Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
3 June 2021 to 30 August 2021
Reliability:
1 (reliable without restriction)

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)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
1-ethoxy-2-(2-methoxyethoxy)ethane
EC Number:
213-690-5
EC Name:
1-ethoxy-2-(2-methoxyethoxy)ethane
Cas Number:
1002-67-1
Molecular formula:
C7H16O3
IUPAC Name:
1-ethoxy-2-(2-methoxyethoxy)ethane
Test material form:
liquid
Details on test material:
CAS No.: 1002-67-1
Molecular formula: C7H16O3
Molecular weight: 148.2 g/mol
Purity: >99.9 %
Appearance: water clear, liquid
Expiry date: 28 March 2023
Storage condition: at room temperature, protected from light
Specific details on test material used for the study:
Lot number 09511

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Details on test animals or test system and environmental conditions:
Animals
Rat

Strain
Crl:CD(SD)

Rationale for strain selection
This strain is widely used in reproduction/developmental toxicity studies using rodents, there is abundant historical data, and a large number of animals are available.

Supplier
Charles River Laboratories Japan, Inc. (Hino Breeding Center)

Number, age, and sex of animals purchased
47 males aged 11 weeks
47 females aged 10 weeks
47 females aged 11 weeks

Body weight range at the receipt
Males: 365.5 to 411.5 g (acceptable range: 320 to 450 g)
Females: 10 weeks; 210.3 to 236.5 g (acceptable range: 170 to 300 g)
11 weeks; 217.0 to 247.4 g (acceptable range: 170 to 300 g)

Quarantine and acclimation
Upon receipt, the species, strain, age, number, and sex were checked. Furthermore, observation of clinical signs and external appearance, and measurement of body weight were performed. 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. During these periods, all animals were observed for clinical signs once daily and measured for body weight 2 times. As a result, no abnormalities were noted in any animal.


Preparation of pregnant animals
Mating was started at 12 weeks of age or more 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).

Group assignment
The successfully copulated females were allocated to the groups with a computer system so that the mean body weights on GD 0 were comparable between the groups.

Identification of animals
Before group assignment
Upon receipt, animals were identified by marking the identification number (the last 1 or 2 digits of the quarantine animal number) on the tail with an oil-based felt tip pen and a label indicating the study number (computer registration number), quarantine animal number, and sex was attached to the front of each cage. The quarantine animal numbers and color of pen are shown below.
Males: Nos. 1001 to 1047 (black)
Females aged 11 weeks: Nos. 2001 to 2047 (black)
Females aged 10 weeks: Nos. 3001 to 3047 (blue)

After group assignment
The animals were identified by ear tags inscribed with an animal number and a label indicating the study number (computer registration number), animal number, dose level, and sex was attached to the front of each cage.

Handling of remaining animals
After the mating period, all remaining animals (not used for study groups) were euthanized by exsanguination from the lateral iliac artery under anesthesia overdose by tail vein injection of thiopental sodium.

Animal management Animal room
A207


Environmental conditions
Temperature
23.4°C to 24.6°C (acceptable range: 20.0°C to 26.0°C)

Relative humidity
43.0% to 60.5% (acceptable range: 35.0% to 75.0%)

Ventilation
10 to 20 times per hour
Ventilation frequency is measured twice a year at the test facility. The results were confirmed to be within the acceptable limits established by the test facility.

Lighting
7:00 to 19:00 light and 19:00 to 7:00 dark cycles

Housing equipment
Cage racks Stainless racks Sterilization: Autoclave
Replacement: Once every 4 weeks or more frequently

Cages
Hanging-type stainless wire mesh cages (W226 × D346 × H198 mm) Sterilization: Autoclave
Replacement: Once every 2 weeks or more frequently

Feeders Stainless feeders Sterilization: Autoclave
Replacement: Once every 2 weeks or more frequently

Sanitary trays under cages
Aluminum trays Sterilization: Autoclave
Replacement: Once a week or more frequently and once every day for males during mating period


Water supply systems
Automatic water supply systems equipped with cage racks

Environmental enrichment
Equipment for animal enrichment such as nesting materials (HAPPIMATS [Marshall BioResources] and Diamond Twist (spread out) [ENVIGO]), gnawing materials (Diamond Twist [ENVIGO]), and rest board were placed in each cage. Rest board was exchanged once every 2 weeks or more frequently. Nesting materials and gnawing materials were exchanged once every 2 weeks or more frequently for HAPPIMATS and once a week or more frequently for Diamond Twist.

Number of animals per cage
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
Description
Pellet diet for experimental animals (CRF-1, Oriental Yeast Co., Ltd., radiation sterilized)

Lot numbers
201105, 210202, and 210304

Method of feeding
Ad libitum

Analysis
Analysis data for each used lot in this study were provided by Oriental Yeast Co., Ltd., and the contaminants in the diet were confirmed to be within the acceptable limits established by the test facility.

Water
Description
Well water


Method of sanitization
Admixed with sodium hypochlorite (free residual chlorine concentration: about 0.2 ppm)

