Lithium phosphorodifluoridate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

Adopted 25 June, 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

lithium(1+) (difluorophosphoryl)oxidanide

EC Number:

643-080-8

Cas Number:

24389-25-1

Molecular formula:

LiPO2F2

IUPAC Name:

lithium(1+) (difluorophosphoryl)oxidanide

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc.
- Age at study initiation: Mating was started at 12 weeks of age by housing females with males on a one-to-one basis day and night.
- Weight at study initiation: Females 10 weeks 201.8 to 237.2 g and 11 weeks 214.3 to 251 g.
- Fasting period before study: None
- Housing: Hanging-type stainless wire mesh cages (W226 × D346 × H198 mm).
Quarantine and acclimation periods: 2 to 3 animals/cage, same sex
Mating period: 1 male and 1 female/cage
After group assignment: 1 animal/cage
After mating period: 1 male/cage
Remaining animals: 2 to 3 animals/cage
- Diet (e.g. ad libitum): Pellet diet for experimental animals available ad libitum
- Water (e.g. ad libitum): Well water provided ad libitum and analysed twice yearly.
- Acclimation period: The quarantine and acclimation periods were set from animal receipt to the day of the start of mating, including the quarantine period for 6 days after receipt.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23.3°C to 24.6°C
- Humidity (%): 44.2% to 64.0%
- Air changes (per hr): 10 to 20 times per hour
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycles

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The dosing formulations were prepared under UV cut-off fluorescent light. The test substance was weighed in the glove box where air was replaced with nitrogen gas, and the relative humidity was confirmed to be below 30% with a hygrometer (actual value: 7% to 11%). The frequency of preparation of 1 and 3 mg/mL dosing formulations was prepared
once every 3 to 7 days (permissible range: once or more every 8 days). The 0.3 mg/mL dosing formulation was prepared just before use. The vehicle (olive oil) was used as the dosing formulation for the control group. The dosing formulations after preparation were divided into polypropylene tubes for each dosing day and filled with nitrogen gas.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Standard vehicle
- Concentration in vehicle: 0, 0.3, 1 and 3 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability and homogeneity: The test substance formulations at 0.4, 4, and 100 mg/mL were confirmed to be stable and homogenous under refrigeration, in a dark place, and in a tube filled with nitrogen gas for 8 days in another study.

Determination of test substance concentration and homogeneity in dosing formulations: Determination of the test substance concentration and homogeneity in the 0.3, 1 and 3 mg/mL dosing formulations were confirmed utilising ion chromatography as follows:

Ion chromatography system: ICS-1000 (No.1 DIONEX)
Workstation: Chromeleon
Autosampler: AS50
Column: DIONEX IonPac, AS14A, 4 mm i.d. x 250 mm
Guard column: DIONEX IonPac, AS14A, 4 mm i.d. x 50 mm
Column temperature: 35°C
Mobile phase: 8.0 mM NA2CO3/1.0 mM NaHCO3 (aqueous solution)
Flow rate: 1.2 mL/minute
Injection volume: 25 µL
Suppressor: AERS500 4mm

The test substance were adequately determined in dose formulations at 1 and 3 mg/mL (homogeneity also assessed at upper, middle and lower layers). The actual concentration was analysed as 100% at 1 mg/L and 98% at 3 mg/mL. The homogeneity values were within 10% across the layers, indicating successful formulation procedures were employed.

In the analysis of test substance concentration and homogeneity in the dosing formulations performed at the test site the analysis results of the initially prepared dosing formulations had failed to meet the criteria specified in the study protocol (concentration: within 100 ± 10%, homogeneity: CV value within 10%). The cause was insufficient stirring during analysis. Since the analysis results obtained using newly prepared dosing formulation in re-analysis conducted under sufficient stirring met the criteria and re-prepared dosing formulations were used for administration, it was judged that this incident had no effect on the reliability of the study.

Details on mating procedure:

Mating was started at 12 weeks of age by housing females with males on a one-to-one basis day and night. Successful copulation was confirmed by the presence of a vaginal plug or sperm in a vaginal smear taken in the morning; the day of confirmed copulation was designated as Day 0 of gestation (GD 0).

