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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Jan 2021-Dember 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Also according to GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2022
Report date:
2022

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
June 25th 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Sodium diisobutyldithiophosphinate
EC Number:
236-419-2
EC Name:
Sodium diisobutyldithiophosphinate
Cas Number:
13360-78-6
Molecular formula:
C8H18NaPS2
IUPAC Name:
sodium [bis(2-methylpropyl)(sulfanylidene)-lambda5-phosphanyl]sulfanide
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD®[SD] VAF/Plus®/SPF
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Vital River Laboratory Animal Technology Co., Ltd.
- Age at study initiation: 10-11 weeks
- Weight at study initiation: males 319.59-408.30 g; females 192.67-262.06 g
- Fasting period before study: None
- Housing: individually housed in solid bottom cages (43×30×20 cm3 and 34×18×17 cm3) with corncob bedding (except during mating during which the rats were housed on the basis of one male to one or two females)
- Diet (e.g. ad libitum): rodent feed ad libitum; There were no known contaminants (including heavy metals and pesticides) present in the diet expected to interfere with the test results.
- Water (e.g. ad libitum): reverse-osmosis purified and chlorinated water by a water bottle ad libitum; No contaminants were present at levels that would interfere with the outcome of the study.
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.9-24-5 °C
- Humidity (%): 36.6-65.9%
- Air changes (per hr): 10 to 20
- Photoperiod (hrs dark / hrs light):12/12

IN-LIFE DATES: From: 2021-01-05 To: 2021-02-03

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
1% (w/v) CMC-Na in purified water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amount of test item was added to 70% of the final volume of vehicle while stirring. The formulation was stirred until a homogenous formulation was formed by visual inspection. The formation was brought to its final volume. The formulations intended for dosing were stirred at room temperature for more than 30 minutes before dosing and continuously during dosing.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item is not soluble in water. Previous experimental studies have shown that dose preparations are adequately prepared using 1% CMC.
- Concentration in vehicle: 1% in purified water
- Amount of vehicle (if gavage): 10 ml/kg
- Lot/batch no. (if required): c2030035
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Triplicate samples (one for analysis, the other two for backup) were collected from the middle layer of the mid-dose formulations and control formulations for the first and last preparations. For the low- and high-dose formulations, mean results of the homogeneity results were used as concentration verification and no additional samples were collected.

For homogeneity analysis, triplicate samples (one for analysis, the other two for backup) were collected from the low and high-dose formulations from the top, middle, and bottom layer of formulations for the first and last preparations.

The concentration and homogeneity results of the test item in the dosing formulations met the acceptance criteria, which demonstrated the formulations were accurately prepared and homogenous.
The concentrations of the test item in the dosing formulations were within 94.7 to 114.0% of nominal values.
Homogeneity analysis of the low- and high-dose dosing formulations was performed. The concentrations of the top, middle and bottom of samples were within 94.6% to 110% of nominal values. The relative standard deviations (RSD) of top, middle, and bottom of samples were within 2.6% to 3.9%
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1 male to one or two female(s)
- Length of cohabitation: Until copulation has occurred, max 3d
- After 3 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: Not applicable
- Verification of same strain and source of both sexes: Both sexes were the same strain and obtained from the same supplier at a single time.
- Proof of pregnancy: Vaginal plug or sperm in vaginal smear referred to as Day 0 of pregnancy
- Any deviations from standard protocol: No
Duration of treatment / exposure:
Oral administration from Gestation Day 5 to 20 (included)
Frequency of treatment:
Once daily
Duration of test:
From Gestation Day 5 to 20
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Control
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Remarks:
Low-dose
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Mid-dose
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
High-dose
No. of animals per sex per dose:
25 mated females per group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were selected based on the available OECD 422 study (Combined repeated dose and reproduction/developmental screening in rats, 29 days for males, 43-49 days for females, gavage, CMC as vehicle). Treatment with the test material by oral gavage in male and female Wistar Han rats at dose levels of 30, 100 and 300 mg/kg body weight/day revealed parental toxicity and potential developmental toxicity at 300 mg/kg body weight/day. Based on these results, the No Observed Adverse Effect Level (NOAEL) was 100 mg/kg/day for both parental toxicity and developmental toxicity. No reproductive toxicity was observed up to the highest level tested (300 mg/kg). The NOAEL for reproductive toxicity was ≥ 300 mg/kg. Therefore, 300 mg/kg/day was selected as the highest dose level for this study.