Method of water supply
Ad libitum

Analysis
The water is analyzed twice a year by MC Evolve Technologies Corporation. From the analytical data, it was confirmed that the quality of the water met the specifications of the test facility.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
The dosing formulations were administered orally using a disposable syringe attached to a gastric tube. Time of administration 8:21 to 11:42 Rationale for method selection This procedure is widely accepted for oral dosing in rats. Frequency and duration The dosing formulations were administered to females for which copulation was confirmed once daily from GD 6 to GD 19 (for 14 days). Rationale for selection of administration conditions The frequency and duration of administration were set according to the applied guideline (see Section 3.4). Dosing volume 10 mL/kg Individual volume was calculated on the basis of the most recently measured body weights. Dose level and its rationale Dose levels were set at 110, 330, and 1000 mg/kg/day. In a combined repeated dose and reproductive/developmental toxicity study conducted at the test facility (dose level: 0, 50, 250, and 1000 mg/kg/day, vehicle: water for injection) [1], the following effects were noted at 1000 mg/kg: prolongation of gestation period and low values of delivery index in dams and low values of number of litter and birth index, and viability index on postnatal Day 4 and high values of number of stillborn in offspring. Therefore, the high dose level was set at 1000 mg/kg which was expected to develop some toxicity. The middle and low dose levels were set at 330 and 110 mg/kg, respectively, with a common ratio of about 3. A control group (0 mg/kg) dosed with the vehicle (water for injection) alone was also established.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dosing formulations Preparation frequency and method, and storage conditions Dosing formulations were prepared once every 7 days based on the results of a study entitled “Analytical Method Validation for the Determination and Stability test of DEGMEE in Water for Injection (JP) (Study No. P130586)” conducted at the test facility. The dosing formulations after preparation were divided into glass vials for each dosing day, and stored in a cold place (actual temperature: 3.8°C to 6.1°C, permissible range: 1°C to 15°C, storage area: in a medical refrigerator in Dosing Formulation Storage Room A032) for which they were confirmed to remain stable. (Control dosing formulation) The required amount of the vehicle was divided into clear glass vials for each dosing day. (100 mg/mL dosing formulation) (1) A prescribed amount of the test substance was weighed into a beaker. (2) An appropriate amount of the vehicle was added to the beaker and stirred with a magnetic stirrer to dissolve the test substance. After dissolution, the mixture was transferred to a measuring cylinder. (3) The beaker and stirring bar were washed with the vehicle, and the washing was transferred to the measuring cylinder. (4) The final volume was adjusted by adding a proper amount of the vehicle to required concentrations of 100 mg/mL. The dosing formulations after preparation were mixed several times by end-over-end rotation and taken into brown glass vials for each dosing day. (11 and 33 mg/mL dosing formulations) A prescribed amount of the 100 mg/mL formulation was transferred to a measuring cylinder, and diluted with the vehicle to make 11 and 33 mg/mL formulations. The dosing formulations after preparation were mixed several times by end-over-end rotation and taken into brown glass vials for each dosing day. Identification of dosing formulations Vials containing the dosing formulations were labeled with the study number, dosing formulation name, dose level, test substance concentration, preparation date, name of the person preparing the formulations, storage conditions, and expiration period. Handling of remaining dosing formulations The remaining dosing formulations were discarded on each dosing day. Stability confirmation The test substance formulations at 1 and 100 mg/mL were confirmed to be stable for 8 days in a cold place and in brown glass vials and further for 24 hours at room temperature in the analytical method validation for determination and stability test (Study No. P130586) conducted at the test facility. 6.4 Determination of test substance concentration in dosing formulation At the initial preparation, 10 mL for the concentration determination was collected from 1 point of the whole dosing formulation at each concentration (except the dosing formulation for the control group). Test substance concentration was measured according to the method validated in the analytical method validation for determination and stability test (Study No. P130586) and in a study entitled “Analytical Method Partial Validation Study (change in instrument) for the Determination of DEGMEE in Water for Injection (Study No. P210149)” conducted at the test facility. Results All of the results met the criteria. See any other information on materials Reference standard The test substance was used as the reference standard. Reagent see any other information on materials Preparation of standard stock solution Standard stock solution was prepared according to the following table (n=1). The preparation was conducted on the day of determination. See any other information on materials. Preparation of standard solution SS2 was pipetted and diluted according to the following table (n=1) to prepare standard solutions. See any other information on materials Preparation of processed sample Each concentration of the dosing formulation was pipetted from two points and diluted according to the following table to prepare processed samples. See any other information on materials. GC conditions Gas chromatograph: Nexis GC-2030 (Shimadzu Corporation) Detector: Hydrogen flame ionization detector Data processing software: LabSolutions (Shimadzu Corporation) Column: DB-1 (0.25 mm I.D. × 15 mm, Film thickness 0.1 µm, Agilent Technologies, Inc.) Column temperature: 50°C (1 min hold)-25°C /min rate-200°C (1 min hold) Injector temperature: 200°C Detector temperature: 220°C Carrier gas: Helium Carrier gas flow rate: 1 mL/min Make up gas: Helium Make up gas flow rate: 40 mL/min Air flow rate: 450 mL/min H2 flow rate: 40 mL/min Injection method: Split injection (Split ratio 3:1) Injection volume: 2 μL Syringe wash solvent: Acetone Calculation of the concentration The standard solutions (ST-1, ST-2, and ST-3) and processed sample solutions (PS1, PS2, and PS3) were injected into GC under the prescribed conditions. The test substance concentration in each dosing formulation was calculated by the following equation using DEGMEE peak area obtained by integrator automatically. The test substance concentrations in the dosing formulations were calculated from two points of each concentration, and the mean value was used as the test substance concentration in the formulations. Test substance concentration in dosing formulation (mg/mL) = (AT − b)/a × D/1000 AT : DEGMEE peak area in processed sample a : Slope of calibration curve b : Intercept of calibration curve D : Dilution factor: 900 (11 mg/mL), 8000/3 (33 mg/mL), 8000 (100 mg/mL) The regression equation for the calibration curve: Y = aX+b X : Nominal concentration of standard solution (μg/mL) Y : DEGMEE peak area a : Slope b : Intercept Data handling See any other information on materials
Details on mating procedure:
Mating was started at 12 weeks of age or more 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:
The dosing formulations were administered to females for which copulation was confirmed once daily from GD 6 to GD 19 (for 14 days).
Duration of test:
The dosing formulations were administered to females for which copulation was confirmed once daily from GD 6 to GD 19 (for 14 days).
Doses / concentrationsopen allclose all
Dose / conc.:
0 ppm
Dose / conc.:
110 ppm
Dose / conc.:
330 ppm
Dose / conc.:
1 000 ppm
No. of animals per sex per dose:
20
Control animals:
yes, concurrent no treatment
Details on study design:
Dose levels were set at 110, 330, and 1000 mg/kg/day. In a combined repeated dose and reproductive/developmental toxicity study conducted at the test facility (dose level: 0, 50, 250, and 1000 mg/kg/day, vehicle: water for injection) [1], the following effects were noted at 1000 mg/kg: prolongation of gestation period and low values of delivery index in dams and low values of number of litter and birth index, and viability index on postnatal Day 4 and high values of number of stillborn in offspring. Therefore, the high dose level was set at 1000 mg/kg which was expected to develop some toxicity. The middle and low dose levels were set at 330 and 110 mg/kg, respectively, with a common ratio of about 3. A control group (0 mg/kg) dosed with the vehicle (water for injection) alone was also established. See any other information on materials for Group Composition. For all pregnant animals, the blood was sampled upon necropsy (GD 20).