Duration of treatment / exposure:

The dosing formulations were administered to females for which copulation was confirmed once daily from GD 6 to GD 19 (for 14 days).

Frequency of treatment:

Once daily

Duration of test:

GD 6 to GD 19 (14 days dosing).

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control

Doses / concentrations 2

Dose / conc.:

3 mg/kg bw/day (nominal)

Remarks:

Low dose

Doses / concentrations 3

Dose / conc.:

10 mg/kg bw/day (nominal)

Remarks:

Middle dose

Doses / concentrations 4

Dose / conc.:

30 mg/kg bw/day (nominal)

Remarks:

High dose

No. of animals per sex per dose:

20 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were set based on the results of a reproductive/developmental screening assay in rats (dose level: 0, 3, 10, and 30 mg/kg/day, vehicle: olive oil).
In this study, 3 of 12 dams at 30 mg/kg died from Day 23 of gestation to Day 2 of lactation. For these dams, necropsy and histopathological examination results showed abnormalities in the stomach. In addition, trends toward reduced newborns viability were observed at 30 mg/kg. Therefore, the high dose level was set at 30 mg/kg which was expected to develop some toxicity. The middle and low dose levels were set at 10 and 3 mg/kg, respectively, with a common ratio of about 3. A control group (0 mg/kg) dosed with the vehicle (olive oil) alone was also established.
- Rationale for animal assignment (if not random): Randomly assigned
- Fasting period before blood sampling for (rat) dam thyroid hormones: Animals not fasted.
- Time of day for (rat) dam blood sampling: GD 20 at scheduled necropsy. Timings for necropsy and blood sampling were not reported.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Clinical signs and mortality were observed twice a day (before dosing and after dosing) during the dosing period and once a day during the other periods.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were measured on GDs 0, 3, 6, 9, 12, 15, 18, and 20.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Food consumption was weighed on GDs 0 to 1, 2 to 3, 5 to 6, 8 to 9, 11 to 12, 14 to 15, 17 to 18, and 19 to 20. Feeders containing diet were weighed and set in the animal cages in the morning. On the following morning, the feeders were weighed to calculate the food consumption for each day. The day for food consumption was expressed on GDs 0, 2, 5, 8, 11, 14, 17, and 19.

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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: The thoracoabdominal organs and tissues were immediately examined macroscopically after excision of the ovary and uterus. The thyroid (including parathyroid) was fixed in 10 vol% phosphate buffered formalin solution and preserved.

ORGAN WEIGHTS: Yes
After necropsy, the thyroid (including parathyroid) from all animals (except for a dead dam) were weighed (absolute weight) and the ratio of organ weight to body weight (relative weight) was calculated on the basis of body weight measured on the day of necropsy. Paired organs were measured together.

MICROSCOPIC EXAMINATION: Yes
The thyroid (including parathyroid, unilateral) from all dams was embedded in paraffin, sectioned, stained with hematoxylin and eosin (HE), and examined microscopically. As a result, examination could not be conducted in 2 dams. A
non-pregnant animal (No. 539) was not examined.

While preparing parathyroid tissue specimens from 2 animals (Nos. 515 and 522) of the control and 3 mg/kg groups, respectively, the tissue side could not be placed on the paraffin section and therefore, histological examination could not be performed on that specimen. Because toxicological assessment was possible in parathyroids of the remaining animals (18 or 19 animals) in the same group, this incident had no effect on the reliability of the study.

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

After the uterus weight (including the uterine cervix) was measured, the numbers of corpora lutea, implantations, early resorptions, late resorptions, dead fetuses, and live fetuses were counted and the placentas were observed macroscopically. The live fetuses were individually sexed, observed macroscopically for external anomalies, and weighed for body weights. The live fetuses were euthanized by overdose with thiopental sodium via an intraperitoneal injection. Fetuses allocated for skeletal examination were examined for the internal reproductive organs after euthanasia for confirmation of the position and morphology, as well as for their gender for the second time. As a result, no abnormality was found in the position or morphology of the internal reproductive organs, and the results of gender check corresponded to those obtained in the observation of external reproductive organs.

