Registration Dossier

Administrative data

Description of key information

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October 4, 2013 to January 17, 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed in accordance with OECD test guidelines in compliance with GLP.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
yes
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
Species: RatStrain: Crl:CD(SD)Rationale for selection: This strain is widely used in toxicity studies using rodents, and there is abundant historical data and a large number of animals are available.Number and sex of animals purchased: 53 males and 64 femalesSupplier (Breeding facility): Charles River Laboratories Japan, Inc. (Hino Breeding Center)Age: At receipt: 8-week-old (males and females); At the start of administration: 9-week-old (males and females)Body weight range at receipt: 245.2 to 274.3 g (males, permissible range: 240 to 330 g); 166.1 to 204.0 g (females, permissible range: 160 to 230 g)Quarantine and acclimatization: At animal receipt, species, strain, age, number and sex were confirmed, and observation for clinical signs and external appearance and body weight measurement were performed.Quarantine period was set for 5 days and acclimatization period was set for 7 days (between animal receipt and on the day before administration). During these periods, all animals were observed once daily and were checked for body weight gain from animal receipt to the end of quarantine. In addition, detailed clinical observations were performed once before grouping. According to these results, small of testis was noted in one male (No. 1053).Identification of animals during quarantine and acclimatization periods: Upon receipt, animals were identified by marking the ID No. (the last one or two figures of quarantine animal number) on the tail with a black oil-based felt tip pen, and a label indicating the study number for computer system, quarantine animal number and sex was attached to the front of each cage. The quarantine animal numbers were shown below.Males: 1001 to 1053Females: 2001 to 2064Grouping: All animals except for one male (No. 1053) were used for grouping based on the results of the observation for clinical signs, detailed clinical observations and body weight measurements during the quarantine and acclimation periods. Animals were assigned to groups by the stratified randomization on the basis of body weight measured on the day before the first dosing. Animals weighing within the mean body weights ± 20% (calculated for each sex) were used for this study. Body weight range of animals used for this study was shown below.Males: 305.2 to 343.5 g (permissible range: 263.1 to 394.7 g)Female: 207.7 to 235.8 g (permissible range: 177.1 to 265.6 g)Identification of animals after grouping: The animals were identified by ear tags inscribed with animal number, and a label indicating the study number for computer system, animal number, dose level and sex was attached to the front of each cage.Handling of remaining animals: The remaining animals were euthanized by exsanguination under anesthesia by intraperitoneal injection of pentobarbital sodium after initiation of administration.Animal managementAnimal room: A101Environmental conditionsRoom temperature: Actual range: 22.4° to 24°C (permissible range: 20.0° to 26.0°C)To record room temperature, a temperature-humidity supervisory system (minimum digit number: one decimal place) was used; however, a self-recording thermohygrometer (minimum digit number: last one digit of integral number) was used during the system maintenance period.Relative humidity: Actual range: 45.1% to 68.9% (permissible range: 35.0% to 75.0%)Ventilation: 10 to 20 times per hourLighting period: 12 hours per day (7:00 to 19:00)Housing equipmentCages(1) For males and for females except during the gestation and lactation periods Stainless-steel cages (W × D × H: 226 × 346 × 198 mm)(2) For females during the gestation and lactation periods Polymethylpentene cages (W × D × H: 220 × 380 × 195 mm)Cage racks and feeders: Made of stainless-steelSanitary tray under cages: Made of aluminumWatering bottle: PolymethylpenteneNumber of animals per cageBefore grouping: one animal per cageAfter grouping: one male and one female per cage during the mating period, one dam and its litter per cage during the lactation period, one animal per cage for the other periodsFrequency of housing equipment exchange(1) Cage racks: Once within four weeks thereafter(2) Stainless-steel cages and feeders: Once at the first dosing, and once within two weeks thereafter(3) Polymethylpentene cages: Once at the day of successful mating, twice within one week thereafter or no exchange on Day 20 of gestation to Day 4 of lactation(4) Caps for polymethylpentene cages: Once at the day of successful mating, once within two weeks thereafter or no exchange on Day 20 of gestation to Day 4 of lactation(5) Sanitary trays under cages: Once within four days or once daily for males during mating period(6) Watering bottles: Twice within one weekCleaning and sanitation: All housing equipment was sterilized by autoclaving after washing with water. The floor of the animal room was cleaned and wiped every day with a disinfectant-soaked mop (NaClO).BeddingDescription: Wood chip satirized by autoclave (White Flake, Charles River Laboratories Japan, Inc.)Frequency of bedding exchange: Bedding placed in the polymethylpentene cages was exchanged concurrently with the cage exchange.Analysis: Data of bedding were obtained from Charles River Laboratories Japan, Inc., and contaminants in the bedding were confirmed to be within the acceptable limits established by the test facility.DietDescription: Autoclave-sterilized pellet diet (CRF-1, Oriental Yeast Co., Ltd.)Feeding method: Diet was given ad libitum except during fresh urine collection and measurement of motor activity. Animals were fasted from the evening before scheduled necropsy for about 17 hours and above.Analysis: Data for each lot (lot Nos. 130605, 130703 and 130802) of diet were obtained from Oriental Yeast Co., Ltd., and contaminants in the diet were confirmed to be within the acceptable limits established by the test facility.WaterDescription: Well waterAntisepsis: Well water admixed with NaClO (free residual chlorine level: about 2 ppm)Water supply method: Water was given ad libitum except during the measurement of motor activity. Automatic water system was used for males and for females except during gestation and lactation periods. Watering bottles were used for females during gestation and lactation periods.Analysis: Water was analyzed twice a year at Nichigo Kyushu Co., Ltd. The results were confirmed to be in compliance with the SOP of the test facility.