Examinations

Maternal examinations:
Clinical sign 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. Body weights Body weights were measured on GDs 0, 3, 6, 9, 12, 15, 18, and 20. Food consumption 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. Necropsy Necropsy was conducted after blood sampling (see Section 6.10.1) on GD 20. The animals were euthanized by exsanguination from the lateral iliac artery under thiopental sodium anesthesia by tail vein injection (RAVONAL®, Nipro ES Pharma co., Ltd., 30 mg/kg). The thoracoabdominal organs and tissues were immediately examined macroscopically after excision of the ovary and uterus. The thyroid was fixed in 10 vol% phosphate buffered formalin solution and preserved. Spleen with macroscopic lesion (No. 832) was fixed and preserved in the same manner, along with the same organ and tissue from one dam of the control group (No. 817). Organ weights After necropsy, the thyroid from all animals were weighed on both sides together (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. Histopathology The thyroid (unilateral) from all dams was embedded in paraffin, sectioned, stained with hematoxylin and eosin (HE), and examined microscopically. Hormone concentration analysis The total triiodothyronine (T3), total thyroxine (T4), and thyroid-stimulating hormone (TSH) plasma concentrations were measured for pregnant animals. The measurements were conducted at the test site.
Ovaries and uterine content:
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.
Blood sampling:
Approximately 1.5 mL of blood was collected from the subclavian vein of unanesthetized animals using a syringe treated with heparin sodium. Each sample 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 -79.0°C, permissible range: -90°C to -65°C) until shipment to the test site. The plasma sample tubes were labeled with the study number, animal number, and timing of blood sampling
Fetal examinations:
Observation on cesarean section 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 (0.1 to 0.5 mL/body, conc. 25 mg/mL) via an intraperitoneal injection. The placenta with gross lesion and the other normal placentas in the litter (No. 871) were fixed and preserved in 10 vol% phosphate buffered formalin solution, along with the normal placenta as controls from one dam of the control group (No. 812). 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. 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 AGD (Anogenital distance) 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. Allocation of live fetuses for skeletal and visceral examination 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 Bouin’s solution. Skeletal examination The fetuses in the control and high dose groups were examined. In addition, the fetuses in the middle dose group were examined because the results of the cesarean section examination showed a low number of live fetuses in the high dose group, and no evaluable number of fetuses could be obtained. As a result of the examination in the middle dose group, the fetuses in the low dose group were examined because the effects of the test substance treatment. Fetuses fixed in 70% ethanol were stained with alizarin red S according to the method of Staples et al. [2] 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. Visceral examination The fetuses in the control and high dose groups were examined. In addition, the fetuses in the middle dose group were examined because the results of the cesarean section examination showed a low number of live fetuses in the high dose group, and no evaluable number of fetuses could be obtained. As a result of the examination in the middle dose group, the fetuses in the low dose group were examined because the effects of the test substance treatment. Fetuses fixed in Bouin’s solution were observed for visceral anomalies by using the razor blade section method of Wilson [3] for the head, neck, and abdomen, and by the microdissection method of Nishimura [4] for the chest. Gender was checked for the second time at observation of the internal reproductive organs. The results of gender check corresponded to those obtained in the observation of external reproductive organs in all fetuses.
Statistics:
Statistical analysis Statistical analysis was performed with a computer system (tsPharma LabSite, Fujitsu Limited). In any case, two-tailed test was used and levels of p<0.01 and p<0.05 were considered significant. Multiple comparison test The mean and standard deviation were calculated and homogeneity of variance was tested by Bartlett’s method (significance level: 5%). When the groups were accepted as homogeneous, Dunnett’s multiple comparison test was used for comparison of the groups of data. When the groups of data were found to be heterogeneous by Bartlett’s test, Steel’s multiple comparison test was conducted. Body weight, food consumption, gravid uterus weight, absolute and relative organ weights, numbers of corpora lutea, numbers of implantations, numbers of live fetuses, body weights of live male and female fetuses*1, AGD*1, sex ratio of live fetuses (number of live male fetuses / number of live male and female fetuses × 100), and numbers of sternebrae and sacrocaudal vertebral body*2. *1: calculated on a litter basis by sex *2: calculated on a litter basis Two comparison test For numbers of sternebrae and sacrocaudal vertebral body, the results of the control and high dose groups, which were examined first, were analyzed by two comparison test. As a result, additional tests were performed in the middle and low dose groups, resulting in multiple groups; therefore, these parameters were analyzed and evaluated by multiple comparison tests (see Section 6.12.1). Two comparison test was conducted as follows: the mean and standard deviation were calculated and homogeneity of variance was tested by F-test (significance level: 5%). When the groups were accepted as homogeneous, t-test was used for comparison of the groups of data. When the groups of data were found to be heterogeneous, Aspin-Welch’s test was conducted.
Indices:
Wilcoxon’s rank sum test The incidence was calculated by the following equation and then compared with the control group. Pre-implantation loss index: [(Number of corpora lutea - number of implantations) / number of corpora lutea] × 100 Post-implantation loss index: [(Number of implantations - number of live fetuses)/ number of implantations)] × 100 Early resorption index: (Number of early resorptions / number of implantations) × 100 Late resorption index: (Number of late resorptions / number of implantations) × 100 Dead fetus index: (Number of dead fetuses / number of implantations) × 100 Incidence of fetuses with external anomalies (by type of anomaly): [Number of fetuses with anomalies (by type of anomaly) / number of fetuses examined] × 100 Incidence of fetuses with placental anomalies (by type of anomaly): [Number of placentas with anomalies (by type of anomaly) / number of placentas examined] × 100 Incidence of fetuses with skeletal anomalies (by type of anomaly): [Number of fetuses with anomalies (by type of anomaly) / number of fetuses examined] × 100 Incidence of fetuses with skeletal variations (by type of variation): [Number of fetuses with variations (by type of variation) / number of fetuses examined] × 100 Incidence of fetuses with visceral anomalies (by type of anomaly): [Number of fetuses with anomalies (by type of anomaly) / number of fetuses examined] × 100
Historical control data:
Historical control data provided see any other information on materials.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
See any other information on results
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight in the 1000 mg/kg group was significantly lower when compared with the control group on GD 20.
Food consumption and compound intake (if feeding study):
not examined
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:
effects observed, treatment-related
Description (incidence and severity):
Plasma TSH and total T4 concentrations in the 1000 mg/kg group were significantly higher when compared with the control group. No statistically significant differences were observed in total T3 between the control and any test substance treatment group.
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 treatment-related changes were observed in any dam. Although yellowish patch in the spleen was observed in one dam in the 110 mg/kg group, it was not judged to be treatment related because no similar change was observed in any dam in the 330 mg/kg group or higher.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Gravid uterus weight in the 1000 mg/kg group was significantly lower when compared with the control group Post-implantation loss index and early and late resorption index in the 1000 mg/kg group were significantly higher when compared with the control group. The post-implantation loss index was 57.9%, and 7 of 20 dams showed all embryo resorption in the 1000 mg/kg group. Along with these changes, the number of live fetuses in the 1000 mg/kg group was significantly lower when compared with the control group. In addition, body weights of live male and female fetuses in the 330 and 1000 mg/kg groups were significantly lower when compared with the control group. Anal atresia and acaudate were observed in 3 fetuses of 2 dams in the 1000 mg/kg group.
Details on results:
Each group initially consisted of 20 successfully copulated females, and all animals were pregnant. Therefore, the number of pregnant animals in each group was 20. Clinical signs The results are shown in "any other information on results". No abnormalities were observed in all animals. Body weights The results are shown in "any other information on results". Body weight in the 1000 mg/kg group was significantly lower when compared with the control group on GD 20. Food consumption The results are shown in "any other information on results". Mean food consumption in the 1000 mg/kg group was significantly lower or tended to be lower when compared with the control group from GDs 17 to 19. Although food consumption in the 330 and 1000 mg/kg groups was significantly lower section on GD 8, it was considered toxicologically insignificant because the values on GD 11 were comparable to those in the control group without a statistically significant difference and not accompanied by body weight loss. Necropsy The results are shown in "any other information on results". No treatment-related changes were observed in any dam. Although yellowish patch in the spleen was observed in one dam in the 110 mg/kg group, it was not judged to be treatment related because no similar change was observed in any dam in the 330 mg/kg group or higher. Organ weights The results are shown in "any other information on results" No statistically significant difference was observed in either absolute or relative thyroid weight between the control and any test substance treatment group. Histopathological examination The results are shown in "any other information on results". No treatment-related changes were noted in the thyroid. Ectopic thymic tissue and ultimobranchial remnant in the thyroid were observed in each test substance treatment group including the control group; however, these were not judged to be treatment related because they are congenital changes noted occasionally in normal rats. Gravid uterus weights The results are shown in "any other information on results". Gravid uterus weight in the 1000 mg/kg group was significantly lower when compared with the control group. Observation on cesarean section The results are shown in "any other information on results". Post-implantation loss index and early and late resorption index in the 1000 mg/kg group were significantly higher when compared with the control group. The post-implantation loss index was 57.9%, and 7 of 20 dams showed all embryo resorption in the 1000 mg/kg group. Along with these changes, the number of live fetuses in the 1000 mg/kg group was significantly lower when compared with the control group. In addition, body weights of live male and female fetuses in the 330 and 1000 mg/kg groups were significantly lower when compared with the control group. External examination The results are shown in "any other information on results". Anal atresia and acaudate were observed in 3 fetuses of 2 dams in the 1000 mg/kg group (Nos. 869 and 875). Placental examination The results are shown in "any other information on results". No treatment-related changes were observed in any group. Enlargement of the placenta was observed in 1 fetus (No. 871) in the 1000 mg/kg group; however, it was not judged to be treatment related because the value of the incidence was within the range of the historical data at the test facility. Anogenital distance The results are shown in "any other information on results". In the 1000 mg/kg group, AGI in the female fetuses was significantly higher when compared with the control group. In the male fetuses in the 1000 mg/kg group, AGD was significantly lower and AGI was significantly higher when compared with the control group, but these changes were not judged to be treatment related because these were due to the low fetal weight. Skeletal examinations The results are shown in "any other information on results". (1) Skeletal anomalies Skeletal anomalies were observed in 2 fetuses (1.3%), 2 fetuses (1.3%), 2 fetuses (1.3%), and 14 fetuses (23.2%) in the control, 110, 330, and 1000 mg/kg groups, respectively, and the incidence of skeletal anomalies in the 1000 mg/kg group was significantly higher when compared with the control group. A finding by type of skeletal anomalies considered to be treatment related in the 1000 mg/kg group was observed as summarized below. - Hemicentric thoracic centrum in 14 fetuses (23.2%) with a significantly higher difference when compared with the control group The following changes were not considered to be treatment related: hemicentric thoracic centrum in 1 fetus each (0.7 and 0.6%) in the 110 and 330 mg/kg groups, respectively, short rib in 1 fetus (0.6%) in the 110 mg/kg group, fused rib in 1 fetus (0.7%) in the 110 mg/kg group, and hemicentric lumbar centrum in 1 fetus (0.7%) in the 330 mg/kg group; however, there were no dose-dependent incidences, here were no statistically significant differences in the incidences of each type of anomalies compared to the control group, and/or they were limited to one fetus and one litter. (2) Skeletal variations Skeletal variations were observed in 29 fetuses (19.2%), 37 fetuses (25.1%), 57 fetuses (39.2%), and 51 fetuses (78.6%) in the control, 110, 330, and 1000 mg/kg groups, respectively, and the incidence of skeletal variations in the 330 and 1000 mg/kg groups was significantly higher when compared with the control group. Findings by type of skeletal variations considered to be treatment related were observed in the 330 and 1000 mg/kg groups as summarized below. - Bipartite ossification of thoracic centrum in 15 and 33 fetuses (9.6% and 48.2%) in the 330 and 1000 mg/kg groups, respectively, with significantly higher differences when compared with the control group - Wavy rib in 3 fetuses each (both 3.2%) in the 330 and 1000 mg/kg groups - Short supernumerary rib in 29 and 13 fetuses (18.8 and 22.7%) in the 330 and 1000 mg/kg groups, respectively - Full supernumerary rib in 6 fetuses (11.5%) in the 1000 mg/kg group - Bipartite ossification of sternebra in 12 fetuses (17.1%) in the 1000 mg/kg group, with a significantly higher difference when compared with the control group - Bipartite ossification of lumbar centrum in 4 and 8 fetuses (2.7 and 12.5%) in the 330 and 1000 mg/kg groups, respectively, with a significantly higher difference in the 1000 mg/kg group when compared with the control group - 7 lumbar vertebrae in 24 fetuses (44.6%) in the 1000 mg/kg group, with a significantly higher difference when compared with the control group The following changes were not considered to be treatment related: bipartite ossification of thoracic centrum in 4 fetuses in the 110 mg/kg group, full supernumerary rib in 5 fetuses each in the 110 and 330 mg/kg group, bipartite ossification of sternebra in 5 and 4 fetuses in the 110 and 330 mg/kg groups, respectively, and 7 lumbar vertebrae in 2 fetuses in the 330 mg/kg group; however, there were no dose-dependent incidences, there were no statistically significant differences in the incidences of each type of variation compared to the control group, the values of their incidences were almost within the range of the historical data at the test facility, and/or they were limited to one fetus and one litter. As for other findings shown in Table 13 (including aymmetry of sternebra with a significantly higher difference), the values of their incidences were almost within the range of the control group or the range of the historical data at the test facility in this study, there were no dose-dependent increases in their incidences, and/or they were limited to one fetus and one litter. (3) Ossification progress The number of ossification of the sacrocaudal body in the 110, 330, and 1000 mg/kg groups and the number of ossification of the sternebra in the 330 and 1000 mg/kg groups were significantly lower when compared with the control group. Visceral examinations The results are shown in "any other information on results". Visceral anomalies were observed in 9 fetuses (6.5%), 10 fetuses (7.5%), 35 fetuses (30.6%), and 42 fetuses (79.7%) in the control, 110, 330, and 1000 mg/kg groups, respectively, and the incidence of visceral anomalies in 330 and 1000 mg/kg groups was significantly higher when compared with the control group. A finding by type of visceral anomalies considered to be treatment related was observed in the 330 and 1000 mg/kg groups as summarized below. - Thymic remnant in the neck in 32 and 40 fetuses (28.3 and 75.6%) in the 330 and 1000 mg/kg groups, respectively, with significantly higher differences when compared with the control group - Ventricular septum defect in 2 fetuses (3.6%) in the 1000 mg/kg group As for other findings shown in "any other information on results", there were no dose-dependent increases in their incidences, and/or they were limited to one fetus and one litter. Hormone concentration (total T3, total T4, and TSH) analysis The data are shown in Hormone Measurement Report. Plasma TSH and total T4 concentrations in the 1000 mg/kg group were significantly higher when compared with the control group. No statistically significant differences were observed in total T3 between the control and any test substance treatment group.

