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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:
10 August 2021 to 21 December 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021
Reference Type:
study report
Title:
Unnamed
Year:
2022
Report date:
2022

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
25 June 2018
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: “Regulation on Test Methods for Chemical Substances” Notification No. 2020-46 National Institute of Environmental Research, Republic of Korea
Version / remarks:
03 November 2020
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2-phenoxyethyl isobutyrate
EC Number:
203-127-1
EC Name:
2-phenoxyethyl isobutyrate
Cas Number:
103-60-6
Molecular formula:
C12H16O3
IUPAC Name:
2-phenoxyethyl 2-methylpropanoate
Test material form:
liquid
Remarks:
Colourless, clear liquid
Details on test material:
- Storage Conditions: At room temperature.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD(SD), SPF
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: Males were 10 weeks old. Females were 9 weeks old at receipt and 10 - 11 weeks old at start of administration.
- Weight at study initiation: Males were 305.5 – 381.1 g. Females were 195.8 – 234.0 g at receipt and 250 - 311.7 g at start of administration.
- Fasting period before study: No
- Housing: Animals were housed in stainless wire mesh cages, 260 W × 350 D × 210 H (mm) (excluding use of polycarbonate cages). Then from GD 16 to necropsy animals were housed in polycarbonate cages, 260 W × 420 D × 180 H (mm). One – three animals were housed per cage during the quarantine acclimation period and females were housed individually during the gestation period.
- Diet: Powder feed rodent chow was placed in feeders and provided ad libitum.
- Water: Public tap water was filtered and irradiated by a UV water steriliser and then provided ad libitum.
- Acclimation period: 7 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.4 – 22.8 °C.
- Humidity (%): 55.4 – 73.2 % (relative)
- Air changes (per hr): 10 – 15 clean, fresh, filtered air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hour light/ dark cycle (7 AM to 7 PM via automated timer), 150 - 300 Lux.

IN-LIFE DATES:
From: 10 August 2021
To: 12 September 2021

Administration / exposure

Route of administration:
oral: feed
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amount of the test material was weighed and placed in a container. The required amount of vehicle was added and mixed using a vortex mixer until dissolved.
The required amount of powder feed except for the amount of the test material was weighed. The required amount of the test material formulation and a small amount of powder feed were mixed in a container. The mixture was then placed in a ball mill and residual powder feed was added and mixed using the ball mill for approximately 5 – 10 minutes to yield the desired concentration.
The required amounts of corn oil and powder feed were weighed on an electronic balance and mixed using the ball mill for approximately 5 – 10 minutes for the control group.

DIET PREPARATION
- Storage temperature of food: The dosing feed was stored under refrigeration conditions for use within 14 days and used at room temperature (1 – 30 °C) for 6 days.

VEHICLE
- Concentration in vehicle: 10 mL of vehicle for 1 kg of powder feed containing test material.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis for homogeneity and stability of the dosing formulations was conducted. The homogeneity and stability at room temperature (1 – 30 °C) for 3 and 6 days and under refrigeration for 8 and 14 days of dosing formulations were confirmed for the dose levels of 0.04 and 1.5 %.
Samples were taken three times from the middle of each dosing formulation and analysed for verification of dose level concentration prior to the first dosing. As a result, the accuracies at 0.25, 0.5 and 1.0 % were 97.92, 95.60 and 96.30 % prior to dosing, respectively. These results were within the acceptable range (range: ± 15 % of nominal values).

DETAILS ON ANALYTICAL METHODS
- Standard Substance
The test substance was used as the standard substance for the comparison of analytical results.

- Vehicle
Corn oil

- Analytical Instrumentation and Equipment
Gas Chromatography (GC):
System: GC-2010 series, Shimadzu Corp., Japan
Main body: GC-2010AF
Autosampler: AOC-20s
Autoinjector: AOC-20i
Detector: Flame Ionization Detector (FID)
Data processor: GC solution ver. 2.3

- Solution for Dilution
tert-Butyl methyl ether was used as the solution for dilution.

- Preparation of the Standard Solutions
Standard stock solution of the highest concentration was prepared by weighing 0.1 g of the standard substance in a 100 mL volumetric flask and diluting with the solution for dilution (concentration: 0.1 %).
Other standard solutions were prepared by diluting this standard stock solution of high concentration with the solution for dilution yielding the concentrations of 0.001, 0.002, 0.005, 0.01 and 0.02 %.

- Quality Control (QC) Sample
The standard solution at the concentration of 0.005 % was used as a QC sample.

- Preparation of Dosing Formulations
The required amount (14.996 g) of the test material was weighed and placed in a container. The required amount of vehicle (10 mL of vehicle for 1 kg of powder feed containing test material) was added and mixed using a vortex mixer until dissolved. The required amount (1 000.04 and 985.48 g) of powder feed except for the amount of the test material was weighed. The required amount of the test material formulation and a small amount of powder feed were mixed in a bottle. The mixture was then placed in a ball mill and residual powder feed was added and mixed using the ball mill for approximately 5 – 10 minutes to yield the desired concentration. The dosing formulations at concentrations of 0.04 and 1.5 % were prepared for analyses for homogeneity and stability at room temperature (1 – 30 °C) for 3, 6 and 8 days and under refrigeration (at 2 − 8 °C) for 8 and 14 days.

- GC Analytical Conditions
Column: ZB-1ms column (20 m × 0.18 mm, 0.18 μm, Phenomenex, U.S.A); Flow: 1.61 mL/min; Oven: A temperature of 60 °C with a hold time of 3 minutes and a temperature of 240 °C with a hold time of 3 minutes at a rate of 9 °C per minute.
Injector: Temperature 200 °C; Split: 70:1; Injection volume: 1 μL
Carrier gas: Nitrogen
Gas flow rate: Makeup gas (Nitrogen): 30 mL/min; Hydrogen gas: 40 mL/min; Air: 400 mL/min
Run time: 26 min
Detector Temperature: 275 °C

- Treatment of the Dosing Formulations
The required amount (1 g) of the samples was taken from each part of the dosing formulations. The 0.04 and 1.5 % samples were diluted with the solution for dilution by applying dilution factors of 10 and 100, respectively, and filtered through a 0.45 μm PTFE-H syringe. Then, 1 μL was injected into the GC within the concentration range of calibration samples and analysed for homogeneity and stability at room temperature (1 – 30 °C) for 3, 6 and 8 days and under refrigeration (at 2 − 8 °C) for 8 and 14 days.

