Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 November 2009 - 30 August 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): 1,3-Dimethyl-2-imidazolidinone
- Physical state: liquid
- Analytical purity: 99.9%
- Lot/batch No.: CDL2333
- Stability under test conditions: stable
- Storage condition of test material: Room temperature, protected from light

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 9.5 wks
- Weight at study initiation: (P) Males: 288.7-326.0g; Females: 212.5-251.2g
- Housing: Each animal was individually housed in a stainless steel wire mesh cage (W 29.1 × D 26.3 × H 18.0 cm) on a water flushing breeding rack (Toyoriko).
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.2 to 23.5
- Humidity (%): 38.5 to 63.4
- Air changes (per hr): 12 or more
- Photoperiod (hrs dark / hrs light):12/12

IN-LIFE DATES: From: 02 December 2009 - To: 09 February 2010

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle: water was used as the substance is highly soluble in this solvent
- Concentration in vehicle: 20, 4 and 0.8 mg/mL (high, middle and low dose)
- Amount of vehicle (if gavage): 0.5ml per 100g body weight. The volume administered was based on the most recently measured body weight
- Purity: pharmaceutical grade (distilled water for injection)
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 2 weeks
- Proof of pregnancy: vaginal plug and sperm in vaginal smear
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
High-performance liquid chromatography (HPLC) conditions:

Column: CAPCELLPAK C18 UG120 (4.6 mm × 150 mm, 3.0 μm,)
Column temperature: 40°C
Mobile phase: Methanol / distilled water (1/9, v/v)
Flow rate: 1.0 mL/min
Wavelength: UV 227 nm
Injection volume: 10 μL

The analytical results confirmed that the all formulations (the nominal concentrations were 0.8, 4 and 20 mg/mL) met the criterion (ratio to the nominal concentration: 100.0±10%). Moreover, no test substance was detected in the dosing solutions for the control group. Therefore, the dosing formulations were confirmed to be adequately prepared.
Duration of treatment / exposure:
Dosing period for males: for consecutive 42 days (for 14 days before mating, 14-day mating period and for 14 days after the end of the mating period).

Dosing period for females: for consecutive 42 to 54 days (for 14 days before mating, mating period [up to 13 days until copulation] and until Day 4 of lactation after the parturition including the pregnancy period for the copulated females).
Frequency of treatment:
once a day
Details on study schedule:
Prior to the present study, a preliminary 2-week repeated dose toxicity study (Experiment No. B889) was conducted using the doses of 0, 20, 60, 200 and 600 mg/kg.
As a result, all animals (6 males and 6 females, total 12 rats) of the 600 mg/kg group died on day 4 and 5 of dosing. Moreover, 2 females of the 200 mg/kg group died on day 12 and day 13 of dosing. As the changes of the general conditions, these dead animals showed tremor, ptosis, emaciation, decrease in locomotor activity, prone position, irregular respiration, no-feces or loose stool.
The animals of 600 mg/kg group showed these symptoms on day 3 and 4 of dosing, and females of 200 mg/kg group showed the symptoms from day 11 to 13 of dosing. The necropsy for the dead animals revealed atrophy of spleen and thymus, black and red spots on the stomach.
No male rats died in the 200 mg/kg group.

During the dosing period, tremor was observed as a change of the general conditions in males of 600 mg/kg group and females of 200 mg/kg or more dose
groups.

Because all females (6 females) of the 200 mg/kg group showed tremor during the latter half of the dosing period, the effect of the test article on the central nervous system was suggested.

Both males and females showed decreases of the body weights and food consumption in the 200 mg/kg or more dose groups. The animals of the 200 mg/kg showed increases of hematocrit, hemoglobin and red blood cell counts; decreases of reticulocyte ratio and platelet counts;
increases of triglycerides, blood glucose and total cholesterol; decreases of creatinine, albumin and A/G ratio. Moreover, urinalysis for this group revealed decrease of urinary volume and increase of osmotic pressure. In the necropsy at the end of dosing period, atrophy of thymus was observed in the males and females of 200 mg/kg group, and the thymus weights were decreased. Also, atrophy of spleen was observed in the females of the same group, and the spleen weights in males and females were decreased or tended to be decreased.
Moreover, black patch in the stomach and red patch in the duodenum were observed in females of the same group and these findings were thought to be caused by the test substance.

Therefore, 100 mg/kg/day was selected as the high dose for this study, because the dosing period was longer than the preliminary study and the test animals included pregnant dams. The doses at 20 and 5 mg/kg/day were selected using a common ratio of 5 from 100 mg/kg/day.
Doses / concentrationsopen allclose all
Dose / conc.:
4 mg/kg bw/day (nominal)
Dose / conc.:
20 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
No. of animals per sex per dose:
For the main study, 12 males or 12 females were assigned to each group. For the recovery study, each 5 males of control and high dose groups were selected.
For the female recovery study, 5 more rats were added to the control and high dose group, respectively.
Control animals:
yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
All males and females were observed for clinical signs once before dosing and once from 60 to 90 minutes after the dosing every day during the dosing period (once a day during the withdrawal period and once on each necropsy day). All abnormal findings and mortality were recorded. However, the animals which were used for the motor sensory reactivity to various stimuli, determination of grip strength and locomotor activity in the functional observation battery (FOB) were observed for post-dosing clinical signs immediately after the end of FOB on each day for these determinations.

