.9, and 28.3 mg/kg/day of DiPotassium hexafluorotitanate (IV) – 01727 over a period of 14 days during pre-mating. During premating, Days 0-14, females were calculated to receive 0, 14.4, 30.3, and 46.9 mg/kg/day for Groups 1-4, respectively. During gestation, Days 0-21, females were calculated to receive 0, 14.5, 26.7, and 76.0 mg/kg/day for Groups 1-4, respectively. During lactation, Days 0-14, females were calculated to receive 0, 27.6, 44.2, and 59.9 mg/kg/day for Groups 1-4, respectively. These values were in generally good agreement with targeted test substance exposure concentrations in mg/kg/day.

There were no treatment-related mortalities over the course of this study. There were seven Group 4 females that were not pregnant following the two-week mating period, and two with a total litter loss. Adverse test substance-related clinical signs noted over the course of the study include thin appearance and piloerection. These findings generally were consistent in severity and incidence over the course of the study and were primarily observed in Group 4 females.

Mean estrus cycle length was comparable between treated and control groups. Decreases in mean absolute body weight in Groups 2-4 over the course of the study were considered to be attributed to the dietary administration of DiPotassium hexafluorotitanate (IV)-01727, with a dose-dependent trend observed. Body weights were statistically significant from control Group 1 for Groups 4 and to a lesser extent Group 3, and interpreted to be adverse in Group 4 males and females, since weight loss exceeded 15% of control levels. Mean body weight gain generally corresponded to significant changes observed in mean absolute body weight, with a lesser degree of statistical significance across dietary intervals in the low and mid dietary levels. Mean daily food consumption decreased in a dose-dependent manner in males and in females generally across the phases of in-life from pre-mating through lactation. These changes were statistically significant in Group 4 and to a lesser extent Group 3, intermittently across dietary intervals, during female phases. Treatment-related decreases observed in food consumption is considered to be a contributing factor in the lower body weight, declining general health, leading to poor Group 4 male and female reproductive performance. Mean daily food efficiency was significantly decreased in Group 4 males during the premating period, which consisted of all recorded dietary intervals.  Female mean daily food efficiency was significantly decreased upon the initiation of dietary administration of DiPotassium hexafluorotitanate (IV)-01727). In general, food efficiency decreased in a dose-dependent manner reaching statistical significance in Groups 3 and 4 during lactation. Overall, the dietary administration of DiPotassium hexafluorotitanate (IV)-01727 impacted the growth efficiency of male and female rats over the course of the study. There were no adverse test substance-related changes in adult mean T3, T4, and TSH thyroid hormone levels. Significant changes in Group 4 male and female T3 and Group 4 female T4 were not associated with expected changes in TSH. Historically at these levels in a similar general tox dietary administration study of Dipotassium hexafluorotitanate, there were no microscopic findings of thyroid follicular hypertrophy to associate these changes with (PSL Study 58508).  There were no adverse, test substance-related macroscopic or organ weight changes in any of the animals of the parental generation.

Dietary administration of DiPotassium hexafluorotitanate (IV) – 01727 for a minimum of four weeks at exposures of 250, 500, and 1000 ppm did not result in any early deaths, organ weight differences in males or females, or microscopic findings in males. In females, microscopic findings considered related to dietary administration of DiPotassium hexafluorotitanate (IV) – 01727 were present in the ovary and consisted of an increased incidence and severity of mild to moderate atrophy in females at ≥ 250 ppm exposure. Markedly decreased corpora lutea were noted in two females at 1000 ppm. The ovarian findings present in the current study were morphologically consistent with ovarian changes noted in the 90-day Repeat dose toxicity study with this material(PSL study 58508) and considered to be due to stress-related  factors including palatable food accessibility, and resulting in available energy, anorexia, and body weight loss, and not the result of direct toxicity of the test substance. The reproductive stress response in female rats can result in a longer estrus cycle, atrophy of reproductive organs, and decreased corpora lutea. No changes were noted in the testes or epididymides of males, and normal progression of the spermatogenic cycle was present throughout the various stages of spermatogenesis.

Male mating index was decreased in Group 4 when compare to control Group 1. Male and female fertility, and fecundity indices, were decreased in Group 4 animals administered Dipotassium hexafluorotitanate (IV) - 01727, due to a lower number of pregnant females at the highest dietary level. Mating and female mean pre-coital length, were comparable for males and females administered Dipotassium hexafluorotitanate (IV) - 01727 and control animals.

Dipotassium hexafluorotitanate (IV) - 01727 administration resulted in significant decreases in Group 4 corpora lutea counts and implantation sites, compared to control Group 1. These changes are considered to be attributed parental weight loss and general stress and health factors, due to anorexia, and not direct test substance toxicity. There were no changes in gestational length, gestation index, resorptions, and pre-implantation loss. Earlier stage gestational and parturition indicators that were impacted by the dietary administration of Dipotassium hexafluorotitanate (IV) – 01727 include Group 4 lower litter size and decreased female offspring. Group 4 pup viability was significantly decreased and primarily attributed to the total litter loss of two dams within the first four days of lactation. Parental female weight loss and reduced food consumption was attributed to the decreased ability to nourish and care of litters. Earlier stage gestational and parturition indicators such as live birth index, fetuses dead at birth, and number of stillborn pups were comparable to control Group 1.

Due to lower food consumption and decreased body weight in the parental animals, there was increased difficulty in Group 4 dams to nourish and care for their litters. Clinical signs of thin appearance, unthrifty, no milk band present, and moderate to extreme emaciation were primarily observed in the pups where the parental in-life endpoints were the most significant. Group 4 male and Group 3 and 4 female ano-genital distance was significantly reduced from control. Corresponding to litter observations, mean litter weight and litter weight gain were significantly decreased in all treatment groups in a dose dependent manner, with Group 4 noted as the most extreme. There were no adverse test substance-related changes in F1 generation mean T3, T4, and TSH thyroid hormone levels. Significant changes noted in Group 2 T3 and
Group 3 T4 levels did not change in a dose-dependent manner and were not in a consistent trending manner across groups in LD14 samples.  TSH levels in LD4 and LD14 time points were comparable to control Group 1.

Under the conditions of this study and the toxicological endpoints evaluated, the no-observed-adverse-effect level (NOAEL) for Dipotassium hexafluorotitanate (IV) - 01727 was 500 ppm for male and female Sprague-Dawley rats. The NOAEL was determined based on direct test substance-related toxicity. Stress-related, mild to moderate ovarian atrophy is considered to be secondary due to anorexia and body weight loss and not the result of direct toxicity of the test substance. However, these secondary effects and the constellation of general toxicity microscopic and in-life findings suggest the test substance at 1000 ppm administered in the diet does have an effect on male and female rats’ ability to mate and successfully reproduce. At levels below 1000 ppm, there is no observed adverse effect on normal adult reproduction and fertility, as well as fetal and pup development.

 

OECD 414, GLP

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 comparabale 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.

Justification for classification or non-classification

No adverse effects on reproduction or development were observed. Thus, no classification is proposed according to Regulation (EC) No 1272/2008.