- Calculations
Linear regression
The calibration curve was constructed by plotting the peak area of the standard solution versus the concentration of standard solution. The linear regression equation was:

y = ax + b

Where:
y = Peak area generated by each standard solution
x = Concentration of the analyte in each standard solution
a = Slope of the calibration curve
b = Y-intercept of the calibration curve

Sample concentration
The measured concentration of the analyte in the samples was determined using the calibration curve (y = ax + b) and by solving the x variable:

x = [(y - b) / a] x d

Where:
y = Peak area generated by the sample
x = Measured concentration of the analyte in each sample
a = Slope of the calibration curve
b = Y-intercept of the calibration curve
d = Dilution factor

Precision, accuracy and variation were calculated as follows:
Precision (%) = (Standard deviation of determined concentrations / Mean of determined concentrations) × 100
Accuracy (%) = (Mean of determined concentrations / Nominal concentration) × 100
Variation (%) = [(Mean value after storage – Mean value of initial concentrations) / Mean value of initial concentrations] × 100

ANALYSES OF THE DOSING FORMULATIONS
- System Suitability
The QC sample was analysed 6 times to evaluate the precision. The values were considered to be acceptable when the precision of peak area and retention time were 3 % or less.

-Linearity
The standard solutions of concentrations from 0.001 to 0.02 % were analysed. The correlation coefficient between the concentration and the peak area of the standard solutions was calculated. The calibration curve was used to evaluate the results of stability analysis of the dosing formulations.
The results were judged to be acceptable when the correlation coefficient (r) of the calibration curve was more than 0.9950 and the accuracy of each concentration was in the range of 85 − 115 % of theoretical values.

- Specificity
The solutions for dilution and vehicle were analysed to check for interfering peaks at the retention time of the standard substance to confirm assay specificity.
The results were judged to be acceptable when a sufficient number of peaks of the standard substance was observed and there were no interfering peaks at the same retention time as the standard substance.

- Intra-day Variation
The samples from the initial point of the dosing formulations of each batch were collected in triplicate and analysed once each.
The results were judged to be acceptable when the precision was 10 % or less and the accuracy was in the range of 80 – 120 % of theoretical values.

- Stability of the Stock Solution
The stock solution was stored under refrigeration for 3 and 8 days. Samples were prepared in triplicate at the concentration of QC sample and analysed.
The results were judged to be acceptable when the precision was 10 % or less and the accuracy was in the range of 85 − 115 % of theoretical values.

- Stability in Autosampler
The samples analysed for intra-day variation were allowed to stand for a certain period of time in the autosampler and re-analysis conducted to check for stability in the autosampler.
The results were judged to be acceptable when the precision was 10 % or less and the variation of stability was within ± 15 % of the initial concentration.

- Homogeneity
The dosing formulations were randomly divided into 3 points, collected in triplicate from each point and analysed once per sample. The result of the initial point was used as a result of intra-day variation.
The results were judged to be acceptable when the precision was 10 % or less and the accuracy was in the range of 80 − 120 % of theoretical values.

- Stability
Stability following 3 ,6 and 8 days of standing at room temperature (1 – 30 °C): All dosing formulations were allowed to stand for 3, 6 and 8 days at room temperature and three replicate samples from the initial point of dosing formulations of each batch were analysed for stability. The stability of the low dose formulation at room temperature for 8 days was outside the acceptance criteria, thus the analysis for stability at room temperature for 14 days was not performed.
The results of intra-day variation were used as the results of analysis performed immediately after preparation.
The results were judged to be acceptable when the precision was 10 % or less and the variation of stability was within ± 15 % of the initial concentration.
Stability following 8 and 14 days storage under refrigeration: All dosing formulations were stored under refrigeration for 8 and 14 days and three replicate samples from the initial point of dosing formulations of each batch were analysed for stability.
The results of intra-day variation were used as the results of analysis performed immediately after preparation.
The results were judged to be acceptable when the precision was 10 % or less and the variation of stability was within ± 15 % of the initial concentration.

- Quality Control (QC) Sample
The QC sample was analysed 3 times after the completion of other analysis to confirm the condition of equipment and analytical method.
The results were judged to be acceptable when the precision was 10 % or less and the accuracy was in the range of 85 − 115 % of theoretical values.

RESULTS
- System Suitability: The precision results of peak area and retention time of 0.005 % QC sample analysed 6 times were 1.05 and 0.08 %, respectively.
- Linearity: The correlation coefficient of the calibration curves in the concentration range of 0.001 to 0.02 % of the standard solution was 1.0000 and the accuracy of each concentration was 98.98 – 104.10 % on Day 0, 97.40 – 101.10 % on Day 3 and 98.90 – 101.00 % on Day 6, 99.00 – 102.35 % on Day 8 and 98.48 – 106.10 % on Day 14.
- Specificity: The chromatogram of the low standard solution showed enough peaks which were possible to analyse, and chromatograms of the solutions for dilution and vehicle did not reveal any interfering peaks at the same retention time for the standard substance.
- Intra-day Variation: The precision results of intra-day variation were 0.81 and 0.21 % for 0.04 and 1.5 % of dosing formulations, respectively. The accuracy results were 98.30 and 93.87 %, respectively.
- Stability of the Stock Solution: Stock solution was stored under refrigeration for 3 and 8 days. The precision results were 0.04 and 0.16 % at 0.005 % of the standard solution, respectively. The accuracy results were 99.90 and 100.82 %, respectively.
- Stability in the Autosampler: The precision results of reanalysed intra-day variation samples in the autosampler for 9 hours were 1.29 and 0.28 % for 0.04 and 1.5 % of dosing formulations, respectively. The variation results were 0.66 and 0.28 %, respectively.
- Homogeneity: The precision results of homogeneity were 0.89 and 0.35 % for 0.04 and 1.5 % of dosing formulations, respectively. The accuracy results were 98.48 and 94.07 %, respectively.
- Stability
Stability following 3 days of standing at room temperature (1 – 30 ℃): The precision results of dosing formulations for 0.04 and 1.5 % to verify stability were 0.48 and 0.51 %, respectively, for 3 days standing at room temperature. The variation results were -5.06 and -3.05 %, respectively.
Stability following 6 days of standing at room temperature (1 – 30 ℃): The precision results of dosing formulations for 0.04 and 1.5 % to verify stability were 0.29 and 0.39 %, respectively, for 6 days standing at room temperature. The variation results were -11.88 and -8.31 %, respectively.
Stability following 8 days of standing at room temperature (1 – 30℃): The precision results of dosing formulations for 0.04 and 1.5 % to verify stability were 0.97 and 1.26 %, respectively, for 8 days standing at room temperature. The variation results were -16.48 and -9.52 %, respectively.
Stability following 8 days of storage under refrigeration: The precision results of dosing formulations for 0.04 and 1.5 % to verify stability were 0.99 and 0.64 %, respectively, for 8 days stored under refrigeration. The variation results were -2.14 and 0.57 %, respectively.
Stability following 14 days of storage under refrigeration: The precision results of dosing formulations for 0.04 and 1.5 % to verify stability were 0.22 and 0.43 %, respectively, for 14 days stored under refrigeration. The variation results were 3.61 and -1.99 %, respectively.
- Quality Control (QC) sample: At the completion of analysis, three replicate 0.005 % triplicate QC samples were analysed. The precision and accuracy results were 1.24 and 98.72 % on Day 0, 0.45 and 98.36 % on Day 3, 0.88 and 99.46 % on Day 6, 0.76 and 97.84 % on Day 8 and 0.60 and 99.66 % on Day 14.