- Rationale for animal assignment (if not random): Random

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily during gestation except for days on which detailed observations are conducted; Unscheduled observations made when a change in condition or behavior was noted.
- Cage side observations: See details of observations in the tables included as attached background material.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before assignment, Once daily on GD0, and from GD5 to GD21.

BODY WEIGHT: Yes
- Time schedule for examinations: Once prior cohabitation, at least once on GD 0, 3, 5, 6, 9, 12, 15, 18, 20, and 21.
The corrected body weight calculated on the day of necropsy (body weight on GD21 minus weight of the gravid uterus).

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21
- Organs examined: Brain, Thyroid glands with parathyroid gland(s), Gross lesions (See details of observations in the tables included as attached background material).
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Blood sampling:
- Plasma: No sampling, gross evaluation performed
- Serum: Yes
- Volume collected: 3 mL
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter (control and high-dose)
- Skeletal examinations: Yes: half per litter (control and high-dose)
- Anogenital distance of all live rodent pups: Yes
- Sex, body weight, crown-rump length of all live rodent pups: Yes
Indices:
Pre-implantation loss was calculated as a percentage from the formula:
(No. of corpora lutea-No. of implantations)/No. of corpora lutea ×100%

Post-implantation loss was calculated as a percentage from the formula:
(No. of implantations-No. of live fetuses)/No. of implantations ×100%

Sex ratios of the live fetuses were calculated as the percentage of males per litter.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 300 mg/kg/day, 13/25 animals showed abnormal yellow stool (GD10 to 20) and unkempt appearance (GD6 to 10). Pale skin, decreased activity and thinness was observed for one of the females found dead.
At 100 mg/kg/day, 1 animal showed abnormal yellow stool (GD13 to 20).
All other clinical signs observed, including alopecia and scab, were considered incidental due to low incidence and/or similar findings in the control animals, and therefore considered not to be test item-related. There were no test item-related changes in the clinical signs at 30 mg/kg/day.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
At 300 mg/kg/day, two females were found dead on Gestation Day (GD) 8 and 7, respectively.
All other animals survived to the scheduled necropsy.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean body weight in animals receiving 300 mg/kg/day decreased (down to -6.89%) relative to the concurrent control group due to decreased body weight gain or body weight loss during GD 5 to GD 9. The body weight gain was comparable to that of the concurrent control after GD 9. At the end of dosing period (GD 21), the body weight at 300 mg/kg/day was comparable with that of the control group.
The mean body weight gain of animals receiving 100 mg/kg/day decreased (-91.29%) relative to the concurrent control group during GD 5- GD6 but recovered and remained comparable to control for the rest of the study period.
All other variations in body weight were small in magnitude or comparable with the concurrent control group. Therefore, they were considered unrelated to test-item treatment. There were no test item-related changes in the body weight, body weight gain, or gravid uterine weight at ≤ 30 mg/kg/day.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The mean food consumption decreased down to -6.1% and -42.1% during GD 5 to GD 9 at 100 and 300 mg/kg/day, respectively. These changes were correlated with the decreased body weight and body weight gain observed over this period. All food consumption was comparable with the control group after GD 9.
All other variations in food consumption were small in magnitude or comparable with the concurrent control group. Therefore, they were considered unrelated to test-item treatment. There were no test item-related changes in the food consumption observed at ≤ 30 mg/kg/day in this study.
Endocrine findings:
no effects observed
Description (incidence and severity):
No test item-related changes of T3, T4 and TSH were observed at ≤100 mg/kg/day in this study. Decreased T3 and T4 (18.45% and 18.41%), and increased TSH (17.76%) were observed at GD 21 when compared with control group for animals treated with 300 mg/kg/day. All above changes were considered test item related but not adverse due to limited change.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no test item-related organ weight changes at scheduled termination.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test item-related macroscopic findings at scheduled termination.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no test item-related microscopic changes in thyroid glands with parathyroid gland (s) at 300 mg/kg/day.
All microscopic findings present in this study were considered background or incidental changes of rats of this age.