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
Preparation method and frequency, and storage conditionsDosing formulations were prepared once within 7 days based on the results of another 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: 3.5° to 6.3°C, permissible range: 1° to 15°C, 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 taken into clear glass vials for each dosing day.(100 mg/mL dosing formulation)(1) A prescribed amount of the test substance was weighed and transferred to a beaker.(2) An appropriate amount of the vehicle was added and stirred with a magnetic stirrer to dissolve the test substance.(3) After dissolution, the mixture was transferred to a measuring cylinder. 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 quantity 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.(5 and 25 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 5 and 25 mg/mL formulations. The dosing formulations after preparation were mixed several times by end-over-end rotation and taken into brown glass vials.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Determination of the test substance concentrations in dosing formulations: At the initial preparation, about 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 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.Result: All of the results met the criteria.Reference standard: The test substance was used as the reference standard.Preparation of standard stock solution: Standard stock solution was prepared according to the following table (n=1) (see Any other information for the table). The preparation was conducted on the day of determination.Preparation of standard solution: SS2 was pipetted and diluted according to the following table (n=1) to prepare standard solutions (see Any other information for the table).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.GC conditionsGas chromatograph: HP6890 (Agilent Technologies, Inc.)Detector: Hydrogen flame ionization detectorData processing software: ChemStation (Agilent Technologies, Inc.)Column: DB-1 (0.25 mm I.D. × 15 m, 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°CDetector temperature: 220°CCarrier gas: HeliumCarrier gas flow rate: 1 mL/minMake up gas: HeliumMake up gas flow rate: 40 mL/minAir flow rate: 450 mL/minH2 flow rate: 40 mL/minInjection method: Split injection (Split ratio 3:1)Injection volume: 2 μLSyringe wash solvent: AcetoneCalculation of the concentration: 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/1000AT: DEGMEE peak area in processed samplea: Slope of calibration curveb: Intercept of calibration curveD: Dilution factor: 400 (5 mg/mL), 2000 (25 mg/mL), 8000 (100 mg/mL)The regression equation for the calibration curve: Y = aX+bX: Nominal concentration of standard solution (μg/mL)Y: DEGMEE peak areaa: Slopeb: Intercept
Duration of treatment / exposure:
Males: From 15 days before mating (Days 1 to 15) until day before the necropsy through the mating period (42 days in total).Females: From 15 days before mating (Days 1 to 15) until the Day 4 of lactation (day of delivery was Day 0 of lactation) through the mating and pregnancy periods and delivery. The females not successfully mated and those not delivered were kept until day before the necropsy.Recovery females (Satellite females): For 42 days without mating (the same period with that for males)
Frequency of treatment:
Once daily between 8:21 and 12:27 (permissible range: between 8:00 and 15:00)
Remarks:
Doses / Concentrations:The dose levels were set at 0, 50, 250 and 1000 mg/kg.Basis:nominal in water
No. of animals per sex per dose:
Each test group consisted of 12 males (including 5 males for recovery test) and 12 females (5 satellite females each were added for the control and 1000 mg/kg groups in recovery test).
Control animals:
yes, concurrent vehicle
Details on study design:
Dose level and its rationale: As a result of a study conducted at the test facility entitled “A 14-Day Repeated Dose Oral Toxicity Study of DEGMEE in Rats, Study No. P130588” (dose levels: 0, 110, 330 and 1000 mg/kg/day, vehicle: water for injection), the following changes were noted.In the hematology, low values of MCHC and low values of reticulocyte count (and ratio) and platelet count were noted in males of the 330 mg/kg group and above and in males of the 1000 mg/kg group, respectively. In the blood chemistry, low values of ALP were noted in both sexes of the 1000 mg/kg group. High values of liver weight and low values of ovary weight were noted in males of the 1000 mg/kg group and in females of the 1000 mg/kg group, respectively.From the above results, a high dose level in this study was set at 1000 mg/kg, which is expected showing of some toxicity effect, and subsequent doses were set at 250 and 50 mg/kg. A control group (0 mg/kg group) dosed with the vehicle (water for injection) alone was also established.
Positive control:
Positive control not required for this study.
Observations and examinations performed and frequency:
Observation and measurement for repeated dose toxicity: The following items were examined. The initial day of dosing was designated as Day 1, and Day 1 to Day 7 was designated as Week 1. The period after Day 43 was designated as the recovery period. For the test females, the day of successful copulation was designated as Day 0 of gestation and the day of delivery was designated as Day 0 of lactation.Clinical observation: All animals were observed twice a day (before and after dosing) during the dosing period, and once a day in the other periods.Functional observation battery: For all animals, detailed clinical observations were performed once before the start of dosing and once a week in the afternoon during the dosing and recovery periods (day of gestation or parturition was prescind). The function tests and motor activity measurement were performed in 5 males per group with the smaller animal numbers once in the afternoon in the final week (Week 6) of the dosing period. For females, 5 dams from the nearer parturition date were selected from each group and performed once in the afternoon during the lactation period. These examinations were performed following the clinical observation after dosing during the dosing period.As a result, the motor activity measurement for females was performed in the final week (Week 8) of the recovery period, since a low value of motor activity was noted in females of the 1000 mg/kg groups during the dosing period. The function tests were not performed during the recovery period, since no treatment-related changes were noted for the parameters during the dosing period.These examinations were not conducted on a blind test bases.Detailed clinical observations(1) Hand held observation: Animals were removed from the cage for observation by grasping their trunk gently from behind.Parameters: Reactivity to handling, trauma, color of skin, soiled fur, exophthalmos, palpebral closure, color of conjunctiva, secretion, lacrimation, salivation, piloerection and pupil size(2) Observation on open field: The floor of the open field was wiped with a tightly squeezed wet cloth before examination. The cloth was washed every time of examination. Animals were placed in the center of the open field and observed quietly for 2 minutes.Parameters: Rearing, arousal, urination, defecation, posture/body position, respiration, gait, tremor, convulsion, stereotypy and bizarre behaviour.Functional tests(1) Sensory reactivity to stimuli: Sensory reactivity to stimuli was observed on the open field.Parameters: Approach response, touch response, auditory response, tail pinch response and rightingReaction (2) Grip strength measurement Grip strength was measured following sensory reactivity to stimuli. Grip strength was measured twice for both forelimbs and hindlimbs using a grip dynamometer (CPU gauge: Model 9502A, Aikoh Engineering Co., Ltd.), and a higher value was adopted.Motor activity measurement: For measurement of motor activity, animals were acclimated to cages (polymethylpentene, W × D × H: 220 × 380 × 195 mm) after the clinical observation following dosing or before the measurement during the recovery period. Animals were transferred to new cages at the time of measurement (no diet or water was given). The motor activity was recorded individually using a motor activity-measuring device (SCANET SV-40, Melquest Ltd.). Data were calculated every 10 minutes from the start of measurement, while a total of 1 hour was calculated.Body weight measurement: The test and recovery males were weighed on Days 1, 8, 15, 22, 29, 36 and 42. The recovery males were also weighed on Days 43, 50 and 56. The satellite females were weighed on the same manner as the recovery males. The test females were weighed on Days 1, 8 and 15, once every 7 days after initiation of cohabitation, on Days 0, 7, 14 and 20 of gestation, and on Days 0 and 4 of lactation. The final body weight was also measured on the day of the scheduled necropsy.Food consumption measurement: Food consumption was measured between Days 1 to 8, 8 to 14, 22 to 29, 29 to 36 and 36 to 40 for test and recovery males, and between Days 43 to 50 and 50 to 54 for recovery males only. For satellite females, it was measured for Days 1 to 8, 8 to 15, 15 to 22, 22 to 29, 29 to 36, 36 to 42, 43 to 50 and 50 to 56. Food consumption of the test females was measured at the same frequency as body weights. However, food consumption was not measured for either sex during the mating period. After the completion of copulation, the measurement for males was started from the nearest measurement day.Gross weight of each feeder was weighed, and the mean daily food consumption for each period was expressed as the data of the last day of each period.Urinalysis in males: Urinalysis was conducted for 5 males per group with the smaller animal numbers in the final dosing week (Day 40) by a reagent strip method. Fresh urine samples excreted spontaneously on a washed tray under the cage were collected after 8:00 (before the dosing) under fasting conditions (no diet was given, and supplied with water).As a result, no treatment-related changes were detected in any parameters; therefore, urinary sediment and accumulated urine test, and urinalysis during the recovery period were not performed.Hematology: All animals were fasted for about 17 hours and above on Day 43 for the test males, Day 57 for the recovery males and satellite females, and Day 5 of lactation for the test females. Subjected animals were 5 animals per group with the smaller animal numbers for the test males, all recovery males and satellite females, and 5 animals from the earlier parturition date with the smaller animal numbers for the test females.The animals were anesthetized with intraperitoneal injection of sodium pentobarbital (30 mg/kg), and about 2.0 to 2.5 mL of blood was collected from the posterior vena cava. For examination of coagulation system, 0.9 mL of blood was collected into a tube containing 0.1 mL of 3.8 w/v% trisodium citrate, and then plasma was obtained by centrifugation at 1870 × g for 15 minutes (set temperature: 4°C). For examination of the other items, the remaining blood was put into a blood container (SB-41, Sysmex Corporation) containing 2 mg of EDTA-2K as an anticoagulant. The remaining samples were discarded after the hematology examination.Blood chemistry: After blood sampling for hematology, 3 to 5 mL of blood was collected from the posterior vena cava under anesthesia for blood chemistry. The blood was left standing at room temperature for about 60 minutes, and then serum was obtained by centrifugation at 1870 × g for 10 minutes (set temperature: 4°C). The serum samples were stored frozen in a deep freezer (actual temperature: -79.5° to -78.1°C, permissible range: -90° to -65°C) until examination. The remaining samples were stored in a deep freezer and discarded until the completion of the study.