BODY WEIGHT: Yes
All males were weighed on days 0 (initiation of dosing), 7, 14, 21, 28, 35, 41 and 42 (necropsy day or day 0 of recovery) of dosing and the body weight gain between day 0 and day 41 of dosing was calculated. Males of the recovery study were weighed on days 0, 7, 13 and 14 (necropsy day) of recovery after the dosing period and the body weight gain between day 0 and day 13 of recovery was calculated.
All females were weighed on days 0 (initiation of dosing), 7 and 14 of dosing and the body weight gain between day 0 and day 14 of dosing was calculated. The females which did not copulate with males were weighed on the following days 21, 28, 35, 41, 49 and 52 (necropsy day) of doing. In addition, the females which copulated with males were weighed on the days 0, 7, 14 and 20 of pregnancy and the body weight gain between day 0 and day 20 of pregnancy was calculated. The females which delivered were weighed on the days 0, 4 and 5 (necropsy day) of lactation and the body weight gain between day 0 and day 4 of lactation was calculated. Females of the recovery study were weighed on the same days as the males for recovery and the body weight gains from day 0 to day 41 of dosing and from day 0 to day 13 of recovery were calculated.

FOOD CONSUMPTION:
Yes
For the males, the weights of food were measured on days 0 (initiation of dosing), 7, 14, 21, 28, 35 and 41 (the day before necropsy) of dosing. The food consumption from a measurement day to a next measurement day was calculated and mean daily food consumption was subsequently calculated. The cumulative food consumption was calculated from day 0 to 14 and from day 21 to 41 of dosing. For the males of the recovery study, the weights of diets were weighed on days 0, 7 and 13 (the day before necropsy) of recovery after the dosing period. The food consumption from a measurement day to a next measurement day was calculated and mean daily food consumption was subsequently calculated. The cumulative food consumption was calculated from day 0 to 14 of recovery.
For the females, the weights of food were measured on days 0 (initiation of dosing), 7 and 14 of dosing. The food consumption from a measurement day to a next measurement day was calculated and mean daily food consumption was subsequently calculated. The cumulative food consumption was calculated from day 0 to 14 of dosing. The food consumption of females which did not copulate with males were determined on the following days 28, 35, 41, 49 and 52 of doing. The food consumption from a measurement day to a next measurement day was calculated and mean daily food consumption was subsequently calculated. The weights of food of females which copulated with males were weighed on the days 0, 7, 14, 18 and 20 of pregnancy. The weights of food of females which delivered were weighed on the days 0 and 4 of lactation. The food consumption from a measurement day to a next measurement day was calculated and mean daily food consumption was subsequently calculated. The cumulative food consumption was calculated from day 0 to 20 of pregnancy. The weights of food of females of the recovery study were weighed on the same days as the males for recovery. The food consumption from a measurement day to a next measurement day was calculated and mean daily food consumption was subsequently calculated. The cumulative food consumption was calculated from day 0 to 41 of dosing and from day 0 to 13 of recovery.

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
Examinations of the hematological parameters, blood coagulation profile, blood chemistry and serum protein profile were performed for males (using 5 animals in the ascending order of the animal number per group) on the day 42 of dosing and the naturally-delivering females (using 5 animals in the ascending order of the animal number and delivering date order per group) on the day 5 of lactation.

In the recovery study, all males and females were examined for these parameters on the day 14 of recovery period. From the evening on the day before each blood sampling day, the animals were fasted for at least 16 hours, then, blood was collected from the abdominal aorta under ether anesthesia.

CLINICAL CHEMISTRY: Yes
Examinations of the hematological parameters, blood coagulation profile, blood chemistry and serum protein profile were performed for males (using 5 animals in the ascending order of the animal number per group) on the day 42 of dosing and the naturally-delivering females (using 5 animals in the ascending order of the animal number and delivering date order per group) on the day 5 of lactation.

In the recovery study, all males and females were examined for these parameters on the day 14 of recovery period. From the evening on the day before each blood sampling day, the animals were fasted for at least 16 hours, then, blood was collected from the abdominal aorta under ether anesthesia.

