Registration Dossier

Administrative data

Description of key information

28-day repeated dose toxicity (HRC, 1989)

28-day repeated dose toxicity NOAEL(oral) = 30 mg/kg bw/day

28-day repeated dose toxicity (Biosafety researc Centre, 2011)

28-day repeated dose toxicity NOAEL(oral) = 20 mg/kg bw/day

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 January 1989 - 02 March 1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD)BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 28 +/- 1 day
- Weight at study initiation: 62 to 81 g
- Housing: All animals were initially caged, in groups of five according to sex. Each animal was identified within each cage by ear-punch and
individually by tail mark (tattoo).
- Diet (e.g. ad libitum): Biosure LAD1 Diet
- Water (e.g. ad libitum): Free access to tap water
-Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): mean minimum of 21.2 and mean maximum 21.9
- Humidity (%): mean minimum 55.4and mean maximum 58.1%
- Air changes (per hr):20
- Photoperiod (hrs dark / hrs light):12/12

IN-LIFE DATES: From: 05 January 1989 To: 02 March 1989
Route of administration:
oral: gavage
Vehicle:
water
Details on analytical verification of doses or concentrations:
Aliquots (20ml) of the test substance formulations and the vehicle were retained on Days 1, 22 and 26 for concentration analysis by HRC Department of Analytical Chemistry.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Once daily, seven days per week for four weeks
Dose / conc.:
6 mg/kg bw/day (nominal)
Dose / conc.:
30 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Two groups consisting of ten males and ten females and two groups consisting of five males and five females.
Control animals:
yes
Details on study design:
- Dose selection rationale: The dosage levels administered were selected on the basis of acute oral toxicity data available from the Sponsor and a 14 day preliminary dose range-finding toxicity study carried out at HRC (Report No. 89860/MTC)

Control animals received distilled water (10 ml/kg/day).

The test substance was administered by oral gavage to rats of groups 2 to 4 inclusive using a syringe and rubber catheter at a dosage volume of 10 ml/kg/day.
Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were checked early in each working day and again in the late afternoon to look for dead or moribund animals. At weekends a similar procedure was followed except that the final check was carried out at approximately mid-day

BODY WEIGHT: Yes
All rats were weighed prior to dosing and subsequently at weekly intervals throughout the study.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
Daily monitoring by visual appraisal of the water bottles was maintained throughout the study.

HAEMATOLOGY: Yes

The following parameters were analysed with an Ortho ELT-1500 analyser using standard Ortho methodology:
Packed cell volume (PCV)
Haemoglobin (Hb)
Red blood cell count (RBC)
Platelet count (Plts)
Absolute indices:
Mean corpuscular haemoglobin concentrations (MCHC)
Calculated: Hb (g/dl) x 100 + PCV (%)
Mean corpuscular volume (MCV)
Calculated: PCV (%) x 10 ? RBC (x 10 e6/mm3)
Total white blood cell count (WBC)

Analysed were: differential white blood cell count (Diff) - by standard microscopy of a blood smear stained with modified Wright's stain counting 100 cells:
Neutrophils (N)
Lymphocytes (L)
Eosinophils (E)
Basophils (B)
Monocytes (M)

The following parameter was also analysed:
Thrombotest (TT)

CLINICAL CHEMISTRY: Yes
The following parameters were analysed with a Roche Cobas centrifugal analyser:
Glucose
Triglycerides
Glutamic-pyruvic transaminase (GPT), also known as "alanine aminotransferase)
Glutamic-oxaloacetic transaminase (GOT), also known as "aspartate aminotransferase"
Gamma-glutamyltransferase

The following estimations were performed:
Alkaline phosphatase
Total bilirubin
Cholestrol
Urea nitrogen
Total protein
Albumin
Globulin
Sodium
Potassium
Calcium
Chloride
Inorganic phosphorus
Creatinine

URINALYSIS: Yes
Volume
pH
Specific gravity (SG) - by refractometry, compared to water with a value of 1000
Protein

Qualitative tests:
Total reducing substances
Glucose
Ketones
Bile pigments
Urobilinogen
Haem pigments
Microscopic examination





Sacrifice and pathology:
GROSS PATHOLOGY: The following organs from each animal killed after four weeks(Day 29) or six weeks ( after 14- day post- treatment observation period - Day 43):
adrenals
brain
kidneys
liver
ovaries
testes (with epididymides)