Sacrifice and pathology:
Necropsy: All surviving animals were euthanized by exsanguination under anesthesia and subjected to necropsy. Necropsy was similarly performed for 1 female in which mating was not confirmed (No. 101) after euthanasia in the above manner 10 days after completion of mating period, for 1 males (No. 48) without confirmation of copulation at the same time as the scheduled necropsy (Day 57) and for 1 female without parturition (No. 49) on day 26 after copulation.The necropsy of the females with total litter loss was performed immediately after their total litter loss was discovered.Organ weight measurement: After the scheduled necropsy, the organ weights (absolute weight) were measured in 5 animals per group with smaller animal numbers of the test males, all recovery males, all satellite females and 5 animals with earlier parturition date with smaller animal numbers in the test females. The testis and epididymis weights were measured in all males. Moreover, relative organ weight was calculated from the body weight measured on the day of necropsy. Bilateral organs were measured together. The measurement for the female with total litter loss, non-copulated female and non-delivery female was not performed.HistopathologyAll organs and tissues were fixed in 10 vol% phosphate buffered formalin (the testes and epididymides were pre-fixed in Bouin’s solution, and the eyes were pre-fixed in a Davidson’s solution).The samples derived from 5 animals with the smaller animal numbers of the test males and 5 animals from the earlier parturition date with the smaller animal numbers for the test females of the control and high dose groups, and gross lesions were embedded in paraffin, sectioned and stained with hematoxylin and eosin, and were examined by microscopy. The samples derived from females with total litter loss were prepared into hematoxylin-eosin stained samples and examined in the same manner. The ovary derived from non-pregnant animals (Nos. 52, 82, 87 and 93) was prepared into hematoxylin-eosin stained samples and examined.As a result, changes attributable to the test substance treatment were noted in the liver of males and females, and testis, epididymis and adrenal of males. Therefore, these organs were additionally examined in the 5 males and 5 females selected in the above manner from the other dose groups with the samples collected after the dosing period, and in all animals for the recovery period with the samples collected after the recovery period.
Other examinations:
Other examinations are in relation to the reproduction/developmental screening test and information can be found in Chapter 7.8.2
Statistics:
Statistical analysis: Statistical analysis was performed with a computer system (Provantis®, Instem LSS Limited). In all cases, levels of p<0.01 (1%) and p<0.05 (5%) were considered to be significant and two-tailed test was used.The body weight and food consumption of non-pregnant female after confirmation of copulation and the body weight after initiation of mating in the females failed mating were excluded from the evaluation.Multiple comparison: The group mean and standard deviation of the following items were calculated, and homogeneity of the variance was tested by the Bartlett’s test (significant level: 5%).When the variance was homogeneous, the Dunnett’s multiple comparison was applied, and when it was not homogenous, the Steel’s multiple comparison was applied for control group and each test substance group. For the results of urinalysis with reagent strip, Steel’s multiple comparison test was performed after the grades were converted into numeric values.Number of rearing, grip strength, motor activity, body weights, food consumption, urinalysis, hematology, blood chemistry, absolute and relative organ weight.Fisher Exact test: Histopathological findings were tested with the Fisher Exact test for the control and each test substance group.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Effects in high dose animals. Details below.
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
effects observed, treatment-related
Details on results:
Clinical signsNo abnormality was noted in any animals throughout the dosing or recovery period.Functional observation batteryDetailed clinical observations: No treatment-related change was noted in any animals in hand held observation or observation on the open field throughout the dosing or recovery period.High values of number of rearing were noted in satellite females of the 1000 mg/kg group in Weeks 7 and 8. However, these changes were not judged to be treatment-related as the number of rearing was similar to that in pre-treatment (Week -1) and no related changes were noted in any examinations.Function tests(1) Sensory reactivity to stimuli: Reactivity to stimuli was comparable in males and females of all groups, and no abnormality was observed.(2) Grip strength: No significant difference was noted in males or females between the control and test substance groups.Motor activity: At the end of the dosing period, low values or lowering tendencies of motor activity at 20-60 minutes and total motor activity were noted in females of the 1000 mg/kg group.At the end of recovery period, no significant difference was noted in satellite females between the control and 1000 mg/kg groups.Body weightNo significant difference was noted in males or females between the control and test substance groups throughout the dosing or recovery period.Food consumptionNo treatment-related changes were noted.High values of food consumption were noted in satellite females of the 1000 mg/kg group on Day 50. However, the changes were not judged to be treatment-related as the changes were noted at only one measurement time point and no significant difference was noted in the body weight on Day 50.Urinalysis in malesIn the qualitative analysis, no significant difference was noted