1-ethoxy-2-(2-methoxyethoxy)ethane

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

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 for cross-reference:

reference to same study

Qualifier:

according to guideline

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 or test system 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.Room 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 exposure:

Preparation of dosing formulations (Preparation method and frequency, and storage conditions): Dosing 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.

Details on mating procedure:

The test males and females of each dose level were cohabited at one on one basis for 24 hours after 15:00 or later on Day 15 up to 14 days. The vaginal smears were collected in the morning from the day after the initiation of mating, and mating was confirmed with the vaginal plug or sperm in the smear sample. The day of successful mating was regarded as Day 0 of gestation. Based on the results, the following parameters were calculated.(1) Days until copulation: Number of days from the start of mating to the detection of copulation(2) Copulation index (%): (Number of animals with successful copulation / Number of animals cohabited) × 100(3) Fertility index (%): (Number of pregnant animals / Number of females with successful copulation) × 100

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Determination of the test substance concentrations in dosing formulationsAt 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.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: Acetone

Duration of treatment / exposure:

Administration periodMales: 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).

Details on study schedule:

Screening study, so not applicable.

Remarks:

Doses / Concentrations:0, 50, 250 and 1000 mg/kg.Basis:nominal conc.

No. of animals per sex per dose:

Detailed in table form - see Any other information.

Control animals:

yes, concurrent vehicle

Details on study design:

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 the study type.

Parental animals: Observations and examinations:

Clinical observationAll animals were observed twice a day (before and after dosing) during the dosing period, and once a day in the other periods.Functional observation batteryFor 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 behaviorFunctional 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 measurementFor 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 measurementThe 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 measurementFood 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 malesUrinalysis 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.HematologyAll 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 chemistryAfter 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.Observation during delivery and lactation periodsAll copulated females were allowed natural delivery. The observation of delivery was conducted twice daily (9:00 and 16:00) from Days 21 to 24 of gestation. The delivery completed by 9:00 was judged as a delivery on the corresponding day (the delivery day was regarded as Day 0 of lactation). The animals that completed delivery by 16:00 were observed for lactation on the next day (the day after delivery was regarded as Day 0 of lactation). The females that did not deliver by 24 days after copulation were regarded as non-delivered females. The delivered animals (dams) were allowed to nurse offspring until day 4 postpartum (Day 4 of lactation), and postpartum behaviour such as lactation, nesting and presence or absence of cannibalism was observed every day.

Oestrous cyclicity (parental animals):

Vaginal smears were collected with swabs from all test females in the morning (same time everyday) from the initial dosing day to the day of successful copulation or end of the mating period to confirm the estrous cycle. The obtained smears were collected on a plate for each animal, and stained with Giemsa. The estrous cycle was classified into diestrus (D), proestrus (P), estrus (E), and metestrus (M). The mean estrous cycle (number of days from the estrous period to the next estrous period) and the number of the estrous period during the test period were calculated. The data obtained during the mating period were treated as referential data.

Sperm parameters (parental animals):

Sperm parameters not examined in this study.

Litter observations:

Observation of offspringThe number of delivered offspring (numbers of live offspring and stillborns at birth), sex and the presence or absence of external anomalies were examined on Day 0 of lactaion. Thereafter, mortality was observed daily.Body weightAll live offspring were weighed individually on Days 0 and 4 postpartum.External examinationAll live offspring, stillborn and dead offspring were observed for external anomaly on Day 4 of lactation, and subsequently euthanized by intraperitoneal pentobarbital sodium injection (concentrate solution: 0.05 to 0.1 mL/body).

Postmortem examinations (parental animals):

NecropsyAll surviving animals were euthanized by exsanguination under anesthesia and subjected to necropsy in accordance with section 6.10.6. 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 measurementAfter 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 shown in section 6.10.8.1 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.Examination after lactationDams were subjected to necropsy on Day 5 of lactation, and the ovaries and uterus were excised to examine the numbers of corpora lutea and implantation sites. The one female without parturition (No. 49) was also examined on day 26 after copulation. As a result, implantation was confirmed, and thus, this female was judged pregnant

Postmortem examinations (offspring):

External observations only performed on the offspring.