HISTOPATHOLOGY: Yes
Samples of the following tissues from all rats were preserved in 10% buffered formalin:
adrenals*,aort, brain, caecum, colon, duodenum, eyes, femur, Harderian gland, head (to preserve nasal cavity, nasopharynx, middle ear, teeth, lachrymal gland and Zymbal's gland), heart*, ileum, jejunum, kidneys*, larynx, liver*, lungs, lymph nodes, mammary gland, oesophagus, ovaries, pancreas, pharynx, pituitary, prostate, rectum, salivary gland, sciatc nerve, seminal vesicles, skeletal muscle, skin, spinal cord, spleen*, stomach, testes, thymus, thyroid, tongue, trachea, urinary bladder, uterus, vagina, any other abnormal tissue.
* Tissues required for histopathological examination

These fixed tissues were embedded in paraffin wax (m.p. 56°C), sections cut at 4 ~m and stained with haematoxylin and eosin.
In the first instance, microscopic examinations were carried out for tissues marked with an asterisk under 'Terminal studies' from all rats of
Group 1 (Control group) and Group 4 (High dosage group) killed on Day 29. In response to changes noted in organ weights and at initial
histopathological examination, microscopic pathology was subsequently extended to include:
(a) Ovaries from female rats in the control and high dosage groups killed at termination or following the 14-day recovery period.
(b) Kidneys from male rats in the low, intermediate and recovery groups.
(c) Testes from male rats in the control, low, intermediate and recovery groups.
Statistics:
All statistical analyses were carried out separately for males and females.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Pilo-erection was noted on one day only for rats in the low dosage group receiving DMI at 6 mg/kg/day.
For rats receiving DMI at 30 or 150 mg/kg/day, pilo-erection, hunched posture and abnormal gait (waddling) were noted during the treatment period.These signs were accompanied by lethary and body tremors for rats in the high dosage group.
Mortality:
no mortality observed
Description (incidence):
There were no mortalities.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Bodyweight gains for male and female rats receiving DMI at 150 mg/kg/day were lower than those of the controls during the treatment period with statistical significance being achieved in most instances. Lower, but not statistically significant, bodyweight gains were also recorded during week 4 for female rats receiving D.M.I at 30 or 6 mg/kg/day.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption for male rats receiving DMI at 150mg/kg/day was lower than that of the controls throughout the treatment and can be related to the lower bodyweight gains recorded for these animals.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Significantly higher (P < 0.01) neutrophil counts were recorded during Week 4 for female rats receiving D.M.I. at 150 mg/kg/day in comparison with controls.
During Week 6, red blue cell counts, packed cell volume and haemoglobin levels for male and female previously treated with DMI at 150mg/kg/day were significantly lower (P < 0.05, P < 0.01 and P < 0.001) than those of the controls.
Thrombotest times for female rats in the high dosage group were significantly higher (P < 0.01) than those of the controls during Week 6.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Significantly lower (P < 0.05) glutamic-pyruvic transaminase (GPT) levels and higher (P < 0.05) or P < 0.01) triglyceride levels were recorded during Week 4 for both male and female rats receiving DMI at 150 mg/kg/day in comparison with controls. Cholesterol levels for female rats in this high dosage group were significantly lower (P < 0.05) than controls.
Significantly lower (P < 0.05) globulin and total protein levels were also recorded during Week 4 for female rats receiving DMI at 150 mg/kg/day.

Following the two-week recovery period, the above changes in triglyceride, cholesterol, globulin and total protein levels were not observed and GPT levels for female rats in the high dosage group were significantly higher (P < 0.01) than those of the controls.

During Week 4 changes in sodium, potassium and phosphorus levels were recorded amongst male rats receiving DMI. However, these changes were low in magnitude and considered unlikely to be of toxicological importance.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Significantly lower (P < 0.01) urine volumes were collected during Week 4 from female rats receiving DMI at 150 mg/kg/day in comparison with controls. Similar but not statistically significant shifts to lower urine volumes were recorded for the corresponding male rats during Week 4 and for male and female treated rats during Week 6.

For animals in the high dosage group, pH values for male rats during Week 4 and for female rats during Week 6 were significantly lower (P < 0.05 or P < 0.01) than those of the controls.

During Week 4, a slight increase in the incidence of epithelial cells and polymorphonuclear leucocytes was noted for male rats in the high dosage group.

During Week 6, no sperm cells were seen in the urine of male rats previously treated with DMI at 150mg/kg/day. This finding may be related to the histopathological changes noted in the testes of treated male rats.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Significantly lower (P < 0.01 or P < 0.001) testes weights were recorded for male rats in the high dosage group following the four-week treatment period and also following the two week recovery period in comparison with controls. These changes can be related to the macroscopic and microscopic findings.