The uterus of 1 female (No. 539; 3 mg/kg group), in which implantation was not confirmed macroscopically was immersed in 10 vol% ammonium sulfide solution to detect implantation. As a result, this animal was determined to be nonpregnant.

Early resorptions, late resorptions, and dead fetuses were classified based on the following criteria:
Early resorption: Implantation site, placental remnant, and formless embryo.
Late resorption: Showing the form of a fetus, but with remarkable maceration due
to time lapse after death.
Dead fetus: Showing a similar development as a live fetus and with no maceration.

Blood sampling:

For all pregnant animals, the blood was sampled upon necropsy (GD 20).

The animals were anesthetized by tail vein injection of thiopental sodium (30 mg/kg). Approximately 2 mL of blood was collected into a syringe containing heparin sodium from the posterior vena cava. The blood was transferred to a polypropylene tube, immediately cooled on ice, and then promptly centrifuged (approximately 1870 × g, 10 min, approximately 4°C) to obtain plasma (500 μL or more). The collected plasma was stored frozen in a deep freezer (actual range: -81.2°C to -78.1°C, permissible range: -90°C to -65°C).

The total Triiodothyronine (T3), total Thyroxine (T4), and Thyroid-Stimulating Hormone (TSH) plasma concentrations were measured for pregnant animals.

All values of the QC samples were within the range of 100 +/- 25% of nominal value, and variations in duplicate cpm of the standard solutions were within the acceptable range. There were no abnormalities in the procedures for the determination or in the values of the test samples. Therefore, the plasma hormone concentration measurement results were judged to be reliable.

Fetal examinations:

Live fetuses were numbered in order, starting from fetuses near the right and left ovary. Fetuses were allocated, in each sex of each litter, as specimens for skeletal and visceral examinations. Approximately one half of live fetuses in each sex (as specimens for skeletal examination) of each litter was individually identified by tattooing their four limbs and fixed in 70 vol% ethanol. The other half of fetuses (as specimens for visceral examination) were individually identified by a dorsal number inscribed with an oil-based felt pen and fixed in a Bouin’s solution.

- External examinations: Yes: The live fetuses were individually sexed, observed macroscopically for external anomalies, and weighed for body weights.
- Soft tissue examinations: Yes: The fetuses in the control and high dose groups were examined. Fetuses fixed in Bouin’s solution were observed for visceral anomalies by using the razor blade section method of Wilson for the head, neck, and abdomen, and by the microdissection method of Nishimura for the chest. Gender was checked for the second time at observation of the internal reproductive organs. For the low and middle dose groups, only the gender check (observation of internal reproductive organs) was performed. As a result, the results of gender check corresponded to those obtained in the observation of external reproductive organs in all fetuses.
- Skeletal examinations: Yes: The fetuses in the control and high dose groups were examined. Fetuses fixed in 70% ethanol were stained with alizarin red S according to the method of Staples et al. and were observed with a stereomicroscope for skeletal anomalies, variations, and ossification progress. In the ossification progress, the numbers of sternebrae and sacrocaudal vertebral body were regarded as end-points. After skeletal examination was completed, all the skeletal specimens were preserved in 100 vol% glycerin containing thymol.
- Head examinations: Not examined
- Anogenital distance of all live rodent pups: The anogenital distance (AGD: distance between the anus and genital node) was measured for all live fetuses with a micrometer caliper (Mitutoyo). In order to correct variations in measured values due to weight differences among individuals, the AGI (anogenital index)
divided by the cubic root of the body weight on the measurement day was also calculated.

Statistics:

Statistical analysis was performed with a computer system (tsPharma LabSite). As to the sex ratio, SAS 9.4 (SAS Institute Japan [CAC Croit Corporation]) was used. In any case, two-tailed test was used and levels of p<0.01 and p<0.05 were considered significant.

The body weight and food consumption of the non-pregnant female were excluded from the evaluation. Concerning the dead dam, statistical analysis was conducted on the body weight and food consumption while the animal was alive.