URINALYSIS: Yes
At the final week (from days 37 to 38 of dosing) of dosing period, urinalysis was carried out for each 5 male rats (animal ID Nos. 1001 to 1005, 1101 to 1105, 1201 to 1205, 1301 to 1305) of each group. Because the positive tendency of the transitional epithelial cells was observed in the 100 mg/kg group, urinalysis was also carried out for the male rats (animal ID Nos. 1008 to 1012, 1308 to 1312) of the recovery groups at the final week (from days 9 to 10 of recovery) of recovery period.
Prior to urine collection, animals were orally given tap water (20 mL/kg) by gavage.
Urine was collected from the animals under the conditions of fasting and water-deprivation in urine-collection cages overnight (from 4:00 PM to AM 9:00 of the next day).
After the determination of the urinary volume and visual observation of color, the following parameters were determined using Ames test strips (N-Multistix SG, Siemens Healthcare Diagnostics) and an automatic strip reader (CLINITEK 500, Bayer); pH, occult blood, ketone bodies, glucose, protein, bilirubin and urobilinogen.

NEUROBEHAVIOURAL EXAMINATION: Yes
For all surviving animals, functional observation battery was performed. Among the following FOB parameters, detailed clinical observations were conducted once before the grouping, and thereafter, once about a week throughout the treatment and recovery period. However, these observations were conducted on the days 7 and 14 of pregnancy for the females which copulated with males, and on the day 4 of lactation for the females which delivered.
The observation for sensory reactivity to various stimuli, grip strength and locomotor activity were examined using 5 animals in the ascending order of the animal number per group. For males, these examinations were conducted on the 41 day of dosing (selected animals: No. 1001 to 1005, 1101 to 1105, 1201 to 1205, and 1301 to 1305). For females, these examinations were conducted on the 4 day of lactation (selected animals in the delivery completion day order: No. 2002, 2004, 2006, 2008, 2010, 2102, 2104, 2106, 2107, 2108, 2204, 2205, 2206, 2208, 2211, 2303, 2305, 2307, 2308, 2311). Also, these examinations were conducted for the males and females of the recovery groups on the 41 day of dosing and on the 13 day of recovery.
Before the grouping, the detailed clinical observations were conducted for all animals. During the dosing period, these examinations were started from 30 minutes after each dosing and we paid attention to prevent the deflections of the examination time periods among the groups.
- Detailed clinical examination
The animals were observed for posture in each cage and examined for the presence of writhing, circling, biting, convulsion and abnormal vocalization and these findings were recorded. Each rat was removed from its cage and observed for ease of removal, ease of handling, abnormal vocalization, muscle tone, piloerection, staining hair, eyelids closure, bite wound, lacrimation, salivation, respiration, eyeballs, visible mucous membrane, urinary incontinence and catalepsy. These findings were recorded. Moreover, to observe for air righting reflex, each animal was dropped on the center of floor of an open-field device from about 30 cm of height. During 3 minutes immediately after the landing, animals were observed for respiration, coordination of movement, convulsion, grooming, gait, abnormal gait, eyelids closure, stereotypical behavior, abnormal behavior, abnormal vocalization, arousal and movement in the open-field device. The numbers of rears (supported or unsupported), defecation and urination were counted. All these findings were recorded.
- Sensory reactivity to various stimuli
The pupillary reflex, approach contact, touch response, auditory response and pain response were examined. All these reactions were recorded.
- Grip strength (forelimbs and hindlimbs)
The animals’ grip strengths were determined using a Digital Push Pull Gauge (Aikoh Engineering). Two measurements were taken for the forelimbs, and 2 for the hindlimbs. The mean of the 2 measurements was calculated for each limb.
- Locomotor activity
The animals’ locomotor activity was individually determined using LOCOMO (Melquest). Locomotor activity determination was started after the examinations from the above-described sections 13.4.1. to 13.4.3. (at about 40 minutes after each dosing).
The data of locomotor activity were collected at 1-minute intervals and measured for 1 hour. These collected data were calculated as activity at 10-minute intervals and total activity (activity of 1 hour).
The locomotor activity was determined in the 7-108 animal room (W 6.4 × D 10.3 × H 2.6 m). The room was maintained with lights on. The noise level was set at about 70 dB using the white noise generator (PA-1, Nagashima Medical Instruments), and measured using the sound level meter (NA-20, RION), and recorded.

OTHER:
All pregnant rats were allowed to deliver naturally. They were checked for the presence of delivery from 8:30 to 10:00 from day 20 to 25 of gestation. For the animals which had delivered within the above-described period of time and which had begun to deliver within the same period of time as above and completed delivery afterwards, the day when delivery was completed was designated as day 0 of lactation. About the animals which had started delivery after 10:00 AM, we confirmed the completion of delivery of these dams on the next day.