DISCUSSION AND CONCLUSION
The gas chromatography (GC) method for the analysis of the dosing formulations was validated. The stability of the low dose formulation at room temperature for 8 days was outside the acceptance criteria, thus the analysis for stability at room temperature for 14 days were not performed. In conclusion, the 0.04 and 1.5 % dosing formulation was homogeneous in corn oil and stable after standing at room temperature (1 – 30 °C) for 3 and 6 days and under refrigeration for 8 and 14 days.
Details on mating procedure:
- Impregnation procedure: Cohoused
- If cohoused:
- M/F ratio per cage: 1:1. During the mating period, virgin female rats were cohabitated with male breeder rats (one male rat per one female rat) in the home cages housing sexually mature breeder males. Female rats without evidence of mating were separated from the male rats in the morning and individually housed until that afternoon. Then, the male and female rats were cohabitated again in the afternoon.
- Length of cohabitation: The mating period was performed for seven days. Then, mating was ended when 20 female rats per group are copulated.
- Proof of pregnancy: Confirmation of pregnancy was conducted the next morning. Mating was evaluated daily during the cohabitation period. The day when female rats were observed with spermatozoa in a smear of the vaginal contents and/or the presence of a copulatory plug was observed in situ was considered to be GD 0.
Duration of treatment / exposure:
Gestation Day (GD) 5 to GD 19.
Frequency of treatment:
Daily. Mixed test material formulation and powder feed were placed in feeders and provided ad libitum.
Duration of test:
GD 5 to GD 19, total 15 days.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
0 %
Dose / conc.:
221.3 mg/kg bw/day (nominal)
Remarks:
0.25 %
Dose / conc.:
454.6 mg/kg bw/day (nominal)
Remarks:
0.5 %
Dose / conc.:
925.1 mg/kg bw/day (nominal)
Remarks:
1.0 %
No. of animals per sex per dose:
20 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: As a result of the dose range finding study, no treatment-related changes were observed in any doses tested. In addition, no deaths were observed at a dose of 1.5 %. And, mean intake test material in the high dose group (1.5 %) was observed at 1 446.7 mg/kg/day. The expected mean intake test material in the high dose group is approximately 1 000 mg/kg/day, 1.0 % was selected as the high dose of this study. The mid and low doses were selected at 0.5 and 0.25 %, respectively. Control animals were received the standard basal powder feed with corn oil (vehicle).
- Rationale for animal assignment: On the day following mating, copulated females with body weights close to the mean body weight were selected and assigned to 4 groups (20 animals/ group). Selected animals were randomly assigned to achieve an even distribution of mean body weight of each group.
- Fasting period before blood sampling for (rat) dam thyroid hormones: No fasting.
- Time of day for (rat) dam blood sampling: Blood sampling took place after necropsy.

Examinations

Maternal examinations:
The parameters listed below were evaluated for all copulated females. However, pregnancy was only finally confirmed at the time of caesarean section; non-pregnant animals were not reflected in the final report.

CAGE SIDE AND CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were observed once daily for clinical signs and twice daily for mortality, moribundity, abortion and premature birth during the observation period.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded on GDs 0, 3, 5, 8, 11, 14, 17, 19 and 20 (the day of necropsy) using an automatic animal balance.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: For the determination of food consumption for individual animals, feed of measured quantity was offered on GDs 0, 2, 4, 5, 7, 10, 13, 16 and 18, and the amount of feed left was measured on the respective following day.
- Food consumption: Individual food consumption data was determined by subtracting the amount of feed left from the given quantity.
- Test substance intake: The test substance intake was calculated as follows and designated by mg/kg/day:
Test substance intake (mg/kg/day) = [Food consumption (mg/day) × Concentration (%)] / Body weight (kg)

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20. All animals were sacrificed by exsanguination from the abdominal aorta under isoflurane anaesthesia on the day of necropsy.
- Gross pathology examinations: Complete gross post-mortem examinations were performed on all animals including the external surface and internal organs. All grossly visible abnormalities were recorded.
- Organ weights: The following organs of all pregnant animals were harvested and wet weight was measured, and the relative ratio of organ weight to terminal body weight of dams (excluding gravid uterus with cervix weight) was calculated: Gravid uterus with cervix, thyroid gland with parathyroid gland (weighed paired and after fixation) and liver. Paired organs were weighed together.
- Histopathology: At necropsy, the following organs and tissues from all pregnant animals were harvested and preserved in 10 % neutral buffered formalin: Brain, pituitary gland, ovary, uterus with cervix, liver, parathyroid gland and thyroid gland.
Histopathological examination of the samples of the following organs and tissues from all pregnant animals was performed: Liver, thyroid gland and parathyroid gland. The parathyroid specimens were examined histopathologically only if present in routine sections.
Ovaries and uterine content:
The ovaries and uterine content were 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

At necropsy, the ovaries and uterus of all females were removed and implantations, early and late resorptions and dead/live foetuses were examined; the number of corpora lutea and implantation sites were counted. The pre- & post-implantation loss rates, early & late resorptions rates and embryofoetal mortality rate were calculated.
Blood sampling:
- Thyroid hormone analysis: Blood samples from the abdominal aorta of animals were taken within a short time (two hours) on the morning of the day of necropsy. Collected blood samples were centrifuged at 3 000 rpm for 10 minutes to obtain serum. The Triiodothyronine (T3), Total thyroxine (T4) and Thyroid stimulating hormone (TSH) from separated serum were analysed using an immunoassay analyser.
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: No
- Anogenital distance of all pups: Yes

- Sex ratio: The sex of each foetus was determined by comparing the distance between the anus and the genital tubercle (short for females, long for males). The sex ratio was calculated as follows:

- External examination of foetuses and observation of placenta: All foetuses were examined for external findings including the eyes, ears, nose, mouth, palate, absence of limbs and tail, position and size, shape etc. In addition, the shape and colour of the placenta were observed.