Indices:

Sex ratio of live fetuses: Number of live male fetuses / number of live male and female fetuses

Pre-implantation loss index, Post-implantation loss index, Early resorption index, Late resorption index, Dead fetus index, Incidence of fetuses with external anomalies (by type of anomaly), Incidence of fetuses with placental anomalies (by type of anomaly), Incidence of fetuses with skeletal anomalies (by type of anomaly), Incidence of fetuses with skeletal variations (by type of variation) and Incidence of fetuses with visceral anomalies (by type of anomaly).

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In the surviving animals, a decrease in locomotor activity was observed in 1 dam (No. 564) in the 30 mg/kg group from GDs 19 to 20 and soiled fur (chest or abdomen) was observed in 2 dams (Nos. 564 and 577) in the 30 mg/kg group on GD 20.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

In the 30 mg/kg group, 1 dam (No. 572) was found dead on GD 20. In the
clinical observation, no remarkable change was observed in this animal throughout the experimental period. The food consumption was low (0.4 g) at death. In the body weights, necropsy, and histopathological examination of the thyroid, no abnormalities were observed in this animal.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weight in the 30 mg/kg group was significantly lower when compared with the control group on GD 20. Individually in the 30 mg/kg group, a continuous decrease in body weight was observed in 1 dam in the mid to late stages of gestation (No. 564: -13.4 g from GDs 15 to 18 and -23.6 g from GDs 18 to 20) and a decrease in body weight was observed in 4 dams in the late stage of gestation (No. 565 : -14.2 g, No.567 : -7.0 g, No.577 : -38.3 g, No. 580: -1.6 g from GDs 18 to 20).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Mean food consumption in the 30 mg/kg group was significantly lower when compared with the control group from GDs 19 to 20. Individually in the 30 mg/kg group, an apparent decrease in food consumption was continuously observed in 1 dam in the late stage of gestation (No. 564: 2.5 g from GDs 17 to 18; 1.2 g from GDs 19 to 20) and was observed in 5 dams (No. 565: 2.5 g, No. 567: 7.1 g, No. 577: 0.1 g, No. 579: 9.0 g, No. 580: 4.0 g from GDs 19 to 20).

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

no effects observed

Description (incidence and severity):

No statistically significant differences were observed in the total T3, total T4, and TSH between the control and any test substance treatment group.

Tabulated results can be found in the section 'Any other information on results incl. tables'.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No statistically significant difference was observed in either absolute or relative thyroid weight between the control and any test substance treatment group.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No macroscopic abnormalities were observed in any dam.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes were noted in the thyroid (including parathyroid).
In the surviving animals, ectopic thymic tissue and/or ultimobrancial remnant in the
thyroid were observed in each test substance treatment group; however, these were not judged to be treatment related because they are noted occasionally in normal rats and the same changes were also observed in the control group.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Maternal developmental toxicity

Number of abortions:

not specified

Description (incidence and severity):

Not applicable in rats

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

No statistically significant effects observed

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

No statistically significant effects observed

Early or late resorptions:

no effects observed

Description (incidence and severity):

No statistically significant effects observed

Dead fetuses:

no effects observed

Description (incidence and severity):

No statistically significant effects observed

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

not examined

Other effects:

not examined

Details on maternal toxic effects:

Death occurred in 1 dam in the 30 mg/kg group on GD 20. This animal showed no
abnormalites that directly led to the death in clinical signs, body weights, or necropsy although low food consumption was noted; thus, the direct cause of death was unknown. However, the cause of death was judged to be effects of the substance treatment by the following reasons:
(1) the surviving dams in the 30 mg/kg group showed worsening of general
conditions and lower body weight and food consumption and
(2) deaths occurred at 30 mg/kg in the end of the gestation period in reproduction/developmental toxicity screening test of the substance.