Oestrous cyclicity (parental animals):
Female animals were observed for their estrous cycles for 14 days prior to mating.
Except for the recovery groups, estrous cycle of each female was observed from the initiation day of dosing to the day of copulation. The mean estrous cycle was determined using the days of estrous cycle based on the number of days from a estrous day [the first estrous day for the female(s) showing consecutive estrous days] to the day before next estrous day. Also, the incidence of abnormal estrous cycle (the days of estrous cycle are not 4 or 5 days) was calculated [(number of females showing abnormal estrous cycle/number of females observed) × 100].
Litter observations:
The offspring were observed for their sex on day 0 of lactation and the sex ratio of each group (number of live male offspring per group/total number of offspring per group) and the sex ratio of each litter (number of live male offspring/total number of offspring) were calculated. The sex ratios at birth (day 0 of lactation) were calculated for the live offspring and for the offspring including the dead offspring, respectively. However, the dead offspring which were not distinguished for their sex due to the partial body loss by cannibalism were excluded from the counting for the sex ratios. Moreover, the live offspring were observed for the external anomalies and the incidences of external anomalies per litter [(number of live offspring with external anomalies/number of observed live offspring) × 100] were calculated. Live offspring were weighed individually at birth (day 0 of lactation) and on day 4 of lactation using the electronic balances (PM400, PG2002-S). The mean body weight for each sex in each litter was calculated.
The numbers of live pups and dead pups among the live offspring were counted daily until day 4 of lactation, and the live pups were observed for their general conditions.
Postmortem examinations (parental animals):
The dams were autopsied pathologically on day 5 of lactation, and the numbers of corpora lutea and implantation sites was counted and examined for the macroscopic abnormalities.
The females which had not delivered until 9:00 AM on day 25 of gestation (animal ID Nos. 2109, 2201, 2301, 2309) were necropsied and their uteri were stained with 10 vol% ammonium sulfide solution. Because no dams showed any stained implantation sites, these rats were judged to be non-pregnant.
Postmortem examinations (offspring):
All live offspring were euthanized by exsanguination under ether anesthesia on day 4 of lactation and their organs and tissues were macroscopically observed. The cannibalized offspring were discarded throughout the lactation period and the dead offspring were fixed in in Bouin’s solution (Polysciences) and their organs and tissues were macroscopically observed.
Statistics:
The data on the body weight, body weight gain, food consumption, cumulative food consumption, mean estrus cycle, numbers of corpora lutea and implantation sites, gestation length, number of offspring delivered, number of dead offspring and number of cannibalism on day 0 of lactation, sex ratio per litter, implantation index, delivery index, incidence of external anomalies, live birth index on day 0 and 4 of lactation, FOB data (grip strength, locomotor activity, number of rears, frequencies of defecation and urination), hematological parameters, blood coagulation factors, blood chemical parameters, serum protein
electrophoresis parameters, urinalysis (volume, electrolytes and osmotic pressure), organ weights and relative organ weights were subjected to automatic analysis by Bartlett’s test for homogeneity of variance. Homogenous data were analyzed by Dunnett’s multiple comparison test for the significant differences between the control group and each dose group. Heterogeneous data by Bartlett’s test were subjected to Steel’s test for the significant differences between the control group and each dose group.
Reproductive indices:
The copulation index [(number of successfully copulated animals/number of mated animals) × 100] was calculated from the results of mating of each group.
The gestation period (the number of days which the date of day 0 of gestation was subtracted from the date of day 0 of lactation), fertility index [(number of pregnant animals/number of successfully copulated animals) × 100], gestation index [(number of dams with live newborns/number of pregnant females) × 100], implantation index [(number of implantation sites/numbers of corpora lutea) × 100], and delivery index [(number of newborns/number of implantation sites) × 100] were calculated.
Offspring viability indices:
Each litter was examined after delivery to count the total number of offspring delivered (live offspring number + dead offspring number + number of cannibalism) and live birth index on day 0 of lactation [(number of live offspring/total number of offspring delivered) × 100] were calculated.