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Basis for effect level:
body weight and weight gain
clinical signs
mortality

Results (fetuses)

Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Anogenital distance of all rodent fetuses:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
One of 310 fetuses at 30 mg/kg/day was observed with a mouth malformation (lower jaw and tongue absent). This external malformation was considered a spontaneous finding and not test item-related due to low incidence and within the range of historical control data of this laboratory.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The skeletal variations, including interrupted costal cartilage, unossified hyoid, incomplete ossification of sternebra, unossified sternebra, short supernumerary rib, and dumbbell-shaped and bipartite ossification of thoracic centrum were observed in treated and/or control groups. These findings were considered not test item-related due to similar findings occurring in the treatment and control groups and/or because of the low incidence (the incidence was within the range of historical control data of this laboratory).
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related visceral abnormalities including malformations and variations were observed at ≤ 100 mg/kg/day in this study.

Increases in total incidence of fetuses and litters with dilated renal pelvis (6.1% and 21.74%) were observed at 300 mg/kg/day when compared with the control group (the total incidence of fetuses and litter were 1.3% and 9.09%, respectively). This visceral variation was considered treatment-related but not adverse due to the incidence being only slightly outside the range of historical control data in this laboratory and due to the absence of a dose-response relationship (the data for the animals given 30 and 100 mg/kg/day was comparable to that of the control animals).

The other visceral variations including convoluted ureter and dilated ureter were observed at treated and/or at control groups, and were considered not test item-related due to similar findings being present in the control, and/or low incidence (the incidence was within the range of historical control data in this laboratory).

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
> 300 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
fetal/pup body weight changes
changes in litter size and weights
changes in postnatal survival
external malformations
skeletal malformations
visceral malformations

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Any other information on results incl. tables

See tables available in the attached background document.

Applicant's summary and conclusion

Conclusions:
The test item was administered daily by oral gavage to pregnant rats from gestation day (GD)-5 up to and including GD20 according to the OECD Test Guideline 414 (June 2018) and following GLP principles.

Administration of the test item resulted in deaths of two females at 300 mg/kg/day and non-adverse findings at 100 and 300 mg/kg/day in clinical signs, body weight, food consumption.
There were no toxicologically significant differences, or test item related-changes in the reproductive parameters examined up to and including 300 mg/kg bw/day.

There were no test item effects on the external, visceral and/or skeletal development of foetuses in the study.

The following no-observed-adverse-effect (NOAEL) levels were derived:

NOAEL maternal toxicity: 100mg/kg bw/day
NOAEL embryotoxicity: 300 mg/kg bw/day
NOAEL foetotoxicity: 300 mg/kg bw/day
NOAEL teratogenecity: 300 mg/kg bw/day
Executive summary:

The purpose of this study was to investigate the effects of the test item on embryo-fetal development in rats when administered orally from Gestation Day (GD) 5 up to and including GD 20 and to characterize the dose-response relationship of any observed toxicity according to the OECD TG 414 and following GLP. 


One hundred (100) mated female rats were randomly assigned to 4 groups with 25 animals in the control and treatment groups.  Animals were administered the test item at dose levels of 0 (control formulation, 1% (w/v) CMC-Na in purified water), 30, 100, and 300mg/kg/day by oral gavage once daily from GD 5 to 20. The dose volume was 10 mL/kg. At initiation of mating, male and female rats were approximately 10 to 11 weeks of age and the male animal body weights were 319.59 to 408.30 g and the female animal body weights were 192.67 to 262.06 g.  Females were nulliparous and non-pregnant before cohabitation.


The study animals were observed daily for mortality and clinical signs, and measured for body weight and food consumption during this study.  On GD 21, all study animals were necropsied and examined for gross pathology, organ weights, and histopathology (control and high dose groups).  Blood samples were collected to conduct thyroid stimulating hormone (TSH) and thyroid hormones T3 and T4 analysis from all study animals at the scheduled necropsy. The ovaries and uteri were examined for determination of litter data. Uteri without visible implantation were placed in ammonium sulfide solution for detection of early resorptions. The placenta of live fetuses was examined macroscopically. All the live fetuses were weighed and examined for external abnormalities; their crown-lump lengths were measured and sexes were determined. Approximately 1/2 of the live fetuses in each litter were fixed with modified Davidson’s fixative for soft tissue examination and the remaining fetuses were stained with Alcian Blue Solution and Alizarin Red Solution for subsequent skeletal examination. The visceral and skeletal examination of fetuses was conducted for the high dose and control groups.