between the control and test substance groups.HematologyAt the end of the dosing period, low values of platelets, white blood cell count, neutrophils, basophils and large unstained cells were noted in females of the 1000 mg/kg group.At the end of the recovery period, the above-mentioned changes were not noted.Besides, at the end of the dosing period, low values of MCHC and eosinophils (and ratio), and high values of monocyte ratio were noted in males of the 1000 mg/kg group.However, these changes were not judged to be treatment-related as the changes were slight and no related changes were noted in any examinations. High values of large unstained cell ratio were noted in females of the 250 mg/kg group. However, the changes were not judged to be treatment-related as there was no dose-dependency.At the end of the recovery period, low values of MCH, MCHC and MCV were noted in males of the 1000 mg/kg group. However, these changes were not judged to be treatment-related as the changes were not noted at the end of the dosing period and no related changes were noted in any examinations. Prolongations of APTT, and high values of red blood cell count and low values of lymphocytes were noted in both sexes of the 1000 mg/kg group and females of the 1000 mg/kg group, respectively. However, these changes were not judged to be treatment-related as the changes were slight and no related changes were noted in any examinations.Blood chemistryNo treatment-related changes were noted.At the end of the dosing period, low values of ALP were noted in males of the 1000 mg/kg group. However, the changes were not judged to be treatment-related as the changes were opposite to the toxicity effect. Low values of Na and Cl were noted in males of the 1000 mg/kg group. However, these changes were not judged to be treatment-related as the changes were slight and no related changes were noted in any examinations. High values of ALP and total bile acid were noted in each one female of the 1000 mg/kg group (Nos. 90 and 91, respectively). However, these changes were not judged to be treatment-related as no histopathological related changes were noted. High values of inorganic phosphorus and K were noted in males of the 50 mg/kg group and females of the 250 mg/kg group, respectively. However, these changes were not judged to be treatment-related as there was no dose-dependency.At the end of the recovery period, high values of total protein, albumin and triglyceride were noted in males of the 1000 mg/kg group. However, these changes were not judged to be treatment-related as the changes were not noted at the end of the dosing period and no related changes were noted in any examinations.NecropsyAt the end of dosing period: No abnormality was noted in any animals.At the end of recovery period: No treatment-related changes were noted.Dilatation of common bile duct was noted in one satellite female of the 1000 mg/kg group (No. 105). However, the change was not judged to be treatment-related for its incidence and no related changes were noted in any examinations.Non-copulated female (No. 101): No abnormality was noted.Non-pregnant animals (Nos. 52, 82, 87 and 93): No treatment-related changes were noted.Remnant of transverse septum in vagina was noted in each one female of the 250 and 1000 mg/kg groups (Nos. 82 and 93, respectively). However, the changes were not judged to be treatment-related for its spontaneous occurrence.Organ weightAt the end of the dosing period, treatment-related changes were noted as follows: high values or highly tendencies of absolute or relative liver weight in both sexes of the 1000 mg/kg group, and low values of relative thymus weight and absolute and relative epididymides weights in males of the 1000 mg/kg group.At the end of the recovery period, treatment-related changes were noted as follows: high values of relative liver weight and low values of absolute and relative testes and epididymides weights in males of the 1000 mg/kg group. The other above-mentioned changes were not noted.Besides, the changes were noted at the end of the recovery period as follows: low values of absolute brain weight and high values of absolute and relative heart weights in males of the 1000 mg/kg group, and high values of absolute and relative thyroids weights and absolute adrenals weight in satellite females of the 1000 mg/kg group. However, the changes were not judged to be treatment-related as the changes were not noted at the end of the dosing period or no related changes were noted in any examinations.Histopathological examinationAt the end of the dosing period, in the liver, minimal or mild hypertrophy of centrilobular hepatocyte was noted in 4 males and 3 females of the 1000 mg/kg group.In the testis, minimal or mild degeneration/necrosis of spermatocyte/spermatid was noted in 3 males of the 1000 mg/kg group. In 1 male of these animals, mild decrease of spermatocyte/spermatid was noted. Moreover, minimal or mild decrease of sperm and cell debris in the duct of epididymis were noted in 2 males of the 1000 mg/kg group.At the end of the recovery period, in the testis, minimal or mild degeneration/necrosis of spermatocyte/spermatid was noted in 4 males of the 1000 mg/kg group. In 3 males of these animals, minimal or mild decrease of spermatocyte/spermatid was noted.Moreover, minimal, mild or moderate of sperm and cell debris in the duct of epididymis was noted in 4 males of the 1000 mg/kg group.The other above-mentioned changes were not noted.In addition to above changes, various histopathological changes were noted in both sexes of among the groups including the control group; however, these changes were not judged to be treatment-related since they are observed nonspecifically in rats and there was no clear dose-dependency in the incidence. Eosinophilic substance in common bile duct with eosinophil infiltration to lamina propria was noted as histopathological change corresponding to dilatation of common bile duct in 1 satellite female of the 1000 mg/kg group (No. 105) observed at necropsy.
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Critical effects observed:
not specified