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
No females showed any changes of general conditions during the dosing period.
Neither males nor females showed any changes of general conditions during the recovery period.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
None of the animals died during the study.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
In males, no statistical difference was detected among the body weight values between the control group and each test substance treated group on each measuring day. The gains in the body weights of each dose group were not statistically different from the control group during the dosing period (from day 0 to day 41 of dosing), but it tended to be lower in the 100 mg/kg group.
During the recovery period, the body weight of male 100 mg/kg group was statistically significantly lower than the control group on day 0 of recovery. However, the gain in the body weight of the same dose group was statistically significantly higher than the control group during the recovery period (from day 0 to day 13 of recovery).
In females, the gain in the body weight of 100 mg/kg group was statistically significantly lower than the control group during the pre-mating period (from day 0 to day 14 of dosing). During the gestation period, the body weights of 100 mg/kg group were statistically significantly lower than the control group on days 7, 14 and 21 of gestation. The gain in the body weights of the same dose group was not statistically different from the control group during the gestation period, but it tended to be lower. Also, the gain in the body weight of 4 mg/kg group was statistically significantly lower than the control group during the gestation period. However, this change was judged to not be due to the dosing of test material, because it lacks dose-dependent trend. During the lactation period, the body weights of 100 mg/kg group were statistically significantly lower than the control group on days 4 and 5 of lactation and the gain in the body weight of same dose group was statistically significantly lower than the control group during the lactation period. In the recovery groups, no statistical difference was detected among the body weight values between the control group and each test substance treated group on each measuring day. However, the gain in the body weight of 100 mg/kg group was statistically significantly higher than the control group during the recovery period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
In males, no statistical difference was detected between the control group and 100 mg/kg group on each measuring day during the dosing and recovery periods. Also, no statistical difference was detected in the cumulative food consumption.
In females, the mean daily food consumption of the 100 mg/kg/day group was significantly lower than that of the control group from day 0 to 4 of lactation. During the other experimental periods, no statistical difference was detected between the control group and each dose group on any measuring day and no statistical difference was detected in the cumulative food consumption. In the recovery groups, no statistical difference was detected between the control group and 100 mg/kg group on each measuring day, also no statistical difference was detected in the cumulative food consumption.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
In the males at the end of the dosing period, no difference was observed in the parameters between the control group and each test material treated group.
In the males at the end of the recovery period, a statistically significantly lower mean corpuscular haemoglobin concentration was observed in the 100 mg/kg group as compared with the control group. However, this change was judged to be toxicologically meaningless because no changes were observed in the haematocrit, haemoglobin and red blood cell counts. Also, a statistically significantly higher reticulocyte ratio was observed in the 100 mg/kg group as compared with the control group, but this change was very slight.
In females on the day 5 of lactation, haematocrit, haemoglobin and red blood cell count were statistically significantly higher in the 100 mg/kg group as compared with the control group and reticulocyte ratio tended to be lower, but not statistically significant, in the same dose group.
In the females of recovery groups, a statistically significantly lower mean corpuscular haemoglobin concentration was observed in the 100 mg/kg group. However, this change was judged to be toxicologically meaningless because no changes were observed in the haematocrit, haemoglobin and red blood cell counts.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In the males at the end of the dosing period, triglyceride level was statistically significantly higher in the 100 mg/kg group as compared with the control group and total cholesterol level tended to be higher, but not statistically significant, in the same dose group.
In the males at the end of the recovery period, total protein level was statistically significantly lower in the 100 mg/kg group as compared with the control group, but this change was very slight.
In the females on the day 5 of lactation, total protein level and inorganic phosphorus level were statistically significantly lower in the 100 mg/kg group as compared with the control group.
In the females of the recovery groups, potassium level was statistically significantly higher in the 100 mg/kg group, but this change was very slight.
In the males at the end of the recovery period, α1-globulin concentration, β-globulin concentration, γ-globulin fraction and concentration were statistically significantly lower and α2-globulin fraction and A/G ratio were statistically significantly higher in the 100 mg/kg group as compared with the control group. However, because these changes were very slight and judged to be toxicologically meaningless.
In the females on the day 5 of lactation, albumin fraction and concentration, γ-globulin fraction and concentration and A/G ratio were statistically significantly lower and β-globulin fraction was statistically significantly higher in the 100 mg/kg group as compared with the control group.
In the females of the recovery groups, no statistical difference was observed in the parameters between the control group and each test material treated group.
Urinalysis findings:
no effects observed
Description (incidence and severity):
At the final week of dosing period, a statistically significantly higher urine potassium concentration was observed in the 100 mg/kg group as compared with the control group. However, this change was judged to be not due to the dosing of test material because the potassium total excretion value did not change. The 1+ (5-14 cells/µL) of transitional epithelial cells were found in the urinary sediments of 1/5 rats of 100 mg/kg group, but the cells were not observed in the sediments of control group. However, this change was judged to be toxicologically meaningless because no changes were observed in the relating blood chemical parameters and in the histopathological examination.
At the final week of the recovery period, no difference was observed in the parameters between the control group and each test material treated group.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
- Detailed clinical observations and sensory reactivity to various stimuli
In the detailed clinical observations in males in the cages during the dosing period, a statistically significant decrease in the number of posture as “sitting” was observed in the 100 mg/kg group as compared with the control group on day 15 of dosing. In the 100 mg/kg group, 4/12 rats, 5/12 rats and 3/12 rats showed “sitting”, “paid attention to an observer and stood and sat down” and “curled up may be asleep”, respectively. However, these findings were normal behaviours because the intact rats show the same behaviours. In addition, the observed changes in 100 mg/kg group were considered to be spontaneous, because 10/12 rats and 2/12 rats of the control group showed “sitting” and “paid attention to an observer and stood and sat down”, respectively. In the open-field observation, a statistically significant decrease in the number of unsupported rears was observed in the 20 mg/kg group as compared with the control group on day 41 of dosing. However, this change was judged to not be due to the dosing of test substance, because it lacks dose-dependent trend. During the recovery period, no abnormal findings were detected by the in- and out-of-cage observation and open-field observation.
In females, no abnormal findings were detected by the in- and out-of-cage observation during the dosing period. In the open-field observation, statistically significant decreases in the number of supported rears were observed in the 100 mg/kg group as compared with the control group in pre-mating (day 2 of dosing), gestation (day 7 of gestation) and lactation periods. Also, a statistically significant decrease in the number of unsupported rears was observed in the same dose group in lactation period. These changes were judged to be not due to the dosing of test substance because the changes were slight or lacked the changes of movement or locomotor activity.
In the females of recovery groups, no abnormal findings were detected during the dosing and recovery periods.
In the observation for sensory reactivity to various stimuli, no behavioral changes due to the dosing of test substance were observed in the males and females during the dosing period. During the recovery period, significant increase in the number of pain response as “walk away from stimulus” was observed in the 100 mg/kg group as compared with the control group. However, this change was judged to be not due to the dosing of test substance because it was not observed at the end of the dosing period. In the females of recovery groups, no behavioral changes due to the dosing of test material were observed.