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
Post-implantation loss index and early and late resorption index in the 1000 mg/kg group were significantly higher when compared with the control group. The post-implantation loss index was 57.9%, and 7 of 20 dams showed all embryo resorption in the 1000 mg/kg group. Along with these changes, the number of live fetuses in the 1000 mg/kg group was significantly lower when compared with the control group. In addition, body weights of live male and female fetuses in the 330 and 1000 mg/kg groups were significantly lower when compared with the control group.
Total litter losses by resorption:
effects observed, treatment-related
Description (incidence and severity):
The post-implantation loss index was 57.9%, and 7 of 20 dams showed all embryo resorption in the 1000 mg/kg group.
Early or late resorptions:
effects observed, treatment-related
Description (incidence and severity):
Post-implantation loss index and early and late resorption index in the 1000 mg/kg group were significantly higher when compared with the control group. The post-implantation loss index was 57.9%, and 7 of 20 dams showed all embryo resorption in the 1000 mg/kg group. Along with these changes, the number of live fetuses in the 1000 mg/kg group was significantly lower when compared with the control group. In addition, body weights of live male and female fetuses in the 330 and 1000 mg/kg groups were significantly lower when compared with the control group.
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not specified
Changes in number of pregnant:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
(1) Skeletal anomalies Skeletal anomalies were observed in 2 fetuses (1.3%), 2 fetuses (1.3%), 2 fetuses (1.3%), and 14 fetuses (23.2%) in the control, 110, 330, and 1000 mg/kg groups, respectively, and the incidence of skeletal anomalies in the 1000 mg/kg group was significantly higher when compared with the control group. A finding by type of skeletal anomalies considered to be treatment related in the 1000 mg/kg group was observed as summarized below. - Hemicentric thoracic centrum in 14 fetuses (23.2%) with a significantly higher difference when compared with the control group (2) Skeletal variations Skeletal variations were observed in 29 fetuses (19.2%), 37 fetuses (25.1%), 57 fetuses (39.2%), and 51 fetuses (78.6%) in the control, 110, 330, and 1000 mg/kg groups, respectively, and the incidence of skeletal variations in the 330 and 1000 mg/kg groups was significantly higher when compared with the control group. Findings by type of skeletal variations considered to be treatment related were observed in the 330 and 1000 mg/kg groups as summarized below. - Bipartite ossification of thoracic centrum in 15 and 33 fetuses (9.6% and 48.2%) in the 330 and 1000 mg/kg groups, respectively, with significantly higher differences when compared with the control group - Wavy rib in 3 fetuses each (both 3.2%) in the 330 and 1000 mg/kg groups - Short supernumerary rib in 29 and 13 fetuses (18.8 and 22.7%) in the 330 and 1000 mg/kg groups, respectively - Full supernumerary rib in 6 fetuses (11.5%) in the 1000 mg/kg group - Bipartite ossification of sternebra in 12 fetuses (17.1%) in the 1000 mg/kg group, with a significantly higher difference when compared with the control group - Bipartite ossification of lumbar centrum in 4 and 8 fetuses (2.7 and 12.5%) in the 330 and 1000 mg/kg groups, respectively, with a significantly higher difference in the 1000 mg/kg group when compared with the control group - 7 lumbar vertebrae in 24 fetuses (44.6%) in the 1000 mg/kg group, with a significantly higher difference when compared with the control group (3) Ossification progress The number of ossification of the sacrocaudal body in the 110, 330, and 1000 mg/kg groups and the number of ossification of the sternebra in the 330 and 1000 mg/kg groups were significantly lower when compared with the control group. The results are shown in Table 14 and Appendix 14. Visceral anomalies were observed in 9 fetuses (6.5%), 10 fetuses (7.5%), 35 fetuses (30.6%), and 42 fetuses (79.7%) in the control, 110, 330, and 1000 mg/kg groups, respectively, and the incidence of visceral anomalies in 330 and 1000 mg/kg groups was significantly higher when compared with the control group. A finding by type of visceral anomalies considered to be treatment related was observed in the 330 and 1000 mg/kg groups as summarized below. - Thymic remnant in the neck in 32 and 40 fetuses (28.3 and 75.6%) in the 330 and 1000 mg/kg groups, respectively, with significantly higher diferences when compared with the control group - Ventricular septum defect in 2 fetuses (3.6%) in the 1000 mg/kg group
Details on maternal toxic effects:
No maternal death occurred at any dose level.
Effects of DEGMEE treatment were observed at 1000 mg/kg as follows. Body weight
and food consumption were low on GD 20 and GD 17 or later, respectively. Gravid uterus
weight at necropsy was low due to embryo resorption described below. In the hormone
concentration analysis, plasma TSH and total T4 levels were high.
No effects of DEGMEE treatment were observed in pathology including thyroid weights
or histopathological examination (thyroid gland), or plasma hormone levels (total T3) at
any dose level.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
330 ppm
Based on:
test mat.
Basis for effect level:
body weight and weight gain
early or late resorptions
gross pathology