Statistics:

Statistical analysis was performed with a computer system (Provantis®, Instem LSS Limited). Sex ratio was tested with the chi-square test using the software SAS 9.1.3. 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 data of offspring were handled on a litter-basis. Multiple comparisonThe 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, estrous cycle, number of estrus, days until copulation, gestation length, number of corpora lutea, number of implantations, number of delivered offspring, number of live offspring, body weight of offspringFisher Exact testHistopathological findings were tested with the Fisher Exact test for the control and each test substance group.Chi-square testThe following items were tested with the chi-square test for comparison between the control and each test substance group. Copulation index, fertility index, gestation index, delivery index, sex ratioWilcoxon’s rank sum testThe following items were tested with the Wilcoxon’s rank sum test for comparison between the control and each test substance group. Implantation index, stillborn rate, external anomaly index, external anomaly typing index, live birth index, viability index on Day 4

Reproductive indices:

Gestation length: Days until completion of delivery from Day 0 of gestationImplantation index (%): (Number of implantations / Number of corpora lutea) × 100Gestation index (%): (Number of pregnant animals delivered live offspring / Number of pregnant animals) × 100Delivery index (%): (Number of delivered offspring / Number of implantations) × 100

Offspring viability indices:

Live birth index (%): (Number of live offspring at birth / Number of implantations) × 100Stillborn index (%): (Number of stillborns / Total number of delivered offspring) × 100Viability index on Day 4 (%): (Number of live offspring on Day 4 / Number of live offspring at birth) × 100Sex ratio: (Number of male live offspring / Number of female live offspring)External anomaly index (%): (Number of offspring with external anomaly / Number of observed offspring) × 100External anomaly typing index (%): (Number of offspring with external anomaly by each type / Number of observed offspring) × 100

Clinical signs:

no effects observed

Description (incidence and severity):

Clinical signsNo abnormality was noted in any animals throughout the dosing or recovery period.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food efficiency:

no effects observed

Description (incidence and severity):

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.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

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.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

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.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Urinalysis in malesIn the qualitative analysis, no significant difference was noted between the control and test substance groups.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

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 activityAt 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.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

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 non-specifically 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.

Histopathological findings: neoplastic:

not specified

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Reproductive performanceEstrous cycleNo significant difference was noted in the mean estrous cycle or count of estrus, indicating no effect of the test substance on prolongation or shortening of the estrous cycle.Irregular estrous cycle was noted in 3 animals of the 50 mg/kg group (Nos. 66, 69 and 70). However, the changes were not judged to be treatment-related as the copulation and conception were confirmed and 2 animals of the control group (Nos. 51 and 60) showed similar changes.

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

MatingNo significant difference was noted in the days until copulation, copulation index or fertility index between the control and test substance groups.In 1 female in the 1000 mg/kg group (No. 101), no mating was confirmed. No abnormality was noted in the ovary of this animal, and no abnormality was noted histopathologically in the testis or epididymis of the male counterpart (No. 48). Although the cause of non-copulation was not clear, the change was not judged to be treatment-related for its incidence. Non pregnancy was noted in 2 animals of the 250 mg/kg group (Nos. 82 and 87) and 1 animal of the 1000 mg/kg group (No. 93). In 2 animals of these animals (Nos. 82 and 93), remnant of transverse septum in vagina was noted, indicating spontaneous occurrence. As the results, non-pregnancy was not judged to be treatment-related, as there was no clear dose-dependency in incidence and 1 animal in the control group (No. 52) showed similar change.Observation during delivery and lactation periodsThe total litter loss was observed in 5 dams of the 1000 mg/kg group. It occurred in 1 dam each on Days 1 and 3 of lactation, and in 3 dams on Day 2 of lactation. Their offspring died mostly due to cannibalism by dams and had a loss of suckling. No abnormality was noted in delivery or nursing.Examination after lactationProlongation of gestation period and lowering tendencies of delivery index were noted in the 1000 mg/kg group.No significant difference was noted in number of corpora lutea or implantations, implantation index or gestation index between the control and test substance groups.