- Grip strength (forelimbs and hindlimbs)
Including the recovery groups, no statistical difference was detected among the grip strength of forelimbs and hindlimbs between the control group and each test material treated group.
- Locomotor Activity
In males, no statistical difference was detected in any interval time between the control group and each test substance treated group at each end of the dosing or recovery period.
In females on the day 4 of lactation, no statistical difference was detected in any interval time between the control group and each test substance treated group.
In the females of recovery groups, a statistically significant decrease in the counts of locomotor activity from 10 to 20 minutes after the start of measurement was observed in the 100 mg/kg group as compared with the control group. However, this change was judged to be not due to the dosing of test material because no changes suggesting the decrement of activity were observed in the routine observation for general conditions and detailed clinical observation. At the end of the recovery period, no statistical difference was detected in any interval time between the control group and each test material treated group.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
In males, absolute and relative weights of the testes and epididymides were statistically significantly lower in the 100 mg/kg group as compared with the control group at each end of the dosing and recovery periods. Also, relative weight of the kidneys was statistically significantly higher in the 100 mg/kg group at the end of dosing period. However, this change was judged to be not due to the dosing of test material because of no change of kidney absolute weight and no clear histopathological changes. The relative weight of the spleen was statistically significantly lower in 20 mg/kg group at the end of dosing period. However, this change was judged to be not due to the dosing of test material because this change lacked dose-relationship and was very slight.
In the females on the day 5 of lactation, absolute and relative weights of the spleen were statistically significantly lower in the 100 mg/kg group as compared with the control group. Also, absolute weights of the heart, liver and adrenal glands were statistically significantly lower and relative weights of the brain and kidneys were statistically significantly higher in the 100 mg/kg group as compared with the control group. However, these changes were judged to be caused by the lower body weights because the mean body weight of this dose group was statistically significantly lower at the necropsy.
In the females of the recovery groups, no statistical difference was observed in the organ weights between the control group and each test material treated group.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
As the effect of the administration of the test material, atrophy of testis and epididymis was observed in the male 100 mg/kg group at the end of the dosing period and atrophy of testis was observed in the same dose group at the end of the recovery period. In addition, atrophy of thymus was observed in the females which showed all dead pups.
Other findings were considered as the spontaneous lesions because these were not different from the incidences, severity and morphological characteristics of the often observed lesions in the SD strain, same week-of-age and intact rats.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
As the effect of the administration of the test material, atrophy and vacuolation of seminiferous tubule and interstitial cell hyperplasia was observed in testis of all males (7/7 males including the males which did not fertilize females) of the 100 mg/kg group at the end of the dosing period. The incidences of these changes were statistically significantly higher than the control group. The diagnostic criteria for atrophy of seminiferous tubule are; “slight” is the atrophic tubules are less than 25% of all seminiferous tubules, “moderate” is the atrophic tubules are 25% or more and less than 50%, “marked” is the atrophic tubules are 50% or less. The 6/7 males including the males which did not fertilize females showed “marked” atrophy of seminiferous tubule and 1/7 male showed “slight” atrophy. In the rat (Animal ID No. 1306) which had “slight” atrophy of seminiferous tubule, the sequence of the germ cells was comparatively normal, but one to several slightly larger vacuoles were found from basement to pars intermedia of the tubules and the vacuoles showed sloughing of normal or slightly degenerated germ cells in the lumen (Photo 1). In the 6 rats (Animal ID Nos. 1301, 1302, 1303, 1304, 1305 and 1307) which had “marked” atrophy of seminiferous tubule, most of germ cells disappeared and formation of multinucleated giant cell was found (Photo 2). At the end of the recovery period, all males (5/5 males) showed atrophy and vacuolation of seminiferous tubule and interstitial cell hyperplasia and 4/5 rats showed formation of multinucleated giant cell. The incidences of these changes were statistically significantly higher than the control group. In addition, Sertoli cell-only syndrome (most of germ cells disappeared and only Sertoli cell remain in the seminiferous tubule)6, 7) was found in 3/5 males (Photo 3). In the other 2/5 males which did not show Sertoli cell-only syndrome, the germ cells remained partly. In the 100 mg/kg group, decrease of sperm and cell debris in the lumen were observed in the epididymis at the end of the dosing period and decrease of sperm was observed at the end of the recovery period. The incidences of decrease of sperm in the epididymis were statistically significantly higher than the control group at each end of the dosing and recovery periods. The males which did not fertilize females showed the same findings in their epididymides as the males which examined at the end of the dosing period. At the end of the dosing period, as the effect of dosing of test substance was detected in the 100 mg/kg group, the each 5 males in the 4 and 20 mg/kg groups were examined histopathologically. However, none of these animals of the lower dose groups showed the same findings as the 100 mg/kg group. Other findings in the test substance-treated groups were detected in few rats or observed in the rats of control group.
In the examination of spermatogenesis stages at the end of the dosing period, as 5/6 males of 100 mg/kg group could not be examined due to the severe atrophy of seminiferous tubule, the spermatogenesis stages of each 5 males in the 4 and 20 mg/kg groups were examined. In the 4 and 20 mg/kg groups, the effect of test substance was not detected, because each number of examined cell types per Sertoli cell was as same as the control group. In 1 male (Animal ID No. 1306) which could be examined for the spermatogenesis stages in 100 mg/kg group, each number of examined cell types per Sertoli cell was as same as each mean number of control group. At the end of the recovery period, in 2 males (Animal ID Nos. 1310 and 1311) which could be examined for the spermatogenesis stages in 100 mg/kg group, the numbers of round spermatid and pachytene spermatocyte per Sertoli cell tended to be lower, but the numbers of spermatogonia and preleptotene spermatocytes were as same as each mean number of control group.
In the females which showed all dead pups and other females on the day 5 of lactation and in the recovery groups, no findings due to the dosing of test substance were observed. In the infertile females, no rats showed abnormal findings.
Histopathological findings: neoplastic:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
In observation of the estrous cycle, an abnormal estrous cycle was observed in 1, 2 and 1 animals in the control, 4 and 100 mg/kg groups, respectively. However, there were no significant differences in the incidences of irregular estrous cycles among the groups. In addition, the mean estrous cycle days were 4.0, 4.1, 4.0 and 4.0 in the control, 4, 20 and 100 mg/kg groups, respectively, and no statistical difference was observed between the control group and each test substance treated group.
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
At the mating, 1 pair failed to copulate in the control group and the copulation index was 91.7%. On the other hand, all pairs successfully copulated in the test material treated groups and all copulation indices were 100%. Infertility was not observed in control group and the female fertility index was 100%. Infertility was observed in 1, 1 and 2 females in the 4, 20 and 100 mg/kg groups, respectively, and these female fertility indices were 91.7, 91.7 and 83.3%, respectively. But there were no significant differences in female fertility indices among the groups.
Total 4 dams of 100 mg/kg group showed all dead pups (total litter death) from day 0 to 3 of lactation (Animal ID Nos. 2306 and 2312, day 0 of lactation; ID No. 2302, day 2 of lactation; ID No. 2310, day 3 of lactation). Among these dams, 1 dam (Animal ID No. 2306) which showed all dead pups on day 0 showed abnormal nesting.
The gestation length, numbers of corpora lutea and implantation sites and total numbers of live offspring delivered were comparable among the control group and all of the test substance treated groups. There were no significant differences in the implantation index, delivery index or sex ratio between the control group and any of the test substance treated groups. In 100 mg/kg group, the delivery index, number of live offspring and live birth index (day 0 of lactation) tended to be lower, but no statistically significant differences were detected. Also, the number of live offspring and of live birth index on day 4 of lactation were statistically significantly lower in the 100 mg/kg group as compared with the control group.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
20 other: mg/kg/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
gross pathology
histopathology: non-neoplastic