Maternal abnormalities

Key result
Abnormalities:
effects observed, treatment-related
Localisation:
uterus
Description (incidence and severity):
Gravid uterus weight at necropsy was low due to embryo resorption . All embryo resorption was noted in 7 of 20 dams, indicating higher early and late resorption index and post-implantation loss index.

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
body weights of live male and female fetuses in the 330 and 1000 mg/kg groups were significantly lower when compared with the control group.
Reduction in number of live offspring:
effects observed, treatment-related
Description (incidence and severity):
The number of live fetuses in the 1000 mg/kg group was significantly lower when compared with the control group
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
body weights of live male and female fetuses in the 330 and 1000 mg/kg groups were significantly lower when compared with the control group.
Anogenital distance of all rodent fetuses:
effects observed, treatment-related
Description (incidence and severity):
In the 1000 mg/kg group, AGI in the female fetuses was significantly higher when compared with the control group. In the male fetuses in the 1000 mg/kg group, AGD was significantly lower and AGI was significantly higher when compared with the control group, but these changes were not judged to be treatment related because these were due to the low fetal weight.
Changes in postnatal survival:
not examined
External malformations:
effects observed, treatment-related
Description (incidence and severity):
external anomalies, anal atresia and acaudate were noted in 3 fetuses.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
incidence of skeletal anomalies in the 1000 mg/kg group was significantly higher when compared with the control group. A finding by type of skeletal anomalies considered to be treatment related in the 1000 mg/kg group was observed as summarized below. - Hemicentric thoracic centrum in 14 fetuses (23.2%) with a significantly higher difference when compared with the control group Findings by type of skeletal variations considered to be treatment related were observed in the 330 and 1000 mg/kg groups as summarized below. - Bipartite ossification of thoracic centrum in 15 and 33 fetuses (9.6% and 48.2%) in the 330 and 1000 mg/kg groups, respectively, with significantly higher differences when compared with the control group - Wavy rib in 3 fetuses each (both 3.2%) in the 330 and 1000 mg/kg groups - Short supernumerary rib in 29 and 13 fetuses (18.8 and 22.7%) in the 330 and 1000 mg/kg groups, respectively - Full supernumerary rib in 6 fetuses (11.5%) in the 1000 mg/kg group - Bipartite ossification of sternebra in 12 fetuses (17.1%) in the 1000 mg/kg group, with a significantly higher difference when compared with the control group - Bipartite ossification of lumbar centrum in 4 and 8 fetuses (2.7 and 12.5%) in the 330 and 1000 mg/kg groups, respectively, with a significantly higher difference in the 1000 mg/kg group when compared with the control group - 7 lumbar vertebrae in 24 fetuses (44.6%) in the 1000 mg/kg group, with a significantly higher difference when compared with the control group (3) Ossification progress The number of ossification of the sacrocaudal body in the 110, 330, and 1000 mg/kg groups and the number of ossification of the sternebra in the 330 and 1000 mg/kg groups were significantly lower when compared with the control group.
Visceral malformations:
effects observed, treatment-related
Description (incidence and severity):
A finding by type of visceral anomalies considered to be treatment related was observed in the 330 and 1000 mg/kg groups as summarized below. - Thymic remnant in the neck in 32 and 40 fetuses (28.3 and 75.6%) in the 330 and 1000 mg/kg groups, respectively, with significantly higher diferences when compared with the control group - Ventricular septum defect in 2 fetuses (3.6%) in the 1000 mg/kg group

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
110 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
external malformations
skeletal malformations
visceral malformations

Fetal abnormalities

Key result
Abnormalities:
effects observed, treatment-related
Localisation:
external: tail
external: anus
skeletal: rib
skeletal: supernumerary rib
skeletal: vertebra
Description (incidence and severity):
At 1000 mg/kg, the following changes were noted; anal atresia and acaudate were noted in 3 fetuses. These changes are occasionally seen in normal rats; however, the frequency of occurrence suggests that they were caused by the test substance In skeletal examination, hemicentric thoracic centrum was noted as skeletal anomalies, and bipartite ossification of thoracic centrum, wavy rib, short supernumerary rib, full supernumerary rib, bipartite ossification of sternebra, bipartite ossification of lumbar centrum, and 7 lumbar vertebrae were observed as skeletal variations. In the visceral examination, ventricular septum defect was observed. The above-mentioned external, skeletal, and vesceral anomalies are teratogenic indicators; however, teratogenic effects could not be accurately evaluated at the dose in which severe toxicity to cause high embryo-fetal lethality (about 60%) occurred.

Overall developmental toxicity

Key result
Developmental effects observed:
no
Lowest effective dose / conc.:
110 mg/kg bw/day
Treatment related:
no

Any other information on results incl. tables

Historical data of placental anomalies in the test facility






























































 



Since 2014



Dams a



20



20



20



20



20



20



20



19



18



19



Fetuses b



283



287



290



272



286



289



287



262



261



263



Total c



0



0



0



0



0



2(0.6)



2(0.8)



0



2(0.7)



1(0.4)



Enlargement d



0



0



0



0



0



0



0



0



0



1(0.4)



Values in parentheses indicate percentages to the number of fetuses examined.


a: Number of examined dams, b: Number of examined fetuses, c: Number of fetuses with placental anomalies, d: Enlargement of placenta


Historical data of skeletal variations in the test facility





































































































 



Since 2014



Dams a



20



20



20



20



20



20



20



20



20



18



Fetuses b



145



137



146



149



150



141



148



154



155



139



Total c



22


(14.35)



20


(14.40)



26


(18.44)



14


(9.39)



22


(15.74)



8


(6.27)



23


(15.58)



32


(20.4)



20


(12.4)



16


(10.8)



Thoracic d



2(1.25)



0



2(1.63)



0



3(1.96)



2(1.55)



3(2.14)



4(2.9)



1(0.6)



2(1.2)



Full e



4(2.59)



2(1.34)



8(6.03)



0



0



0



3(2.05)



6(3.5)



4(2.2)



0



Sternebra f



2(1.34)



2(1.43)



2(1.25)



4(2.77)



1(0.71)



0



2(1.34)



0



1(0.6)



2(1.3)



Lumbar g



0



0



1(1.00)



0



0



0



0



2(1.2)



0



1(0.7)



Values in parentheses indicate percentages to the number of fetuses examined.


a: Number of examined dams, b: Number of examined fetuses, c: Number of fetuses with skeltal variations, d: Bipartite ossification thoracic centrum, e: Full supernumerary rib, f: Bipartite ossification of sternebra,


g: 7 lumbar vertebrae


 


 


The summary of hormone measurements is tabulated below.