Absolute and relative organ weights – Week 6

Day(s): 43 Relative to Start Date

Sex: Male

Organ Weights (Rats)

Final Body Weight (g)

Brain

(g)

Brain Ratio

 (%)

Thyroids (mg)

Thyroids Ratio

(x 10^-3%)

Thymus (mg)

Thymus Ratio

 (x 10^-3%)

Heart

 (g)

Heart Ratio

 (%)

Liver

 (g)

Liver Ratio

(%)

Spleen

(g)

Spleen Ratio

(%)

Control

Mean

SD

N

426.50

13.84

7

2.066

0.073

5

0.483

0.013

5

23.08

2.96

5

5.40

0.76

5

282.4

38.6

5

65.80

7.08

5

1.438

0.049

5

0.336

0.018

5

10.940

0.315

5

2.559

0.157

5

0.774

0.146

5

0.180

0.029

5

50 mg/kg

Mean

SD

N

434.18

26.60

12

2.112

0.049

5

0.479

0.024

5

22.24

4.20

5

5.05

1.09

5

322.0

52.2

5

73.17

14.02

5

1.394

0.151

5

0.315

0.026

5

10.910

1.010

5

2.464

0.097

5

0.720

0.079

5

0.163

0.019

5

250 mg/kg

Mean

SD

N

439.42

27.09

12

2.136

0.092

5

0.484

0.035

5

21.02

3.73

5

4.75

0.77

5

299.8

77.3

5

67.36

15.24

5

1.502

0.136

5

0.340

0.036

5

12.100

2.106

5

2.721

0.346

5

0.706

0.113

5

0.159

0.019

5

1000 mg/kg

Mean

SD

N

443.91

19.12

7

2.054

0.038

5

0.461

0.020

5

22.84

2.55

5

5.13

0.62

5

209.0

39.9

5

46.65d1

7.52

5

1.514

0.118

5

0.340

0.030

5

14.116s2

1.257

5

3.162dd3

0.195

5

0.822

0.101

5

0.184

0.022

5

1[d – Test: Dunnett 2 Sided p<0.05]                               2[s – Test: Steel 2 Sided p<0.05]                     3[dd – Test: Dunnett 2 Sided p<0.01]

 

Absolute and relative organ weights – Week 6

Day(s): 43 Relative to Start Date

Sex: Male

Organ Weights (Rats)

Kidneys (g)

Kidneys Ratio

(%)

Adrenals

(mg)

Adrenals Ratio

(x 10^-3%)

Testes

(g)

Testes Ratio

(%)

Epididymides

(g)

Epididymides Ratio

(%)

Seminal vesicle

(g)

Seminal vesicle Ratio

(%)

Prostrate

(g)

Prostate Ratio

(%)

Control

Mean

SD

N

2.974

0.190

5

0.695

0.054

5

62.60

6.95

5

14.62

1.49

5

3.220

0.408

7

0.755

0.093

7

1.253

0.136

7

0.293

0.025

7

2.538

0.180

5

0.593

0.041

5

0.760

0.236

5

0.178

0.057

5

50 mg/kg

Mean

SD

N

2.906

0.299

5

0.656

0.043

5

60.94

11.35

5

13.71

1.88

5

3.217

0.266

12

0.744

0.087

12

1.277

0.071

12

0.295

0.021

12

2.638

0.254

5

0.597

0.058

5

0.758

0.107

5

0.171

0.020

5

250 mg/kg

Mean

SD

N

2.990

0.413

5

0.673

0.069

5

60.86

13.97

5

13.70

2.70

5

3.393

0.242

12

0.773

0.051

12

1.288

0.094

12

0.294

0.023

12

2.552

0.184

5

0.576

0.0030

5

0.840

0.165

5

0.189

0.028

5

1000 mg/kg

Mean

SD

N

3.108

0.124

5

0.697

0.028

5

52.36

9.98

5

11.81

2.65

5

3.013

0.429

7

3.013

0.429

7

0.983dd1

0.124

7

0.222dd1

0.033

7

2.366

0.167

5

0.532

0.052

5

0.620

0.062

5

0.140

0.019

5

1[dd – Test: Dunnett 2 Sided p<0.01]

 

Absolute and relative organ weights – Week 6

Day(s): 5 Relative to Littering (A)

Sex: Female

Organ Weights (Rats)

Final Body Weight (g)

Brain

(g)

Brain Ratio

 (%)

Thyroids (mg)

Thyroids Ratio

(x 10^-3%)

Thymus (mg)

Thymus Ratio

 (x 10^-3%)

Heart

 (g)

Heart Ratio

 (%)

Liver

 (g)

Liver Ratio

(%)

Spleen

(g)

Spleen Ratio

(%)

Control

Mean

SD

N

294.88

14.21

5

1.994

0.118

5

0.677

0.050

5

17.92

3.68

5

6.07

1.16

5

212.8

67.5

5

71.52

19.39

5

1.048

0.149

5

0.356

0.053

5

9.614

0.659

5

3.266

0.274

5

0.676

0.072

5

0.229

0.024

5

50 mg/kg

Mean

SD

N

316.08

24.70

5

1.994

0.036

5

0.634

0.048

5

16.70

2.10

5

5.30

0.66

5

289.8

99.2

5

90.77

27.06

5

1.102

0.077

5

0.349

0.015

5

10.560

1.080

5

3.340

0.199

5

0.728

0.085

5

0.231

0.027

5

250 mg/kg

Mean

SD

N

285.38

26.51

5

1.982

0.054

5

0.699

0.064

5

18.54

4.29

5

6.59

1.97

5

264.8

74.2

5

91.88

19.58

5

1.000

0.085

5

0.355

0.062

5

10.086

0.739

5

3.559

0.422

5

0.752

0.229

5

0.270

0.107

5

1000 mg/kg

Mean

SD

N

308.90

28.36

5

1.986

0.082

5

0.646

0.039

5

17.22

3.42

5

5.57

0.97

5

297.0

69.3

5

96.08

21.38

5

1.016

0.098

5

0.329

0.019

5

11.338d1

1.170

5

3.673

0.258

5

0.628

0.058

5

0.204

0.018

5

1[d – Test: Dunnett 2 Sided p<0.05]               