Target system / organ toxicity (P0)

Critical effects observed:
not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
The external observation of the newborns revealed no external abnormalities among the control group and all of the test material treated groups. In the observation for general conditions of newborns, one pup showed wound in the left forelimb.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
On day 0 of lactation, 2, 2, 2 and 23 offspring died in control, 4, 20 and 100 mg/kg groups, respectively. Among the dead offspring in 100 mg/kg group, 20 pups were born from 2 dams which showed all dead pups (Animal ID Nos. 2306 and 2312). There was no significant difference in the number of dead offspring per litter on day 0 of lactation between the control group and any of the test material treated groups.
Only in the 100 mg/kg group, 4 cannibalized offspring were observed and all of these were born from 1 dam which showed all dead pups (Animal ID No. 2306) on day 0 of lactation.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In the body weights, male and female weights were statistically significantly lower in the 100 mg/kg group as compared with the control group.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
In the necropsy of the dead offspring during the lactation period, thymic remnant in neck was observed in each 1 male of 4 and 100 mg/kg groups and 3 females of 100 mg/kg group, and dilatation of renal pelvis was observed in 1 female of 20 mg/kg group. However, these lesions were unrelated to the cause of death. In addition, 1 pup which showed external abnormalities (short trunk and short tail) in control group also showed anophthalmia, renal agenesis and renal malposition. Other dead offspring did not show any abnormal findings.
In the necropsy of the offspring on day 4 of lactation, no abnormal findings caused by the dosing of test substance were observed. The observed findings were dilatation of renal pelvis in each 1 female of control and 20 mg/kg groups, unilateral renal agenesis in 1 female of 20 mg/kg group and loss of forelimb in 1 female of control group. No abnormal findings were observed in the male offspring.
Histopathological findings:
not examined
Other effects:
no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
20 other: mg/kg/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
other: growth inhibition