 









































Group



Dose (mg/kg)



TSH


(ng/mL)



total T4 (nmol/L)



total L


(ng/mL)



Control



0



4.09 ± 1.41



14.5 ± 2.2



0.52 ± 0.08



Low dose



110



4.13 ± 0.97



13.8 ±2.8



0.51 ± 0.10



Middle dose



330



4.24 ± 0.85



14.9 ± 2.9



0.52 ± 0.06



High dose



1000



4.98 ± 0.90*



20.2 ± 4.1**



0.49 ± 0.15



 


*: p<0.05, Significantly different from control group by Dunnett's test


**: p<0.01, Significantly different from control group by Steel test


(Mean± SD)


Clinical signs






























































Test article


Dose (mg/kg)



Control


0



DEGMEE


110



DEGMEE


330



DEGMEE


1000



No. of animals



20



20



20



20



Pre-treatment Gestation period


No. of animals



 


 


20



 


 


20



 


 


20



 


 


20



Normal



20



20



20



20



 


Treatment


Gestation period No. of animals



 


 


 


20



 


 


 


20



 


 


 


20



 


 


 


20



Normal



20



20



20



20



 


Post-treatment Gestation period


No. of animals



 


 


 


20



 


 


 


20



 


 


 


20



 


 


 


20



Normal



20



20



20



20



2  Body weights (g) (Mean ± S.D.)











































































































































































Test article


Dose  (mg/kg)



Control


0



DEGMEE


110



DEGMEE


330



DEGMEE


1000



No. of animals



20



20



20



20



Pre-treatment



 



 



 



 



Gestation period



0



265.85



±



11.20



265.56



±



9.47



262.69



±



9.53



266.78



±



8.87



 



3



284.69



±



10.77



284.54



±



8.93



281.68



±



11.98



288.59



±



8.89



Treatment



 



 



 



 



 



 



 



 



 



 



 



 



 



Gestation period



6



295.49



±



12.13



298.30



±



10.88



292.80



±



13.17



301.82



±



11.96



 



9



307.83



±



12.73



310.86



±



10.34



304.77



±



13.68



309.42



±



12.41



 



12



325.59



±



14.06



328.93



±



12.59



324.04



±



17.12



333.47



±



13.78



 



15



342.53



±



15.12



346.36



±



13.86



340.27



±



18.47



347.51



±



14.19



 



18



386.47



±



18.21



389.07



±



15.66



377.66



±



21.60



374.04



±



19.00



Post-treatment Gestation period



 


20



 


421.71



 


±



 


20.70



 


425.64



 


±



 


16.82



 


411.33



 


±



 


22.28



 


386.69



 


±



 


27.02 ##



Significantly different from the control group ; ##:p<0.01 (Dunnett test)


 


Food consumption (g) (Mean ± S.D.)



























































































































































Test article


Dose  (mg/kg)



Control


0



DEGMEE


110



DEGMEE


330



DEGMEE


1000



No. of animals



20



20



20



20



Pre-treatment



 



 



 



 



Gestation period



0



17.80



±



1.61



17.71



±



1.79



17.31



±



2.61



19.00



±



2.14



 



2



22.55



±



1.85



21.85



±



2.38



22.19



±



2.17



23.72



±



2.09



 



5



23.56



±



2.39



23.51



±



2.63



23.46



±



2.36



24.70



±



2.77



Treatment


Gestation period



 


8



 


24.22



 


±



 


1.84



 


24.25



 


±



 


2.27



 


22.57



 


±



 


2.17 #



 


22.50



 


±



 


2.21 #



 



11



25.24



±



2.74



24.77



±



2.34



24.93



±



2.03



25.67



±



3.38



 



14



26.26



±



2.90



25.82



±



2.47



25.35



±



2.91



25.50



±



1.97



 



17



29.64



±



3.41



28.13



±



2.36



27.55



±



3.48



26.80



±



3.28 #



 



19



26.82



±



3.35



26.78



±



3.05



26.15



±



2.82



24.76



±



3.76



 













































Necropsy findings



 



Test article



Control



DEGMEE



DEGMEE



DEGMEE



Dose (mg/kg)



0



110



330



1000



No. of animals



20



20



20



20



 


Not remarkable



 


20



 


19



 


20



 


20



HEMATOPOIETIC SYSTEM


Spleen


Yellowish patch



 


 


0



 


 


1



 


 


0



 


 


0



 


Absolute organ weights (Mean ± S.D.)





























































Sex        Test                  article



Dose (mg/kg)



No. of animals



Terminnal body weight (g)



 



 



Thyroids (mg)



 



 



Female  Control



0



20



421.71



±



20.70



23.13



±



4.66



DEGMEE



110



20



425.64



±



16.82



21.94



±



2.55



DEGMEE



330



20



411.33



±



22.28



20.76



±



3.38



DEGMEE



1000



20



386.69



±



27.02



21.54



±



3.47



Relative organ weights (Mean ± S.D.)






























































Sex



Test article



Dose (mg/kg)



No. of animals



Terminal Body Weight (g)



Thyroids (x10-3%)



Female



Control



0



20



421.71



±



20.70



5.48



±



1.05



 



DEGMEE



110



20



425.64



±



16.82



5.16



±



0.56



 



DEGMEE



330



20



411.33



±



22.28



5.07



±



0.92



 



DEGMEE



1000



20



386.69



±



27.02



5.59



±



0.97



Not significantly different from the control group


 


Histopathological findings

































































Test Article


Dose (mg/kg)



 



Control


0



 



DEGMEE


110



 



DEGMEE


330



 



DEGMEE


1000



 



Number of animals



 



20



 



20



 



20



 



20



ENDOCRINE SYSTEM



 



 



 



 



 



 



 



 



 



Thyroid gland -----------------------------------------------------



Number examined



20



 



20



 



20



 



20



 



Ectopic thymic tissue present


Ultimobranchial remnant


present



 



3


 


2



 


3


 


2



3


 


4



 


3


 


4



2


 


8



 


2


 


8



1


 


7



 


1


 


7



Gravid uterus weights (Mean ± S.D.)





















































Sex            Test article



Dose (mg/kg)



No. of animals



Terminal Body Weight (g)



Gravid Uterus (g)



Female  Control



0



20



421.71 ±  20.70



83.60 ±       8.52



 



DEGMEE



110



20



425.64



±



16.82



81.59



±      7.91



 



DEGMEE



330



20



411.33



±



22.28



75.50



±  11.68



 



DEGMEE



1000



20



386.69



±



27.02



40.39



±  22.36 $$



Significantly different from the control group ; $$:p<0.01 (Steel test)


9  Observation on cesarean section (Mean ± S.D.)
















































































































































































































































Test article Dose (mg/kg)



 



Control


0



 



DEGMEE 110



 



DEGMEE 330



 



DEGMEE 1000



 



No. of animals



 



20



 



20



 



20



 



20



No. of corpora lutea (A)



Total



323


16.2 ±



 


2.2



307


15.4 ±



 


1.4



306


15.3 ±



 


1.7



308


15.4 ±



 


0.9



 



No. of implants (B)



Total



306


15.3 ±



 


1.2



299


15.0 ±



 


1.3



287


14.4 ±



 


1.9



294


14.7 ±



 


1.0



 



Pre-implantation loss



Total



17



 



8



 



19



 



14



 



 



(A-B)/(A)



(%)



4.5 ±



7.1



2.5 ±



3.7



6.1 ±



11.0



4.4 ±



6.4



 



No. of dead implants


Early resorptions



 


Total



 


12



 



 


9



 



 


14



 



 


118



 



 



 


Late resorptions



(%)


Total (%)



4.1 ± 0


0.0 ±



5.2


 


0.0



3.0 ± 0


0.0 ±



4.1


 


0.0



5.1 ± 1


0.7 ±



6.1


 