Ovary weights for female rats in the high dosage group were higher than those of the controls at the terminal and recovery kills with statistical significance being achieved (P < 0.05) for animals at the recovery kill.

At terminal sacrifice significantly lower (P < 0.05) brain weights were recorded for female rats in the high dosage group.

At recovery sacrifice, adjusted kidney weights for male rats in the treatment group were significantly higher (P < 0.01) than those of the controls.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Small testes and pallor of the kidneys were noted at termination for all male rats in the high dose group.
Following the two-week recovery period, testes from male rats in the treatment group were similarly small and blue discoloured.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Kidneys:
At terminal sacrifice, eosinophilic droplets were seen in the cortical tubular epithelium of all male rats receiving DMI., 30 or 150 mg/kg/day.
Focal tubular basophilia was also noted in all male rats receiving DMI at 150mg/kg/day and was associated with a focus of cortical scarring in one rat.
Following the recovery period, eosinophilic droplets in the cortical tubular epithelium were seen in three males previously treated with DMI at 150mg/kg/day. Focal tubular basophilia was observed in four male rats in the treatment group.

Testes and epididymides:
For all male rats in the high dosage group killed at terminal or recovery sacrifice, testicular atrophy and numbers of multinucleate spermatogenic cells were noted. In two rats killed at recovery sacrifice, there was evidence of early spermatogenesis in a proportion of a tubules and this may represent an early stage in the recovery of testicular function. Focal mineralisation was associated with the testicular atrophy in one rat at recovery sacrifice.
The epididymides of most male rats in the high dosage group showed a reduced number of spermatozoa (terminal sacrifice) or an absence of spermatozoa (recovery sacrifice), with or without numbers of abnormal spermatogenic cells.

Ovaries:
In view of an apparent increase in ovary weights from rats receiving DMI at 150 mg/kg/day, these tissues were included at microscopic examination. There was no change noted to be of toxicological significance.
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Dose descriptor:
NOEL
Effect level:
6 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The NOEL of 1,3 -Dimethyl-2 -Imidazolidinone is considered to be 6 mg/kg/day under the conditions of the subjected study after taking account of the clinical signs noted in male and female rats receiving 30mg/kg/day for periods of several days.
Key result
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Histopathology: Testicular atrophy and reduced epididymus sperm at 150 mg/kg/day
Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: The observed changes were considered unlikely to be of toxicological importance
Critical effects observed:
not specified
Conclusions:
On the basis of above results, the No Observed Adverse Effect Level (NOAEL) of the test substance for the males was judged to be 30 mg/kg/day each in the conditions of this study because of the effects on the testis and epididymis of the 150 mg/kg group.

For treated male rats, the presence of eosinophilic droplets in the cortical tubular epithelium of the kidneys also extended to rats in the intermediate dosage group receiving DMI at 30 mg/kg/day. However, this change is commonly found following treatment with low molecular weight hydrocarbons and is species and sex specific to the male rat. The finding is not therefore considered to be a significant finding in respect to the test substance toxicity.

Changes in general health and in bodyweight gains were also noted for rats receiving DMI at 6 or 30 mg/kg/day. These changes were not severe in nature and in the absence of any other significant morphological or physiological changes, were not considered to be of major toxicological importance.

Executive summary:

The repeated dose toxicity of the test material was investigated in a GLP study which was conducted accoding to a methodology similar to the standardised guideline OECD 407.

During the study, groups of rats received DMI, formulated as a solution, at dose levels of 6, 30 and 150 mg/kg bw/day, daily, for a period of 28 consecutive days, by oral gavage. Clinical findings, bodyweight changes, food consumption, haematology parameters, biochemistry parameters and urinalayses were conducted throughout the study. Following treatment on day 28, the animals were sacrificed. Organs were weighed and macroscopic and microscopic pathology investigation ensued.

Under the conditions of the study, the No Observed Adverse Effect Level (NOAEL) of DMI for the males was judged to be 30 mg/kg/day each in the conditions of this study because of the effects on the testis and epididymis of the150 mg/kg group.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD) strain [SPF]
Sex:
male/female
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
distilled
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
The dosing formulations were administered orally to animals by gavage throughout the dosing period. The control group was administered with only the solvent (water for injection) in the same manner.
Frequency of treatment:
once a day
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:
5
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
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.