Target system / organ toxicity (F1)

Critical effects observed:
not specified

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
On the basis of above results, the No Observed Effect Level (NOEL) and No Observed Adverse Effect Level (NOAEL) of the test substance for the males were judged to be 20 mg/kg/day each in the conditions of this study because of the effects on the testis and epididymis of 100 mg/kg group.

For dams, the NOEL and NOAEL of the test substance were judged to be 20 mg/kg/day each because of the lower body weight gains of
100 mg/kg group from the initiation of dosing during the pregnant and lactation periods and the lower mean daily food consumption of the same group during the lactation period.

For the F1 animals, the NOEL and NOAEL of the test substance were judged to be 20 mg/kg/day each because of the growth inhibition and lower viability index on day 4 of lactation of 100 mg/kg group.
Executive summary:

The reproductive toxicity of the substance was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guideline OECD 422.

During the study the test material was administered at 0 (treated with the solvent, water for injection, only), 4, 20 or 100 mg/kg/day by gavage to rats from 14 days prior to mating to the end of the 14-day mating period.  Males were further dosed for 14 days from the end of the mating period.  Daily dosing of the parental females continued throughout pregnancy and up to day 4 of lactation period. Five males from the main groups and an additional 5 females (satellite groups) were used for the observation of reversibility for 14 days post treatment.

In any dose group, there were no effects of the test substance on the estrus cycle, copulation index and fertility index. At each end of the dosing and recovery periods, histopathological eaxamination revealed the marked decrease of spermatogenesis. However, it is considered that the 2-week dosing of test substance before mating did not affect the male fertility, because no histological changes of the male reproductive organs had been found in the 2-week repeated dose preliminary study. 

The dosing of test substance did not affect the gestation length, numbers of corpora lutea and implantation sites, implantation index and delivery index of dams. At the observation of delivery, 2 dams of 100 mg/kg group showed all dead pups on day 0 of lactation and 1 of these 2 dams showed abnormal nesting. Moreover, each 1 dam of 100 mg/kg group showed all dead pups on day 2 and 3 of lactation. These results indicate that treatment of the test substance could affect the lactating behavior of dams. Atrophy of the thymus was found in the 3 of 4 dams which showed all dead pups. However, this atrophy is considered not to be due to the dosing of test substance, because no histopathological changes were found in these organs. 

For the offsprings, no effect(s) of the test substance was observed on the total numbers of live offspring delivered and sex ratios. However, 23 offspring of 100 mg/kg group died on day 0 of lactation, and delivery index, number of live offspring (day 0) and live birth index (day 0) tended to be lower in the same dose group. One of the reasons for these findings, the test substance had inhibited the fetal growth and it induced the immaturity of offspring indicated as the lower body weights of male and female live offsprings (day 0). Also, the lower body weights of male and female live offsprings were found in the same dose group on day 4 of lactation and the body weights did not increase so much from the day 0. It is considered that the decreases of number of live offspring and of live birth index on day 4 of lactation were caused by the growth inhibition effect of test substance after birth. The external examination and necropsy of newborns on day 4 of lactation revealed no abnormalities caused by the dosing of the test substance. 

On the basis of above results, the No Observed Effect Level (NOEL) and No Observed Adverse Effect Level (NOAEL) of the test substance for the males were judged to be 20 mg/kg/day each in the conditions of this study because of the effects on the testis and epididymis of 100 mg/kg group. For dams, the NOEL and NOAEL of the test substance were judged to be 20 mg/kg/day each because of the lower body weight gains of 100 mg/kg group from the initiation of dosing during the pregnant and lactation periods and the lower mean daily food consumption of the same group during the lactation period. For the F1 animals, the NOEL and NOAEL of the test substance were judged to be 20 mg/kg/day each because of the fetal growth inhibition and lower viability index on day 4 of lactation of 100 mg/kg group. 

Based on effects seen in this study it is considered appropriate to classify this substance as Category 2 reproductive toxicant under the CLP Regulation.