- Body weights of live foetuses and placenta weights: Live foetuses removed from the uterus were numbered sequentially from the left ovary to the right ovary on the dorsal surface with an indelible pen. The weights of surviving foetuses and placental weights were recorded. Foetal body weights and placental weights were based on the average value of the foetuses of the same litter. All live foetuses were euthanised by hypothermia.

- Anogenital distance: AGD of each foetus was measured after caesarean section using a Digimatic calliper.

- Visceral examination of live foetuses: For the visceral examination of foetuses, specimens of the preserved odd number foetuses were prepared by FA solution. Then, microdissections were examined for the visceral examination using a stereomicroscope. All specimens and sections were individually preserved in 10 % neutral buffered formalin.

- Skeletal examination of live foetuses: For the skeletal examination of foetuses, specimens of the preserved even number foetuses were prepared. The skeletons were stained using Alizarin Red S by the single staining technique. Subsequently, these foetuses were evaluated using a stereomicroscope and/or macroscopic examination for skeletal anomalies and ossification variations. These specimens were then stored in 98 - 100 % glycerin.
Statistics:
Statistical analysis was performed for the body weights of live pregnant animals, food consumption, thyroid hormone values, organ weights, number of corpora lutea, number of implantations, pre- & post- implantation rates, body weights and placenta weights of live foetuses, number of live foetuses, sex ratio, number of ossifications, and AGD index using the SAS program (version 9.4, SAS Institute Inc., U.S.A.).
The data was analysed utilizing Bartlett’s test for homogeneity of variance (significance level: 0.05). One-way analysis of variance (ANOVA) was employed on homogeneous data (significance level: 0.05); then, if significant, Dunnett's test was applied for multiple comparisons (significance levels: 0.05 and 0.01, two-tailed). The Kruskal-Wallis test was employed on heterogeneous data (significance level: 0.05); then, if significant, DSCF (Dwass-Steel-Critchlow-Fligner) was applied for multiple comparisons (significance levels: 0.05 and 0.01, two-tailed).
The results of other data types such as malformation and anomalies through skeletal examination, visceral examination and external findings, early resorption rate, late resorption rate and embryofoetal mortality rate were analysed utilising the Kruskal-Wallis test (significance level: 0.05). If significant, DSCF (Dwass-Steel-Critchlow-Fligner) was applied for multiple comparisons (significance levels: 0.05 and 0.01, two-tailed).
Indices:
- Pre-implantation loss rate (%) = [(No. of corpora lutea – No. of implantations) / No. of corpora lutea] × 100
- Post-implantation loss rate (%) = [(No. of implantations – No. of live foetuses ) / No. of implantations] × 100
- Early resorptions (%) = (No. of early resorptions / No. of implantations) × 100
- Late resorptions (%) = (No. of late resorptions / No. of implantations) × 100
- Foetal mortality (%) = ((No. of resorptions + No. of dead foetuses) / No. of implantations) × 100
- Anogenital Distance index: AGD index was calculated as follows: AGD index = AGD / cubed root of body weight
- Sex ratio (%) = (No. of live male foetuses / No. of total live foetuses) × 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Crushing of teeth was observed in one animal and overgrown teeth were observed in another in the 0.25 % group. It was not considered to be a test material-related effect since these were low incidence, not dose-related, or comparable to the vehicle control animals, and considered incidental or spontaneous.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There were no deaths in the animals in the control group, 0.25, 0.5 and 1.0 % groups during the observation period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No test material-related effects in body weights and body weight gains were observed in the 0.25, 0.5 and 1.0 % groups during the study period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No test material-related effects in food consumption and relative food consumption were observed in the 0.25, 0.5 and 1.0 % groups during the study period.
The mean total test material intakes in the 0.25, 0.5 and 1.0 % groups were 221.3, 454.6 and 925.1 mg/kg/day, respectively.
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 test material-related effects in thyroid hormone analysis were observed in the 0.25, 0.5 and 1.0 % groups.
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 test material-related effects in absolute and relative organ weights were observed in the 0.25, 0.5 and 1.0 % groups.
No test material-related effect in placental weights was observed in the 0.25, 0.5 and 1.0 % groups.
Gross pathological findings:
no effects observed
Description (incidence and severity):
At necropsy, no noticeable findings were observed in the control, 0.25, 0.5 and 1.0 % groups.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Microscopic examination did not reveal treatment-related changes in all pregnant females in the 0.25, 0.5 and 1.0 % groups. All microscopic findings seen in the liver, thyroid gland and parathyroid gland did not show significant difference in the incidence compared to controls or were normal background lesions observed at the testing facility or were found with low/ isolated frequency.
Histopathological findings: neoplastic:
not examined

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
Other effects:
no effects observed
Description (incidence and severity):
No test material-related changes in caesarean section parameters were observed in the 0.25, 0.5 and 1.0 % groups.
No test material-related effect in placentas was observed in the 0.25, 0.5 and 1.0 % groups. The changes including shared, large and small placentas in the control, 0.25 and 0.5 % groups were observed without dose-dependence. Therefore, these changes were not considered to be test material related.
No test material-related effect in placental weights was observed in the 0.25, 0.5 and 1.0 % groups.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
925.1 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: No adverse effects were observed at the 1.0 % dose level, the highest tested.