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 activityAt 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.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.Necropsy[At 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.[Total litter loss (Nos. 94, 95, 97, 99 and 100)]: No abnormality was noted in any animals.[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.[Non-delivery animal (No. 49)]: No abnormality was noted.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 non-specifically 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.Reproductive performanceEstrous cycleNo significant difference was noted in the mean estrous cycle or count of estrus, indicating no effect of the test substance on prolongation or shortening of the estrous cycle.Irregular estrous cycle was noted in 3 animals of the 50 mg/kg group (Nos. 66, 69 and 70). However, the changes were not judged to be treatment-related as the copulation and conception were confirmed and 2 animals of the control group (Nos. 51 and 60) showed similar changes.MatingNo significant difference was noted in the days until copulation, copulation index or fertility index between the control and test substance groups.In 1 female in the 1000 mg/kg group (No. 101), no mating was confirmed. No abnormality was noted in the ovary of this animal, and no abnormality was noted histopathologically in the testis or epididymis of the male counterpart (No. 48). Although the cause of non-copulation was not clear, the change was not judged to be treatment-related for its incidence. Non pregnancy was noted in 2 animals of the 250 mg/kg group (Nos. 82 and 87) and 1 animal of the 1000 mg/kg group (No. 93). In 2 animals of these animals (Nos. 82 and 93), remnant of transverse septum in vagina was noted, indicating spontaneous occurrence. As the results, non-pregnancy was not judged to be treatment-related, as there was no clear dose-dependency in incidence and 1 animal in the control group (No. 52) showed similar change.Observation during delivery and lactation periodsThe total litter loss was observed in 5 dams of the 1000 mg/kg group. It occurred in 1 dam each on Days 1 and 3 of lactation, and in 3 dams on Day 2 of lactation. Their offspring died mostly due to cannibalism by dams and had a loss of suckling. No abnormality was noted in delivery or nursing.Examination after lactationProlongation of gestation period and lowering tendencies of delivery index were noted in the 1000 mg/kg group.No significant difference was noted in number of corpora lutea or implantations, implantation index or gestation index between the control and test substance groups.

Dose descriptor:

NOAEL

Remarks:

Reproductive Toxicity

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Prolongation of gestation period and low values of delivery index. Low values of number of litter, high values of number of stillborn (and ratio) and low values of birth index. Low values of viability index and body weight.

Clinical signs:

not examined

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Examination of external fetusesMalrotated paw was noted in 1 offspring and 1 stillborn of the 1000 mg/kg group (same dam) on postnatal Day 0. However, the change was not judged to be treatment-related for its incidence.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Observations of offspringAt birth, lowering tendencies of number of litter and live newborn, highly tendencies of number of stillborn (and ratio) and low values of birth index were noted in the 1000 mg/kg group. And low values of viability index on postnatal Day 4 were noted in the 1000 mg/kg group.No significant difference was noted in sex ratio between the control and test substance groups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weightLowering tendencies of body weight were noted in offspring of the 1000 mg/kg group on postnatal Day 4.No significant difference was noted in body weight of live newborns between the control and test substance groups on postnatal Day 0.

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Dose descriptor:

NOAEL

Remarks:

Reproductive Toxicity

Generation:

F1

Effect level:

250 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Prolongation of gestation period and low values of delivery index. Low values of number of litter, high values of number of stillborn (and ratio) and low values of birth index. Low values of viability index and body weight.

Clinical signs:

not examined

Reproductive effects observed:

not specified

Terminal delivery

Day(s): 0 Relative to Littering (A)

Sex: Female		Control	50 mg/kg	250 mg/kg	1000 mg/kg
Gestation Length  (Days)	Mean SD N	22.30 0.48 10	22.25 0.45 12	22.70 0.48 10	23.20^dd1 0.42 10
Number of Corporal Lutea	Mean SD N Sum	16.00 1.41 10 160.0	16.67 1.56 12 200.0	16.10 2.77 10 161.0	15.50 2.22 10 155.0
Number of Implantations	Mean SD N Sum	14.20 1.69 10 142.0	15.17 1.27 12 182.0	13.70 3.83 10 137.0	14.00 2.75 10 140.0
Implantation Index (%)	Mean N	88.70 10	91.13 12	83.65 10	89.75 10
No. of Litter (T)	Mean SD N Sum	13.0 2.0 10 130	14.4 1.6 12 173	12.7 4.1 10 127	11.0 2.2 10 110
Delivery  Index (%)	Mean N	91.868 10	95.046 12	90.998 10	79.530 10
Gestation  Index	% ProA	100.0 10/10	100.0 12/12	100.0 10/10	100.0 10/10