In the surviving dams, effects of the substance treatment were observed in the 30 mg/kg group as follows. In the clinical signs, decrease in locomotor activity was observed in 1 dam from GDs 19 to 20 and soiled fur (chest or abdomen) in 2 dams on GD 20. Decreased body weight and food consumption were observed in the late phase of the gestation period (GD 15 or later). No effects of the substance treatment were observed in necropsy including gravid uterus weights,organ weights, or histopathological examination (thyroid gland), or plasma hormone concentration (total T3, total T4, or TSH) at any dose level.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

mortality

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

No statistically significant effects observed

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

No statistically significant effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

No statistically significant effects observed

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

No statistically significant effects observed

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

No statistically significant difference was observed in AGD or AGI between the control and any test substance treatment group.

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

In the 30 mg/kg group, anal atresia and acaudate were observed in 1 fetus (No. 566); however, these were not judged to be treatment related because these were spontaneous changes, which are occasionally seen in normal animals.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Skeletal anomalies:
No anomalies were observed in the 30 mg/kg group.

In the control group, skeletal anomalies were observed in 2 fetuses (1.4%).
With regard to individual types of skeletal anomalies, thoracic hemivertebra was observed in 1 fetus (0.7%) and short 13th rib in 1 fetus (0.7%).

Skeletal variations
No treatment-related changes were observed in the 30 mg/kg group.
Skeletal variations were observed in 23 fetuses (15.1%) and 22 fetuses (14.6%) in the control and 30 mg/kg groups, respectively. With regard to individual types of skeletal variations, the following findings were observed in the control and 30 mg/kg groups: full supernumerary rib in 3 fetuses (1.9%) and 4 fetuses (2.7%), short supernumerary rib in 13 fetuses (8.7%) and 16 fetuses (10.6%), asymmetry of the sternebra in 2 fetuses (1.2%) and 3 fetuses (2.0%), and bipartite ossification of sternebra in 2 fetuses (1.1%) and 2 fetuses (1.3%), respectively. However, no statistically significant differences were observed in their incidences between the control and 30 mg/kg groups. As the findings observed only
in the control group, bipartite ossification thoracic centrum was observed in 1 fetus (0.6%), cervical rib in 3 fetuses (2.1%), and 5 lumbar vertebrae in 1 fetus (0.7%).

Ossification progress
No statistically significant differences were observed in the number of sacrocaudal vertebral bodies or sternebrae between the control and 30 mg/kg groups.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related change were observed in the 30 mg/kg group.

Visceral anomalies were observed in 8 fetuses (5.6%) and 1 fetus (0.8%) in the control and 30 mg/kg groups, respectively. With regard to individual types of visceral anomalies, thymic remnant in the neck was observed in 2 fetuses (1.6%) and 1 fetus (0.8%) in the control and 30 mg/kg groups, respectively. As the findings observed only in the control group, dilated lateral ventricle was observed in 1 fetus (1.0%), dilated renal pelvis in 1 fetus (0.6%), and dilated ureter in 6 fetuses (3.9%).

Other effects:

not examined

Details on embryotoxic / teratogenic effects:

No effects of the test substance were observed in examination of uterine contents (number of corpora lutea, implantations, live fetuses, or resorption, pre- implantation loss, or postimplantation loss) or examination of fetuses (sex ratio, fetal weights, AGD, or visceral, skeletal, external, or placental development).
Therefore, the substance is considered to have had no embryo-fetal lethality, growth retardation, or structural abnormalities in rats in this study.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

30 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

no

Any other information on results incl. tables

Summary or hormone analysis measurements:

Group	TSH (ng/mL)	Total T4 (ng/mL)	Total T3 (ng/mL)
Control	6.04 ± 2.41	15.9 ± 3.3	0.60 ± 0.11
Low dose	5.71 ± 1.93	16.2 ± 3.7	0.62 ± 0.10
Middle dose	6.32 ± 2.28	16.8 ± 3.2	0.58 ± 0.08
High dose	6.52 ± 2.17	16.3 ± 1.8	0.63 ± 0.08

Applicant's summary and conclusion

Conclusions:

In conclusion, the NOAEL of the test substance was considered to be 10 mg/kg for general toxicity and reproductive function in dams, and 30 mg/kg for embryonic/fetal development under the conditions of this study.