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
No test material-related effect in foetal body weights was observed in the 0.25, 0.5 and 1.0 % groups.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
not specified
Anogenital distance of all rodent fetuses:
no effects observed
Description (incidence and severity):
No test material-related effect in AGD index of male and female foetuses was observed in the 0.25, 0.5 and 1.0 % groups.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
No test material-related effect in external examinations of foetuses was observed in the 0.25, 0.5 and 1.0 % groups.
Skeletal malformations:
no effects observed
Description (incidence and severity):
No test material-related effects were observed in the skeletal examination of foetuses in the study.
No skeletal malformation was observed in any groups.
The skeletal variations observed in the study were not considered to be test material-related effects because these were not statistically significant and/ or were not dose dependent.
No test material-related effects in the number of ossification centres were observed in this study.
Visceral malformations:
no effects observed
Description (incidence and severity):
No test material-related effects were observed in the visceral examination of foetuses in the study.
No visceral malformation was observed in any groups.
The visceral variations observed in the study were not considered to be test material-related effects because these were not statistically significant and/ or were not dose dependent.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
925.1 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects were observed at the 1.0 % dose level, the highest tested.

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

Summary of Clinical Signs: Females

Group/ Dose

(%)

No. of Animals

Clinical Sign

Gestation Day

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

G1: 0

19

NOA

19

19

19

19

19

19

19

19

19

19

19

19

19

19

19

19

19

19

19

19

G2: 0.25

19

NOA

19

19

19

19

19

19

19

19

19

19

19

19

19

19

17

19

19

19

19

19

Crushing of teeth

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1

 

 

 

 

 

Overgrown teeth

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1

 

 

 

 

 

G3: 0.5

17

NOA

17

17

17

17

17

17

17

17

17

17

17

17

17

17

17

17

17

17

17

17

G4: 1.0

20

NOA

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

NOA: No Observable Anomaly.

 

Mean Body Weights: Females (g)

Group/ Dose

(%)

Gestation Day

0

3

5

8

11

14

17

19

20

G1: 0

Mean

249.9

271.8

282.1

294.5

314.0

332.2

363.2

395.2

412.8

S.D

9.9

12.0

12.8

14.8

15.4

16.9

18.7

23.0

23.5

N

19

19

19

19

19

19

19

19

19

G2: 0.25

Mean

249.6

270.8

279.4

292.7

311.3

331.1

356.9

389.1

405.9

S.D

11.9

11.8

12.3

13.4

15.3

15.0

22.6

26.2

32.2

N

19

19

19

19

19

19

19

19

19

G3: 0.5

Mean

250.9

271.7

281.1

295.8

315.5

332.4

362.4

395.8

416.0

S.D

12.5

12.1

11.8

12.1

14.3

12.7

17.6

21.4

22.9

N

17

17

17

17

17

17

17

17

17

G4: 1.0

Mean

249.3

272.3

282.8

297.5

316.1

333.1

365.8

398.2

416.6

S.D

12.0

132

13.7

15.5

18.2

18.0

18.9

21.6

23.7

N

20

20

20

20

20

20

20

20

20

Significantly different from control by Dunnett's t-test: * p<0.05, ** p><0.01. Significantly different from control by DSCF: # p><0.05, ## p><0.05, ** p<0.01.

Significantly different from control by DSCF: # p<0.05, ## p <0.01.

 

Mean Body Weight Gains: Female (g)

Group/ Dose

(%)

Gestation Day

3

5

8

11

14

17

19

20

G1: 0

Mean

21.8

10.4

12.4

19.4

18.2

31.1

32.0

17.6

S.D

5.6

3.6

5.0

5.0

4.6

6.1

6.2

5.1

N

19

19

19

19

19

19

19

19

G2: 0.25

Mean

21.1

8.7

13.3

18.6

19.9

25.8

32.1

16.8

S.D

6.7

3.9

4.8

5.7

5.5

14.1

12.8

7.7

N

19

19

19

19

19

19

19

19

G3: 0.5

Mean

20.8

9.5

14.7

19.6

16.9

30.1

33.4

17.2

S.D

4.3

2.8

3.5

4.9

5.5

7.9

6.5

7.2

N

17

17

17

17

17

17

17

17

G4: 1.0

Mean

23.0

105

14.7

18.6

17

32.7

32.4

18.4

S.D

4.8

3.8

4.7

6.2

6.4

7.7

5.3

10.9

N

20

20

20

20

20

20

20

20

Significantly different from control by Dunnett's t-test: * p<0.05, ** p><0.01. Significantly different from control by DSCF: # p><0.05, ## p><0.05, ** p<0.01.

Significantly different from control by DSCF: # p<0.05, ## p <0.01.

 

Mean Food Consumption: Female (g/day)

Group/ Dose

(%)

Gestation Day

1

3

5

6

8

11

14

17

19

G1: 0

Mean

23.4

25.1

26.3

27.2

27.4^

28.4

27.8^

33.7^

32.4^

S.D

3.1

3.3

3.0

3.3

3.8

3.1

2.6

5.4

3.7

N

19

19

19

19

18

19

18

16

16

G2: 0.25

Mean

21.8

25.2

25.1^

26.3^

27.1^

28.5

28.2^

30.4^

30.9^

S.D

2.8

3.2

2.9

3.5

3.6

3.4

3.7

4.9

4.3

N

19

19

18

18

18

19

18

17

17

G3: 0.5

Mean

32.2

25.5^

26.5^

27.1^

27.5

29.4

29.4^

32.6^

33.2

S.D

4.6

4.3

3.7

4.5

3.4

3.5

2.7

5.1

3.9

N

17

16

16

16

17

17

16

16

17

G4: 1.0

Mean

23.1^

26.6^

27.8^

29.2^

28.7^

29.1

29.5^

32.8^

33.8^

S.D

4.3

4.6

4.2

5.1

4.3

3.7

2.9

4.8

4.1

N

19

19

19

16

18

20

17

15

19

Significantly different from control by Dunnett's t-test: * p<0.05, ** p><0.01. Significantly different from control by DSCF: # p><0.05, ## p><0.05, ** p<0.01.

Significantly different from control by DSCF: # p<0.05, ## p <0.01.

^: The animals that abandoned most of the feed from the feeder was excluded from the data.