The concentrations of the test item in dosing formulations (which were used to dose) were within 94.7 to 114.0% of nominal values. Homogeneity analysis of the low and high-dose formulations was performed. The concentrations of the top, middle and bottom of samples (in dosing formulation which were used to dose) were within 94.6% to 110% of nominal values. The relative standard deviations (RSD) of top, middle, and bottom of samples were within 2.6 to 3.9%.


At 300 mg/kg/day, two females were found dead, one on Gestation Day (GD) 8 and one on GD 7. The cause of death of the two females was uncertain based on these limited macroscopic and microscopic findings, but were considered test item related based on the changes in clinical signs, food consumption, and body weight. All other animals survived to the scheduled necropsy. 


There were 23/25, 21/25, 23/25, and 25/25 pregnant animals in 0, 30, 100, and 300 mg/kg/day groups, respectively.


There were no test item-related changes in the clinical signs at 30 mg/kg/day during the study.  Test item-related clinical signs included abnormal yellow stool and unkempt appearance.  Pale skin, decreased activity, and thinness were also observed in one rat at 300 mg/kg/day on GD 7, this animal was found dead on GD 8. 


There were no test item-related changes in the body weight, body weight gain, or gravid uterine weight at 30 mg/kg/day. The mean body weight of animals receiving 300 mg/kg/day decreased (down to -6.89%) relative to the concurrent control group due to decreased body weight gain or body weight loss during GD 5 to GD 9.  The body weight gain was comparable to that of the concurrent control after GD 9. At the end of dosing period (GD 21), the body weight at 300 mg/kg/day was comparable with that of the control group. The mean body weight gain of animals receiving 100 mg/kg/day decreased (-91.29%) relative to the concurrent control group during GD 5- GD6 but recovered and remained comparable to control for the rest of the study period.


There were no test item-related changes in the food consumption observed at 30 mg/kg/day in this study. The mean food consumption decreased down to -6.1% and -42.1% during GD 5 to GD 9 at 100 and 300 mg/kg/day, respectively. These changes were correlated with the decreased body weight and body weight gain observed over this period. All food consumption was comparable with that of the control group after GD 9.


No test item-related changes of T3, T4 and TSH were observed at ≤100 mg/kg/day in this study. Decreased T3 and T4 (18.45% and 18.41%), and increased TSH (17.76%) were observed at GD 21 when compared with control group for animals treated with 300 mg/kg/day. All changes were considered test item related but not adverse due to limited change.


There were no test item-related changes in the gravid uterine weight, gross pathology, organ weights, and histopathology of the dams.


There were no test item-related changes in litter data (number of corpora lutea, implantation sites, live fetuses, fetal death, dead fetuses, resorptions, pre‑ and post- implantation loss, fetal weight and fetal crown-rump length), sex ratio, placental examination, the anogenital distance or fetal skeletal examinations in this study.  Increases in total incidence of fetuses and litters with dilated renal pelvis (6.1% and 21.74%) were observed at 300 mg/kg/day when compared with the control group (the total incidence for fetuses and litter were 1.3% and 9.09%, respectively).  This visceral variation was considered treatment-related but not adverse due to the incidence being only slightly outside of the range of historical control data in this laboratory and due to the absence of a dose-response relationship (the data for the animals given 30 and 100 mg/kg/day was comparable to that of the control animals).


In conclusion, administration of the test item to pregnant female rats once daily during the period of organogenesis (from Gestation Day 5 to 20) by oral gavage at dose levels of 0, 30, 100, and 300 mg/kg/day resulted in deaths at 300 mg/kg/day and non-adverse findings at 100 mg/kg/day for clinical signs, body weight, and food consumption. The No‑Observed-Adverse-Effect-Level (NOAEL) of the test item for maternal toxicity was considered to be 100 mg/kg/day. 


There was no treatment-related adverse embryo-fetal developmental toxicity (including external, and/or visceral and skeletal malformations, decreases in number of live fetuses and increases in the number of embryonic resorptions and nonviable fetuses) observed in this study. At 300 mg/kg/day, slight increases in total incidence of fetuses and litters with dilated renal pelvis (6.1% and 21.74%) were noted but this effect was not considered as adverse. Based on these results, the NOAEL of the test item for embryo-fetal developmental toxicity was considered to be 300 mg/kg/day (the highest dose tested).