1[dd – Test: Dunnett 2 Sided p<0.01]

 

Terminal delivery

Day(s): 0 Relative to Littering (A)

Sex: Female		Control	50 mg/kg	250 mg/kg	1000 mg/kg
No. of Live Newborn (T)	Mean SD N Sum	12.8 2.3 10 128	14.3 1.8 12 172	12.7 4.1 10 127	9.9 2.8 10 99
Birth Index (%)	Mean N	90.049 10	94.491 12	90.998 10	70.460^ww1 10
No. of Live Newborn (M)	N Sum	10 63	12 82	10 59	10 53
No. of Live Newborn (F)	N Sum	10 65	12 90	10 68	10 46
Sex Ratio – Live Newborn	N Sum	1.241 10	0.989 12	0.948 10	2.082 9
No. of Stillborn (M)	N Sum	10 1	12 0	10 0	10 6
No. of Stillborn (F)	N Sum	10 1	12 1	10 0	10 3
No. of Stillborns (N)	N Sum	10 0	12 0	10 0	10 2
No. of Stillborn (T)	N Sum	10 2	12 1	10 0	10 11
% of Stillborn	Mean N	1.818 10	0.694 12	0.000 10	11.126 10

1[ww – Test: Wilcoxon 2 Sided p<0.01]

 

Postnatal course

Sex: Female		Control	50 mg/kg	250 mg/kg	100 mg/kg
Day(s) Relative to Littering (A)
No. of Live Newborn (M) d0	N Sum	10 63	12 82	10 59	10 53
No. of Live Newborn (F) d0	N Sum	10 65	12 90	10 68	10 46
No. of Live Newborn (T) d0	N Sum	10 128	12 172	10 127	10 99
No. of Dead (from 1 to 4) (M) d1 → d4	N Sum	10 0	12 0	10 0	10 8
No. of Dead (from 1 to 4) (F) d1 → d4	N Sum	10 0	12 0	10 0	10 11
No. of Dead (from 1 to 4) (T) d1 → d4	N Sum	10 0	12 0	10 0	10 19
No. of Cannibalized (M) d1 → d4	N Sum	10 1	12 1	10 0	10 20
No. of Cannibalized (F) d1 → d4	N Sum	10 1	12 0	10 1	10 8
No. of Cannibalized (T) d1 → d4	N Sum	10 2	12 1	10 1	10 28
No. of Live Newborns Day 4 (M) d4	N Sum	10 62	12 81	10 59	10 25
No. of Live Newborns Day 4 (F) d4	N Sum	10 64	12 90	10 67	10 27
No. of Live Newborns Day 4 (T) d4	N Sum	10 126	12 171	10 126	10 52
Viability Index on Day 4 (M) (%) d4	Mean N	98.750 10	98.958 12	100.00 10	48.889^w1 10
Viability Index on Day 4 (F) (%) d4	Mean N	99.000 10	100.00 12	99.167 10	54.321^w1 9
Viability Index on Day 4 (T) (%) d4	Mean N	98.745 10	99.510 12	99.333 10	48.167^ww2 10

1 [w – Test: Wilcoxon 2 Sided p<0.05]

 

Body weights (F1, lactation period)

Sex: Female			Control	50 mg/kg	250 mg/kg	1000 mg/kg
Day(s) Relative to Littering (A)
Sum of Count of Pup BW M	0		63	82	59	53
	4		62	81	59	25
Mean Male Pup BW/L	0	Mean SD N	6.918 0.629 10	6.956 0.371 12	7.489 0.854 10	6.643 0.651 10
	4	Mean SD N	11.366 1.148 10	11.262 0.830 12	11.999 2.089 10	9.870 1.366 5
Sum of Count of Pup BW F	0		65	90	68	46
	4		64	90	67	27
Mean Female Pup BW/L	0	Mean SD N	6.576 0.487 10	6.627 0.472 12	6.952 0.693 10	6.440 0.693 9
	4	Mean SD N	10.933 0.947 10	10.933 0.998 12	11.216 1.809 10	9.829 1.232 5

 

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 and was conducted to the following guideline:

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.