 

Mean Relative Food Consumption: Female (g/kg body weight/day)

Group/ Dose

(%)

Gestation Day

1

3

5

8

11

14

17

19

G1: 0

Mean

93.7

92.1

93.0

92.6^

90.4

83.6^

93.5^

83.1^

S.D

12.9

10.9

9.0

11.0

7.5

6.3

16.1

11.5

N

19

19

19

18

19

18

16

16

G2: 0.25

Mean

97.5

93.2

89.8^^

92.3

91.6

85.1^

85.5^

80.3^

S.D

12.6

11.8

9.8

10.8

9.3

10.6

13.0

11.2

N

19

19

18

18

19

18

17

17

G3: 0.5

Mean

92.8

94.1^

94.6^

92.9

93.3

88.8^

90.1^

83.8

S.D

18.5

16.5

13.7

11.1

9.9

8.5

14.0

8.1

N

17

16

16

17

17

16

16

17

G4: 1.0

Mean

92.5^

97.7^

98.1^

96.5^

91.9

88.4^

89.2^

85.0^

S.D

15.5

14.9

122

12.9

9.3

7.6

11.7

9.5

N

19

19

19

18

20

17

15

19

Significantly different from control by Dunnett's t-test: * p<0.05, ** p><0.01. Significantly different from control by DSCF: # p><0.05, ## p><0.05, ** p<0.01.

Significantly different from control by DSCF: # p<0.05, ## p <0.01.

^: The animals that abandoned most of the feed from the feeder was excluded from the data.

Mean Test Material Intake: Female (mg/kg/day)

Group/ Dose

(%)

Gestation Day

Total Mean*

6

8

11

14

17

19

G2: 0.25

Mean

235.0^

230.7^

229.1

212.8^

214.0^

200.7^

221.3

S.D

28.5

27.1

23.2

26.6

32.5

28.1

17.3

N

18

18

19

18

17

17

19

G3: 0.5

Mean

483.4^

464.4

466.5

443.6

450.8

418.9

454.6

S.D

79.7

55.3

49.7

42.8

70.1

40.5

38.3

N

16

17

17

16

16

17

17

G4: 1.0

Mean

1 040.7^

965.4^

918.6

884.0^

891.8^

849.5^

925.1

S.D

158.5

129.1

92.6

76.2

117.3

211.4

64.716

N

16

18

20

17

15

19

20

^: The animals that abandoned most of the feed from the feeder was excluded from the data.

*: The animals that abandoned most of the feed from the feeder was excluded from the total mean data.

 

Mean Thyroid Hormone Value: Female

Group/ Dose

(%)

T4

(μg/dL)

TSH

(μIU/mL)

T3

(ng/dL)

G1: 0

Mean

0.93

0.248

37

S.D

0.41

0.112

9

N

19

19

19

G2: 0.25

Mean

1.14

0.273

40

S.D

0.83

0.243

16

N

19

19

19

G3: 0.5

Mean

1.04

0.186

42

S.D

0.36

0.181

11

N

17

17

17

G4: 1.0

Mean

0.99

0.225

43

S.D

0.60

0.083

21

N

20

20

20

Significantly different from control by Dunnett's t-test: * p<0.05, ** p><0.01. Significantly different from control by DSCF: # p><0.05, ## p><0.05, ** p<0.01.

Significantly different from control by DSCF: # p<0.05, ## p <0.01.

 

Mean Absolute Organ Weights: Female (g)

Group/ Dose

(%)

B.W

Liver

Gravid Uterus with Cervix

Thyroid Gland with Parathyroid Gland

G1: 0

Mean

412.8

16.00

82.88

0.0293

S.D

23.5

1.22

13.93

0.003

N

19

19

19

19

G2: 0.25

Mean

405.9

15.77

77.83

0.315

S.D

32.2

2.11

22.32

0.0050

N

19

19

19

19

G3: 0.5

Mean

413.0

16.05

81.85

0.0288

S.D

22.9

1.24

11.27

0.0055

N

17

17

17

17

G4: 1.0

Mean

416.6

16.39

84.15

0.0311

S.D

23.7

1.66

9.44

0.0056

N

20

20

20

20

Significantly different from control by Dunnett's t-test: * p<0.05, ** p><0.01. Significantly different from control by DSCF: # p><0.05, ## p><0.05, ** p<0.01.

Significantly different from control by DSCF: # p<0.05, ## p <0.01.

 

Mean Relative Organ Weights: Females (g/ 100g body weight)

Group/ Dose

(%)

Correct B.W

Liver

Gravid Uterus with Cervix

Thyroid Gland with Parathyroid Gland

G1: 0

Mean

329.9

3.8757

25.1051

0.0071

S.D

14.6

0.1820

3.9610

0.0012

N

19

19

19

19

G2: 0.25

Mean

328.1

3.8708

23.7007

0.0078

S.D

18.8

0.2782

6.9631

0.0012

N

19

19

19

19

G3: 0.5

Mean

331.1

3.8865

24.7298

0.0070

S.D

16.8

0.2040

3.2280

0.0014

N

17

17

17

17

G4: 1.0

Mean

332

3.9279

25.3167

0.0075

S.D

17.9

0.2510

2.4745

0.0014

N

20

20

20

20

Correct B.W. = Terminal B.W. – Gravid uterus with cervix

Significantly different from control by Dunnett's t-test: * p<0.05, ** p><0.01. Significantly different from control by DSCF: # p><0.05, ## p><0.05, ** p<0.01.

Significantly different from control by DSCF: # p<0.05, ## p <0.01.

 

Mean Caesarean Section Data

Group/ Dose

(%)

No. of Corpus Luteum

No. of Implantation

No. of Embryo Foetal Death

Implantation Loss Rate (%)

Resorption Rate (%)

Embryo Foetal Mortality

(%)

No. of Live Foetuses

Sex Ratio

(%)

Resorption

Dead Foetuses

Pre-

Post-

Early

Late

Sex

Total

Early

Late

Male

Female

G1: 0

Mean

15.4

14.7

0.5

0.0

0.0

5.1

3.6

3.6

0.0

3.6

7.0

7.2

14.2

49.8

S.D

2.0

2.3

0.8

0.0

0.0

7.3

5.8

5.8

0.0

5.8

1.8

2.4

2.6

11.4

N

19

19

19

19

19

19

19

19

19

19

19

19

19

19

G2: 0.25

Mean

14.4

13.7

0.2

0.0

0.0

4.8

6.3

6.3

0.0

6.3

3.9

6.6

13.5

51.2

S.D

3.6

3.7

0.5

0.0

0.0

8.9

22.9

22.9

0.0

22.9

2.6

2.6

3.9

11.5

N

19

19

19

19

19

19

19

19

19

19

19

19

19

19

G3: 0.5

Mean

14.8

14.5

0.7

0.0

0.1

2.1

5.5

5.1

0.0

5.5

7.0

6.7

13.7

51.0

S.D

2.1

1.8

0.8

0.0

0.2

5.3

6.0

5.6

0.0

6.0

1.9

1.6

2.2

10.3

N

17

17

17

17

17

17

17

17

17

17

17

17

17

17

G4: 1.0

Mean

15.0

14.7

0.6

0.0

0.0

2.4

4.0

4.0

0.0.