3.2



39.8 ± 54


18.2 ±



29.1


 


17.1



**


 


**



Dead fetuses



Total (%)



0


0.0 ±



 


0.0



0


0.0 ±



 


0.0



0


0.0 ±



 


0.0



0


0.0 ±



 


0.0



 



 


Post-implantaion loss (C)



 


Total



 


12



 



 


9



 



 


15



 



 



 


172



 



 



 



(C)/(B)



(%)



4.1 ±



5.2



3.0 ±



4.1



5.8 ±



7.9



 



57.9 ±



38.6



**



 



No. of live fetuses



 



14.7 ±



1.6



14.5 ±



1.4



13.6 ±



2.3



 



6.1 ±



5.5



$$



 



Male



 



156



 



170



 



136



 



 



54



 



 



 



Female



 



138



 



120



 



136



 



 



68



 



 



 



Total


Sex ratio M/(M+F) (%)



 



294


53.1 ±



 


13.9



290


58.4 ±



 


12.5



272


50.4 ±



 


15.0



 



122


45.3 ±



 


9.5



 



 


(13)



Weight of fetuses (g)


Male



 



 


3.84 ±



 


0.35



 


3.66 ±



 


0.29



 


3.37 ±



 


0.35



 


##



 


2.55 ±



 


0.21



 


##



 


(13)



Female



 



3.63 ±



0.37



3.42 ±



0.24



3.19 ±



0.24



$$



2.48 ±



0.18



$$



(13)



Significantly different from the control group ; ##:p<0.01 (Dunnett test) Significantly different from the control group ; $$:p<0.01 (Steel test) Significantly different from the control group ; **:p<0.01 (Wilcoxon test) Number in parentheses indicates the number of dams


 


External examinations (Mean ± S.D.)


 




























































































Test article


Dose (mg/kg)



Control


0



 



DEGMEE


110



 



DEGMEE


330



 



DEGMEE


1000



 



No. of animals (F0)



20



 



20



 



20



 



13



No. of fetuses examined



294



 



290



 



272



 



122



Anomalies



 



 



 



 



 



 



 



 



Frequencies (%)



0


0.0 ±



 


0.0



0


0.0 ±



 


0.0



0


0.0 ±



 


0.0



3


2.0 ±



 


5.4



Types and frequencies



 



 



 



 



 



 



 



 



 


Anal atresia (%)



 


0


0.0 ±



 


 


0.0



 


0


0.0 ±



 


 


0.0



 


0


0.0 ±



 


 


0.0



 


3


2.0 ±



 


 


5.4



Acaudate (%)



0


0.0 ±



 


0.0



0


0.0 ±



 


0.0



0


0.0 ±



 


0.0



3


2.0 ±



 


5.4



 


Placental examinations (Mean ± S.D.)









































Test article



Control



DEGMEE



DEGMEE



DEGMEE



Dose (mg/kg)



0



110



330



1000



No. of animals (F0)



20



20



20



13



No. of placentae examined



294



290



272



122



Anomalies



 



 



 



 



 




































Frequencies


(%)



0


0.0 ±



 


0.0



0


0.0 ±



 


0.0



0


0.0 ±



 


0.0



1


0.8 ±  2.8



Types and frequencies



 



 



 



 



 



 



 



 


Enlargement of placenta (%)



 


0


0.0 ±



 


 


0.0



 


0


0.0 ±



 


 


0.0



 


0


0.0 ±



 


 


0.0



 


1


0.8 ±  2.8



Not significantly different from the control group


 


Anogenital distance - Male














































Sex



Test article



Dose (mg/kg)



No. of animals



Anogenital distance


(mm)



Anogenital distance


(mm/3√BW)



Female



Control



0



20



3.675 ± 0.157



2.353 ± 0.128



 



DEGMEE



110



20



3.742 ± 0.243



2.429 ± 0.138



 



DEGMEE



330



20



3.608 ± 0.258



2.409 ± 0.126



 



DEGMEE



1000



13



3.378 ± 0.192 ##



2.477 ± 0.120 #



Significantly different from the control group ; #:p<0.05, ##:p<0.01 (Dunnett test)


 


Anogenital distance - Female














































Sex



Test article



Dose (mg/kg)



No. of animals



Anogenital distance


(mm)



Anogenital distance


(mm/3√BW)



Female



Control



0



20



1.965 ± 0.258



1.281 ± 0.157



 



DEGMEE



110



20



1.915 ± 0.193



1.273 ± 0.125



 



DEGMEE



330



20



1.862 ± 0.257



1.267 ± 0.173



 



DEGMEE



1000



13



1.921 ± 0.236



1.425 ± 0.181 #



Significantly different from the control group ; #:p<0.05 (Dunnett test)


 


Skeletal examinations (Mean ± S.D.)






























































































































































































































































































































































































































































Test article



Control



DEGMEE



DEGMEE



DEGMEE



Dose    (mg/kg)



0



110



330



1000



No. of animals (F0)



20



20



20



13



No. of fetuses examined



157



155



146



69



Anomalies



 



 



 



 



Frequencies



2



 



2



 



2



 



14



 



(%)



1.3 ±



5.6



1.3 ±



4.1



1.3 ±



4.1



23.2 ±



19.0 **



Types and frequencies



 


Hemicentric thoracic centrum



 


0



 



 


1



 



 


1



 



 


14



 



(%)



0.0 ±



0.0



0.7 ±



3.2



0.6 ±



2.8



23.2 ±



19.0 **



Short rib



2



 



1



 



0



 



0



 



(%)



1.3 ±



5.6



0.6 ±



2.8



0.0 ±



0.0



0.0 ±



0.0



Fused rib



0



 



1



 



0



 



0



 



(%)



0.0 ±



0.0



0.7 ±



3.2



0.0 ±



0.0



0.0 ±



0.0



Hemicentric lumbar centrum



0



 



0



 



1



 



0



 



(%)



0.0 ±



0.0



0.0 ±



0.0



0.7 ±



3.2



0.0 ±



0.0


 



Variations



Frequencies



29



 



37



 



57



 



51



 



(%)



19.2 ± 



24.2



25.1 ± 



25.6



39.2 ± 



22.7 **



78.6 ± 


 



24.9 **



Types and frequencies



Bipartite ossification thoracic centrum



3



 



4



 



15



 



33



 



(%)



2.1 ±



6.8



3.1 ±



7.9



9.6 ±



11.7 **



48.2 ± 



33.7 **



Cervical rib



0



 



3



 



3



 



0



 



(%)



0.0 ±



0.0



2.3 ±



7.0



2.1 ±



7.0



0.0 ±



0.0



Wavy rib



0



 



0



 



3



 



3



 



(%)



0.0 ±



0.0



0.0 ±



0.0



3.2 ±



8.5



3.2 ±



8.4



Full supernumerary rib



3



 



5



 



5



 



6



 



(%)



1.7 ±



5.4



3.4 ±



7.5



3.2 ±



7.0



11.5 ± 



19.4



Short supernumerary rib



22



 



11



 



29



 



13



 



(%)



14.9 ±



21.4



7.1 ±



8.7



18.8 ±



18.3



22.7 ±



28.1



Asymmetry of the sternebra



2



 



13



 



5



 



3



 



(%)



1.1 ±



3.3



9.3 ±



14.7 **



4.1 ±



8.5



4.7 ±



9.2



Bipartite ossification of sternebra



0



 



5



 



4



 



12



 



(%)



0.0 ±



0.0



3.2 ±



5.7 *



3.5 ±



8.4 *



17.1 ±



22.5 **



Bipartite ossification of lumbar centrum



1



 



1



 



4



 



8



 



(%)



0.5 ±



2.2



0.6 ±



2.8



2.7 ±



5.5



12.5 ±



18.0 *



Supernumerary lumbar arch



0



 



0



 



1



 



0



 



(%)