 

Absolute and relative organ weights – Week 6

Day(s): 5 Relative to Littering (A)

Sex: Female

Organ Weights (Rats)

Kidneys (g)

Kidneys Ratio

(%)

Adrenals (mg)

Adrenals Ratio

(x 10^-3%)

Ovaries

(mg)

Ovaries Ratio

(x 10^-3%)

Control

Mean

SD

N

1.846

0.122

5

0.627

0.051

5

68.72

6.99

5

23.30

2.11

5

103.78

20.09

5

35.25

6.94

5

50 mg/kg

Mean

SD

N

1.948

0.254

5

0.614

0.040

5

69.80

7.33

5

22.09

1.59

5

103.54

12.91

5

32.66

1.87

5

250 mg/kg

Mean

SD

N

1.918

0.156

5

0.674

0.051

5

78.48

15.13

5

27.84

7.08

5

102.54

14.64

5

36.01

4.60

5

1000 mg/kg

Mean

SD

N

2.066

0.196

5

0.669

0.035

5

73.84

11.93

5

23.83

2.21

5

107.24

10.00

5

34.72

1.12

5

 

Absolute and relative organ weights – Week 8

Day(s): 57 Relative to Start Date

Sex: Male

Organ Weights (Rats)

Final Body Weight (g)

Brain

(g)

Brain Ratio

 (%)

Thyroids (mg)

Thyroids Ratio

(x 10^-3%)

Thymus (mg)

Thymus Ratio

 (x 10^-3%)

Heart

 (g)

Heart Ratio

 (%)

Liver

 (g)

Liver Ratio

(%)

Spleen

(g)

Spleen Ratio

(%)

Control

Mean

SD

N

470.90

27.10

5

2.212

0.068

5

0.471

0.030

5

27.04

4.05

5

5.77

1.00

5

311.2

127.3

5

65.50

25.27

5

1.476

0.111

5

0.314

0.020

5

11.474

0.506

5

2.439

0.092

5

0.802

0.189

5

0.169

0.033

5

1000 mg/kg

Mean

SD

N

452.72

13.24

5

2.006dd1

0.082

5

0.444

0.030

5

21.90

3.13

5

4.84

0.70

5

295.0

76.2

5

65.41

17.94

5

1.662d2

0.118

5

0.367dd1

0.026

5

12.426

0.964

5

2.745d2

0.211

5

0.712

0.081

5

0.157

0.017

5

1[dd – Test: Dunnett 2 Sided p<0.01]             2[d – Test: Dunnett 2 Sided p<0.05]                               

 

Absolute and relative organ weights – Week 8

Day(s): 57 Relative to Start Date

Sex: Male

Organ Weights (Rats)

Kidneys (g)

Kidneys Ratio

(%)

Adrenals (mg)

Adrenals Ratio (x 10^-3%)

Testes

(g)

Testes Ratio

(%)

Epididymides

(g)

Epididymides Ratio

(%)

Seminal vesicle

(g)

Seminal ves.

Ratio

(%)

Prostate (g)

Prostate Ratio

(%)

Control

Mean

SD

N

2.958

0.167

5

0.630

0.047

5

53.98

3.84

5

11.51

1.27

5

3.302

0.196

5

0.704

0.071

5

1.298

0.055

5

0.276

0.013

5

2.640

0.401

5

0.560

0.072

5

0.782

0.200

5

0.166

0.040

5

1000 mg/kg

Mean

SD

N

2.930

0.285

5

0.647

0.058

5

54.20

4.26

5

11.99

1.16

5

2.444dd1

0.467

5

0.530d2

0.099

5

0.892dd1

0.089

5

0.197dd1

0.023

5

2.286

0.102

5

0.505

0.031

5

0.656

0.146

5

0.145

0.033

5

1[dd – Test: Dunnett 2 Sided p<0.01]             2[d – Test: Dunnett 2 Sided p<0.05]               

 

Absolute and relative organ weights – Week 8

Day(s): 57 Relative to Start Date

Sex: Female

Organ Weights (Rats)

Final Body Weight (g)

Brain

(g)

Brain Ratio

 (%)

Thyroids (mg)

Thyroids Ratio

(x 10^-3%)

Thymus (mg)

Thymus Ratio

 (x 10^-3%)

Heart

 (g)

Heart Ratio

 (%)

Liver

 (g)

Liver Ratio

(%)

Spleen

(g)

Spleen Ratio

(%)