4.0

8.0

6.1

14.1

56.4

S.D

1.2

1.4

0.7

0.0

0.0

4.1

4.6

4.6

0.0

4.6

2.3

2.1

1.4

15.2

N

20

20

20

20

20

20

20

20

20

20

20

20

20

20

Pre-implantation loss rate (%) = ((No. of corpora lutea – no. of implantation sites) / No. of corpora lutea) x 100

Post-implantation loss rate (%) = ((No. of implantations – No. of live foetuses) / No. of implantations) x 100

Early resorption rate (%) = (No. of early resorptions / No. of implantations) x 100

Late resorption rate (%) = (No. of late resorptions / No. of implantations) x 100

Embryo foetal mortality rate (%) = (No. of resorptions + No. of dead foetuses / No. of implantations) x 100

Sex ration (%) = (No. of live male foetuses / No. of total live foetuses) x 100

Significantly different from control by Dunnett's t-test: * p<0.05, ** p><0.01. Significantly different from control by DSCF: # p><0.05, ## p><0.05, ** p<0.01.

Significantly different from control by DSCF: # p<0.05, ## p <0.01.

 

Mean External Findings of Foetuses and Placentas

Group/ Dose

(%)

No. of Dams

 

Foetuses

Placentas

Abnormal

Shared

Large

Small

G1:0

19

Total

0.00 (0)

0.00 (0)

 

0.00 (0)

Incidence (%)

0.00

0.00

 

0.00

G2: 0.25

18

Total

0.00 (0)

0.13 (2)

0.00 (0)

0.00 (0)

Incidence (%)

0.00

0.72

0.00

0.00

G3: 0.5

17

Total

0.00 (0)

0.00 (0)

0.00 (0)

0.08 (1)

Incidence (%)

0.00

0.00

0.00

0.47

G4: 1.0

20

Total

0.00 (0)

0.00 (0)

0.00 (0)

0.00 (0)

Incidence (%)

0.00

0.00

0.00

0.00

( ): No. of found foetuses

Value = No of found foetuses / No. of foetuses per litter

Incidence (%) = (Total value / No. of dams) x 100

Significantly different from control by DSCF: # p<0.05, ## p <0.01.

 

Mean Foetal Body Weights and Placental Weights (g)

Group/ Dose

(%)

Body Weights

Placenta Weights

Total

Male

Female

Total

Male

Female

G1: 0

Mean

3.90

4.00

3.80

0.45

0.46

0.44

S.D

0.25

0.27

0.24

0.04

0.05

0.05

N

19

19

19

19

19

19

G2: 0.25

Mean

3.94

4.05

3.84

0.44

0.45

0.44

S.D

0.52

0.54

0.52

0.04

0.03

0.05

N

18

18

18

18

18

18

G3: 0.5

Mean

3.95

4.04

3.85

0.47

0.47

0.46

S.D

0.27

0.33

0.24

0.06

0.07

0.06

N

17

17

17

17

17

17

G4: 1.0

Mean

3.97

4.06

3.87

0.46

0.47

0.45

S.D

0.19

0.20

0.20

0.03

0.03

0.03

N

20

20

20

20

20

20

N: No. of dams.

Significantly different from control by Dunnett's t-test: * p<0.05, ** p><0.01. Significantly different from control by DSCF: # p><0.05, ## p><0.05, ** p<0.01.

Significantly different from control by DSCF: # p<0.05, ## p <0.01.

 

Mean Anogenital Distance (mm)

Group/ Dose

(%)

Anogenital Distance

Male

Female

G1: 0

Mean

1.26

0.58

S.D

0.05

0.05

N

19

19

G2: 0.25

Mean

1.26

0.59

S.D

0.07

0.06

N

18

18

G3: 0.5

Mean

1.25

0.58

S.D

0.05

0.05

N

17

17

G4: 1.0

Mean

1.26

0.60

S.D

0.07

0.05

N

20

20

N: No. of dams.

AGD index = AGD/^3√(body weight).

Significantly different from control by Dunnett's t-test: * p<0.05, ** p><0.01. Significantly different from control by DSCF: # p><0.05, ## p><0.05, ** p<0.01.

Significantly different from control by DSCF: # p<0.05, ## p <0.01.

 

Mean Visceral Findings of Foetuses

Group / Dose

(%)

No. of Dams

 

Variation

Ureter

Kidneys

Umbilical Artery

Dilated

Convoluted

Dilated Renal Pelvis

Malpositioned

G1: 0

19

Total

2.31 (6)

4.54 (32)

0.00 (0)

0.13 (1)

Incidence (%)

12.16

23.89

0.0

0.68

G2: 0.25

18

Total

1.10 (8)

3.32 (24)

0.00 (0)

0.13 (1)

Incidence (%)

6.11

18.44

0.00

0.72

G3: 0.5

17

Total

1.28 (8)

4.40 (30)

0.28 (2)

0.27 (2)

Incidence (%)

7.53

25.88

1.65

1.59

G4: 1.0

16

Total

1.32 (9)

4.00 (28)

0.00 (0)

0.26 (2)

Incidence (%)

6.60

20.00

0.00

1.30

( ): No. of found foetuses.

Total value = sum (No. of found foetuses / No. of foetuses per litter).

Incidence (%) = (Total value / No. of dams) x 100.

Significantly different from control by DSCF: # p<0.05, ## p <0.01.