0.0 ±



0.0



0.0 ±



0.0



0.7 ±



3.2



0.0 ±



0.0



Supernumerary lumbar centrum



0



 



0



 



1



 



0



 



(%)



0.0 ±



0.0



0.0 ±



0.0



0.7 ±



3.2



0.0 ±



0.0



5 lumbar vertebrae



1



 



0



 



0



 



0



 



(%)



0.6 ±



2.8



0.0 ±



0.0



0.0 ±



0.0



0.0 ±



0.0



7 lumbar vertebrae



0



 



0



 



2



 



24



 



(%)



0.0 ±



0.0



0.0 ±



0.0



1.3 ±



3.8



44.6 ±



36.8 **



Significantly different from the control group ; *:p<0.05, **:p<0.01 (Wilcoxon test)









































































Skeletal examinations (Mean ± S.D.) (Continued)



 



Test article



Control



DEGMEE



 



DEGMEE



 



DEGMEE



Dose    (mg/kg)



0



110



 



330



 



1000



No. of animals (F0)



20



20



 



20



 



13



No. of fetuses examined



157



155



 



146



 



69



Degree of ossification



 



 



 



 



 



 



No. of sacrocaudal body



8.0 ±     0.3



7.4 ±



0.4 $$



6.6 ±



0.6 $$



3.1 ±     1.2 $$



No. of sternebrae



5.9 ±     0.2



5.7 ±



0.3



4.9 ±



0.7 $$



2.0 ±     1.1 $$



Significantly different from the control group ; $$:p<0.01 (Steel test)


 




























































































































































































































































































































Visceral examination (F1 fetuses) (Mean ± S.D.)



 



Test article



Control



 



 



DEGMEE



 



DEGMEE



 



DEGMEE



 



Dose (mg/kg)



0



 



 



110



 



330



 



1000



 



No. of animals (F0)



20



 



 



20



 



20



 



11



 



No. of fetuses examined



137



 



 



135



 



126



 



53



 



Anomalies



 



 



 



 



 



 



 



 



 



Frequencies



9



 



 



10



 



35



 



42



 



(%)



6.5



±



13.3



7.5 ±



12.1



30.6 ±



36.2 *



79.7 ±



19.4 **



Types and frequencies



 



 



 



 



 



 



 



 



 



 


Thymic remnant in the neck



 


6



 



 



 


4



 



 



 


32



 



 



 


40



 



 



(%)



4.6



±



11.9



3.1



±



6.4



28.3



±



34.7 **



75.6



±



24.7 **



Ventricular septum defect



0



 



 



0



 



 



0



 



 



2



 



 



(%)



0.0



±



0.0



0.0



±



0.0



0.0



±



0.0



3.6



±



8.1



Persistent A-V canal



0



 



 



0



 



 



0



 



 



1



 



 



(%)



0.0



±



0.0



0.0



±



0.0



0.0



±



0.0



2.3



±



7.5



Malpositioned aorta origin



0



 



 



0



 



 



0



 



 



1



 



 



(%)



0.0



±



0.0



0.0



±



0.0



0.0



±



0.0



2.3



±



7.5



Retroesophageal subclavian



0



 



 



1



 



 



0



 



 



1



 



 



(%)



0.0



±



0.0



0.7



±



3.2



0.0



±



0.0



1.5



±



5.0



Dilated renal pelvis



0



 



 



0



 



 



1



 



 



0



 



 



(%)



0.0



±



0.0



0.0



±



0.0



2.5



±



11.2



0.0



±



0.0



Dilated ureter



4



 



 



5



 



 



4



 



 



1



 



 



(%)



2.8



±



5.8



3.7



±



8.0



4.8



±



12.9



1.5



±



5.0



Significantly different from the control group ; *:p<0.05, **:p<0.01 (Wilcoxon test)

Applicant's summary and conclusion

Conclusions:
In conclusion, the no observed adverse effect level (NOAEL) of DEGMEE was considered to be 330 mg/kg for general toxicity and reproductive function in dams, and 110 mg/kg for embryonic/fetal development under the conditions of this study.
Executive summary:

In order to investigate the effects of DEGMEE on dams and embryo-fetal development in rats, DEGMEE at dose levels of 110, 330, and 1000 mg/kg/day was administered orally once daily to pregnant Crl:CD(SD) rats (20 animals per group) from Days 6 to 19 of gestation (the period from implantation to the day before cesarean section). The animals in the control group were given the vehicle (water for injection) alone. The dosing volume of each group was 10 mL/kg. During the gestation period, clinical observations and measurements of the body weights and food consumption were conducted.  On Day 20 of gestation, the animals were euthanized for cesarean sections and necropsy including the measurement of gravid uterus and thyroid weights, and examinations of the embryos/fetuses and placentas were performed. Live fetuses were observed macroscopically for external anomalies and measured for body weights and AGD, and visceral and skeletal examinations were conducted. In addition, plasma hormone concentrations (total T3, total T4, and TSH) were measured and histopathology for the thyroid was conducted for dams.


 


Effects on dams


No maternal death occurred at any dose level.


At 1000 mg/kg, body weight and food consumption were low on GD 20 and GD 17 or later, respectively. Gravid uterus weight at necropsy was low. In the hormone concentration analysis, plasma TSH and total T4 levels were high.


No effects of DEGMEE treatment were observed in pathology including thyroid weights or histopathological examination (thyroid gland), or plasma hormone levels (total T3) at any dose level.


 


Effects on Embryo-fetal development


At 1000 mg/kg, the following changes were noted.  All embryo resorption was noted in 7 of 20 dams, indicating higher early and late resorption index and post-implantation loss index. Along with these changes, the number of live fetuses was low. In the live fetuses, fetal weights were low in both sexes, and several structural abnormalities were noted in external, visceral, and skeletal examinations. In anogenital distance measurement, AGI was high in the female fetuses. In ossification progress, the number of ossification of the sacrocaudal body and sternebra was low. Visceral examinations revealed a thymic remnant in the neck. Consequently, 1000 mg/kg of DEGMEE showed a high embryo- fetal lethality, growth retardation, and structural abnormalities. These changes noted at 1000 mg/kg were due to the effects by treatment of DEGMEE; however, the dose was so toxic that embryo-fetal lethality occurred frequently, and the teratogenic potential and effects on endocrine of DEGMEE could not be accuralately evaluated.


 


 


At 330 mg/kg, the following changes were noted. Body weights of live male and female fetuses were low. In the skeletal examinations, bipartite ossification of thoracic centrum, wavy rib, short supernumerary rib, and bipartite ossification of lumbar centrum were noted as skeletal variations. In ossification progress, the number of ossification of the sacrocaudal body and sternebra was low. No effects of DEGMEE were observed in examination of uterine contents (number of corpora lutea, implantations, live fetuses, or resorption, pre- implantation loss, or post- implantation loss) or examination of fetuses (sex ratio, AGD, or visceral, external, or placental development). Therefore, 330 mg/kg of DEGMEE is considered to have had no embryo-fetal lethality or structural abnormalities (excluding skeletal variations) in rats in this study. In addition, there was no teratogenic effect.


 


At 110 mg/kg, the number of ossification of the sacrocaudal body was low in the ossification progress. No effects of DEGMEE were observed in examination of uterine contents (number of corpora lutea, implantations, live fetuses, or resorption, pre- implantation loss, or post- implantation loss) or examination of fetuses (sex ratio, fetal weights, AGD, or visceral, skeletal, external, or placental development). Therefore, 110 mg/kg of DEGMEE is considered to have had no embryo-fetal lethality or structural abnormalities in rats in this study.  In addition, there was no teratogenic effect.


 


In conclusion, the no observed adverse effect level (NOAEL) of DEGMEE was considered to be 330 mg/kg for general toxicity and reproductive function in dams, and 110 mg/kg for embryonic/fetal development under the conditions of this study.