 

In the dams, prolongation of gestation period and low values of delivery index were noted in the 1000 mg/kg group. The total litter loss was observed in 5 dams of the 1000 mg/kg group. Their offspring died mostly due to cannibalism by dams and had a loss of suckling. No abnormality was noted in delivery or nursing in any dams.

In the offspring, at birth, low values of number of litter, high values of number of stillborn (and ratio) and low values of birth index were noted in the 1000 mg/kg group. And on postnatal Day 4, low values of viability index and body weight were noted in the 1000 mg/kg group.

 

No test substance-related changes were noted in any of the examinations, including estrous cycle, days until copulation, copulation index, fertility index, numbers of corpora lutea or implantations, implantation index, gestation index, parturition or maternal behavior in dams. In the offspring, no changes attributed to the test substance were noted in sex ratio or external features.

 

NOAEL

From the above results, no-observed-adverse-effect level (NOAEL) for reproductive toxicity was judged as follows.

 

The main test substance-related changes were as follows. In the dams, prolongation of gestation period and low values of delivery index were noted in the 1000 mg/kg group. The total litter loss was observed in 5 dams of the 1000 mg/kg group. In the offspring, at birth, low values of number of litter, high values of number of stillborn (and ratio) and low values of birth index were noted in the 1000 mg/kg group. On postnatal Day 4, low values of viability index and body weight were noted in the 1000 mg/kg group.

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

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

250 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

K1; Study performed in accordance with OECD test guidelines in compliance with GLP.

Additional information

The effects expressed were clearly dose related with clear a NOAEL at 250 mg/kg/day; at this dose there was no indication of reproductive effect or other toxicity.

The effects at the high dose of 1000 mg/kg/day were seen in the liver of both sexes and as histological change in the testes and effects on reproductive function in females (prolonged gestation, reduced delivery index, reduced post partum survival). Effects in the liver were minimal to mild but nevertheless indicate systemic effect that may be related to the other changes seen. It is considered

that the effects in the liver are connected to the reproductive effects, which were therefore seen in the presence of toxicity.

Justification for selection of Effect on fertility via oral route:
The data present the only regulkatory standard data currently available.

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

110 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

K1

Justification for classification or non-classification

The following points should be considered when recommending a classification for DEGMEE (CAS 1002-67-1):

• The toxicity of DEGMEE is dose related and includes effects in the liver and gonads with associated changes in haematology, blood chemistry and organ weights.


• Dose related change in the stomach was seen in the 90-day study but not in the shorter treatment period of the first combined toxicity and developmental study.


• In respect of foetal development, there were clear, dose-related effects on weight, ossification and post-natal survival; however, there was no suggestion of an inherent potential for teratogenic effect.


• The NOAEL in both the latter two studies, in which dose levels were 0, 110, 330, and 1000 mg/kg, was 110 mg/kg/day. The higher NOAEL of 250 mg/kg/day, set in the first study, probably reflects the lower low and mid dose levels and shorter duration of treatment.


• It is noted that the effects on the gonads were not associated, in the combined study, with effects on fertility. Again, this may be due to differences in duration and dose levels.


• In the developmental studies, effects such as at the high dose, such as litter loss, poor post-partum survival, reduced ossification and lower foetal weight are consistent with toxicity, as are the other effects noted.


• There was no suggestion of any inherent teratogenic potential that might be expressed at doses that were not maternally toxic.

 As noted above, the guidance (Regulation (EC) No 1272/2008 section 3.7.2.2.1) states:

Classification is made on the basis of the appropriate criteria, outlined above, and an assessment of the total weight of evidence... Classification as a reproductive toxicant is intended to be used for substances which have an intrinsic, specific property to produce an adverse effect on reproduction and substances shall not be so classified if such an effect is produced solely as a non-specific secondary consequence of other toxic effects.

In addition, it is stated elsewhere in the guidance, that effects seen in the presence of toxicity should not lead to classification as toxic to reproduction.

It was concluded that DEGMEE (CAS 1002-67-1) should not be classified as a reproductive and developmental toxin in respect of effects on reproduction. This was based on the presence of a clear NOAEL at 110 mg/kg/day and the observation of dose related effects only in the presence of toxicity at doses of 330 or 1000 mg/kg/day.