 

Mean Skeletal Findings of Foetuses

Group / Dose

(%)

No. of Dams

 

Variation

Skull

Rib

Parietal Bone

Supraoccipital Bone

Interparietal

Short

Bent

Wavy

Lumbar Rib

Incomplete Ossification

Incomplete Ossification

Biparite Ossification

Incomplete Ossification

G1: 0

19

Total

0.29 (2)

0.00 (0)

0.00 (0)

0.00 (0)

0.00 (0)

0.00 (0)

0.00 (0)

1.56 (10)

Incidence (%)

1.53

0.00

0.00

0.00

0.00

0.00

0.00

8.21

G2: 0.25

18

Total

0.00 (0)

0.00 (0)

0.15 (1)

0.00 (0)

0.00 (0)

0.00 (0)

0.00 (0)

3.12 (20)

Incidence (%)

0.00

0.00

0.83

0.00

0.00

0.00

0.00

17.33

G3: 0.5

17

Total

0.63 (4)

0.15 (1)

0.00 (0)

0.00 (0)

0.35 (2)

0.17 (1)

0.00 (0)

2.15 (15)

Incidence (%)

3.71

0.88

0.00

0.00

2.06

1.00

0.00

12.65

G4: 1.0

16

Total

0.57 (4)

0.00 (0)

0.00 (0)

0.25 (2)

0.00 (0)

0.00 (0)

0.00 (0)

2.36 (16)

Incidence (%)

2.85

0.00

0.00

1.25

0.00

0.00

0.00

11.80

 

Group / Dose

(%)

No. of Dams

 

Variation

Vertebra

Pelvic Girdle

Thoracic

Sternebra

Ischiu

Pubis

Dumbbell Shaped

Bipartite Ossification

Bipartite Ossification

Dumbbell Shaped

Incomplete ossification

Incomplete ossification

G1: 0

19

Total

0.20 (1)

0.59 (4)

0.00 (0)

0.00 (0)

0.00 (0)

0.00 (0)

Incidence (%)

1.05

3.11

0.00

0.00

0.00

0.00

G2: 0.25

18

Total

0.30 (2)

1.07 (7)

0.15 (1)

0.00 (0)

0.25 (2)

0.54 (4)

Incidence (%)

1.67

5.94

0.83

0.00

1.39

3.00

G3: 0.5

17

Total

0.33 (2)

0.47 (3)

0.00 (0)

0.00 (0)

0.25 (2)

0.58 (4)

Incidence (%)

1.94

2.76

0.00

0.00

1.47

3.41

G4: 1.0

16

Total

0.15 (1)

0.32 (2)

0.00 (0)

0.00 (0)

0.00 (0)

0.00 (0)

Incidence (%)

0.75

1.60

0.00

0.00

0.00

0.00

( ): No. of found foetuses.

Total value = sum (No. of found foetuses / No. of foetuses per litter).

Incidence (%) = (Total value / No. of dams) x 100.

Significantly different from control by DSCF: # p<0.05, ## p <0.01.

 

Mean Ossification Status of Foetuses

Group / Dose

(%)

No. of Foetuses

OSSB

Pairs of Ribs

Sternebra

Sacro-caudal vertebra

Fore Limb

Hind Limb

Mc

Pp

Mp

Dp

Mt

Pp

Mp

Dp

G1: 0

Mean

6.9

1.9

13.0

5.6

7.9

7.5

0.4

0.0

0.0

8.0

0.0

0.0

0.0

S.D

1.3

0.1

0.1

0.4

0.5

0.6

0.9

0.0

0.0

0.0

0.0

0.0

0.0

N

19

19

19

19

19

19

19

19

19

19

19

19

19

G2: 0.25

Mean

6.9

1.8

13.1

5.6

8.0

7.5

0.7

0.0

0.0

8.1

0.1

0.0

0.0

S.D

1.1

0.2

0.1

0.4

0.7

0.6

1.5

0.0

0.0

0.4

v

0.0

0.0

N

18

18

18

18

18

18

18

18

18

18

18

18

18

G3: 0.5

Mean

6.6

1.9

13.0

5.5

7.9

7.5

0.5

0.0

0.0

8.0

0.0

0.0

0.0

S.D

1.1

0.1

0.1

0.4

0.4

0.5

0.9

0.0

0.0

0.0

0.0

0.0

0.0

N

17

17

17

17

17

17

17

17

17

17

17

17

17

G4: 1.0

Mean

6.9

1.9

13.1

5.6

8.0

7.5

0.4

0.0

0.0

8.0

0.0

0.0

0.0

S.D

0.8

0.2

0.1

0.4

0.4

0.5

0.5

0.0

0.0

0.0

0.0

0.0

0.0

N

20

20

20

20

20

20

20

20

20

20

20

20

20

N: No. of dams.

OSSB: Ossified step of supraoccipital bone.

Mc: Metacarpal, Pp: Proximal phalanx, Mp: Middle phalanx, DP: Distal phalanx, Mt: Metatarsal.

Significantly different from control by Dunnett's t-test: * p<0.05, ** p><0.01. Significantly different from control by DSCF: # p><0.05, ## p><0.05, ** p<0.01.

Significantly different from control by DSCF: # p<0.05, ## p <0.01.

 

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) of the test material for both pregnant rats and embryofoetal development are considered to be at 1.0 % (925.1 mg/kg/day).
Executive summary:

A prenatal developmental toxicity study was carried out in rats in accordance with the standardised guideline OECD 414 and NIER Notification No. 2020-46 under GLP conditions.

The purpose of this study was to investigate the effects of the test material on pregnant females and prenatal development when administered orally (via the diet) to pregnant female Sprague-Dawley rats (20 animals per group) from implantation to the day prior to scheduled caesarean section (from Gestation Day (GD) 5 to GD 19, total 15 days). The administration was carried out at doses of 0 (control), 0.25, 0.5, and 1.0 % (0, 221.3, 454.6 and 925.1 mg/kg/day, respectively).

Evaluated parameters included clinical signs, body weight, food consumption, thyroid hormone analysis, organ weights, gross post-mortem examinations, histopathology, caesarean section, external examination of foetuses and placentas, measurement of body weights of live foetuses and placental weights, anogenital distance (AGD), and foetal visceral and skeletal examination.

No deaths related to the test material occurred in the treatment groups during the study period.

No test material-related changes were observed in the clinical signs, body weight, food consumption, thyroid hormone levels, organ weights, caesarean section, gross pathology and histopathology at any dose tested.

No test material-related changes were observed in the external examination of foetuses and placentas, foetal body weight and placental weight, AGD index, and visceral and skeletal examination at any dose tested.

Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) of the test substance for both pregnant rats and embryofoetal development are considered to be at 1.0 % (925.1 mg/kg/day).