Control

Mean

SD

N

273.94

14.68

5

1.880

0.095

5

0.688

0.055

5

16.40

2.30

5

5.98

0.72

5

366.2

45.8

5

133.45

12.38

5

0.952

0.033

5

0.348

0.022

5

7.178

0.312

5

2.623

0.105

5

0.564

0.121

5

0.206

0.044

5

1000 mg/kg

Mean

SD

N

282.74

21.97

5

1.970

0.120

5

0.701

0.076

5

21.04d1

3.09

5

7.46d1

1.07

5

344.6

57.3

5

121.35

12.02

5

1.040

0.105

5

0.368

0.028

5

7.398

0.948

5

2.610

0.161

5

0.498

0.090

5

0.176

0.025

5

1[d – Test: Dunnett 2 Sided p<0.05]               

 

Absolute and relative organ weights – Week 8

Day(s): 57 Relative to Start Date

Sex: Female

Organ Weights (Rats)

Kidneys (g)

Kidneys Ratio

(%)

Adrenals (mg)

Adrenals Ratio (x 10^-3%)

Ovaries (mg)

Ovaries Ratio (x 10^-3%)

Control

Mean

SD

N

1.824

0.171

5

0.667

0.072

5

63.18

6.66

5

23.18

3.36

5

85.62

4.33

5

31.27

0.87

5

1000 mg/kg

Mean

SD

N

1.884

0.131

5

0.667

0.014

5

76.92d1

10.61

5

27.45

5.21

5

83.70

15.56

5

29.47

3.68

5

1[d – Test: Dunnett 2 Sided p<0.05]               

Conclusions:
From the results, NOAEL was estimated to be 250 mg/kg/day under the conditions of this study.
Executive summary:

The purpose of the study was to examine repeated dose toxicity and reproductive/developmental toxicity after oral administration of DEGMEE in rats in accordance with OECD Guidelines for Testing of Chemicals (No. 422, March 22, 1996).

 

DEGMEE was administered repeatedly by oral gavage at dose levels of 0 (control group), 50, 250 and 1000 mg/kg from 15 days before mating through mating for 42 days in males and satellite females, and 15 days before mating through gestation and parturition until Day 4 of lactation in females to determine the repeated dose toxicity and reproductive and developmental toxicity, as well as reversibility of the changes observed. Each test group consisted of 12 males (including 5 males for recovery test) and 12 females (5 satellite females each were added for the control and 1000 mg/kg groups in recovery test).

The control group was given the vehicle (water for injection) alone.

 

In the motor activity measurement, low values of motor activity at 20-60 minutes and total motor activity were noted in females of the 1000 mg/kg group.

 

In the hematology, low values of platelets, white blood cell count, neutrophils, basophils and large unstained cells were noted in females of the 1000 mg/kg group.

 

In the pathology, treatment-related changes were noted as follows.

As for the liver, high values of liver weight were noted in both sexes of the 1000 mg/kg group at necropsy. Moreover, in the histopathology, minimal or mild hypertrophy of centrilobular hepatocyte was noted in 4 males and 3 females of the 1000 mg/kg group.

As for the testis and epididymis, low values of epididymides weight were noted in males of the 1000 mg/kg group. Moreover, in the histopathology, minimal or mild degeneration/necrosis of spermatocyte/spermatid was noted in 3 males of the 1000 mg/kg group. In 1 male of these animals, mild decrease of spermatocyte/spermatid was noted. Moreover, minimal or mild decrease of sperm and cell debris in the duct of epididymis were noted in 2 males of the 1000 mg/kg group.

Besides, in the organ weight measurement, low values of thymus weight were noted in males of the 1000 mg/kg group.

 

No test substance-related changes were noted in any of the examinations, including clinical signs, detailed clinical observations, function tests, body weight, food consumption, urinalysis in males, blood chemistry and necropsy.

 

At the end of the recovery period, high values of liver weight and low values of testes and epididymides weight were noted in males of the 1000 mg/kg group. Moreover, in the histopathology, in the testis, minimal or mild degeneration/necrosis of spermatocyte/spermatid was noted in 4 males of the 1000 mg/kg group. In 3 males of these animals, minimal or mild decrease of spermatocyte/spermatid was noted. Minimal, mild or moderate of sperm and cell debris in the duct of epididymis was noted in 4 males of the 1000 mg/kg group.

The other changes noted in the dosing period were not noted.

 

The main test substance-related changes were as follows: in the liver, high values of liver weight and minimal or mild hypertrophy of centrilobular hepatocyte in both sexes of the 1000 mg/kg group, in the testis, minimal or mild degeneration/necrosis of spermatocyte/spermatid in males of the 1000 mg/kg group, in the epididymis, low values of epididymides weight and minimal or mild decrease of sperm and cell debris in the duct of epididymis in males of the 1000 mg/kg group.

From the results, NOAEL was estimated to be 250 mg/kg/day under the conditions of this study.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
250 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
K1 quality data, prepared according to OECD guidelines in accordance with GLP

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: liver

Justification for classification or non-classification

The data have established a NOAEL of 250 mg/kg/day. On the basis of this value the substance is not classified according to the CLP Regulation 1907/2006.