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
In the gross necropsy, the external surface of animals, oral cavity, nasal cavity and cranial cavity, skeleton, the external surface and sections of brain and spinal cord, thoracic, abdominal and pelvic cavities including internal organs and cervical tissues and organs were observed. All macroscopic abnormalities were recorded in terms of the location, size, color tone, etc. In all necropsied animals, the following organs and tissues were fixed in an adequate volume of 10 vol% neutral buffered formalin solution and stored; skin, mammary gland, lymph nodes (mesenteric and cervical), salivary glands (sublingual gland and mandibular gland), sternum, femur, bone marrow (sternum and femur), thymus, trachea, lungs (including bronchi), heart, thyroid glands, parathyroid glands, tongue, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, liver, pancreas, spleen, kidneys, adrenal glands, urinary bladder, seminal vesicles, prostate, ovaries, uterus, vagina, eyes (including optic nerve), Harderian glands, brain (including cerebrum, cerebellum and pons), pituitary gland, spinal cord (cervical, thoracic and lumbar parts), skeletal muscle, sciatic nerve, aorta and macroscopic abnormal organs/tissues. The testes and epididymides were pre-fixed in Bouin’s fluid, and then retained in 10 vol% neutral buffered formalin solution and stored.

HISTOPATHOLOGY: Yes
The animals corresponding to the conditions of following (1) to (6) were examined histopathologically.
The hematoxylin-eosin stained specimens and PAS reaction specimens (for testes and epididymides) were prepared using the fixed organs and tissues according to the routine procedure.
All histological abnormalities, including the type, grade, etc., were recorded.
The testes and epididymides were staind with PAS- hematoxylin and with hematoxylin-eosin. The typical abnormalities of these organs were examined using the hematoxylin-eosin stained specimens. The spermatogenesis stages (VII or VIII) were examined using the PAS-hematoxylin stained specimens of the males corresponding to the following (2) , (4), (5) and (6)1). Moreover, as the findings suggesting the effect of dosing of test substance were detected in the 100 mg/kg group, the corresponding organs and tissues of the each 5 males (Animal ID Nos. 1101 to 1105, 1202 to 1206) corresponding to the following (2)in the 4 and 20 mg/kg groups were examined histopathologically.
(1) Females showed all dead pups (Animal ID Nos. 2302, 2306, 2310, 2312)
All fixed organs.
(2) Males fertilized females
All fixed organs of 5 rats (Animal ID Nos. 1001 to 1005) of the control group and high dose group, and the tissues of macroscopic abnormal sites of the other animals (pituitary gland, Animal ID No. 1006; lungs, Animal ID No. 1210; kidneys, Animal ID Nos. 1105, 1108).
(3) Naturally-delivering females
All fixed organs of 5 rats (Animal ID Nos. 2001 to 2005) of the control group and high dose group, and the tissues of macroscopic abnormal sites of the other animals (lungs, Animal ID No. 2008; kidneys, Animal ID Nos. 2007, 2011, 2108, 2110, 2111, 2208; liver, Animal ID Nos. 2110, 2203, 2210; stomach, Animal ID No. 2111).
(4) A male and a female did not copulate (Animal ID Nos. 1009, 2009)
Vagina, uterus, ovaries, testes, epididymides, seminal vesicles, prostate and the tissues of macroscopic abnormal sites.
(5) Males did not fertilize females and infertile females (Animal ID Nos. 1109, 1201, 1301, 1309, 2109, 2201, 2301, 2309)
Vagina, uterus, ovaries, testes, epididymides, seminal vesicles, prostate and the tissues of macroscopic abnormal sites.
(6) Male and female animals of recovery groups
All tissues of macroscopic abnormal sites and all fixed organs.

Statistics:
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.
Detailed clinical observation and sensory reactivity to various stimuli and the incidence of histopathological findings were subjected to Fisher’s exact test.
The numbers of cell type per Sertori cell in the spermatogenesis stages were analyzed by the Mann-Whitney U test.
The findings of general conditions and macroscopic findings at necropsy were not analyzed statistically.

The significance level in Bartlett’s test for homogeneity of variance was 5% and the significance levels in the other tests were two-sided 5% and 1%. If the animal number was 2 or less per group, these data of this group were not analyzed statistically.
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.
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
Dose descriptor:
NOEL
Effect level:
20 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Effects on the testis and epididymis of the 100 mg/kg group.
Key result
Dose descriptor:
NOAEL
Effect level:
20 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Effects on the testis and epididymis of 100 mg/kg group.
Dose descriptor:
NOEL
Effect level:
20 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Low body weight gain and daily food consumption.
Key result
Dose descriptor:
NOAEL
Effect level:
20 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Low body weight gain and daily food consumption.
Critical effects observed:
not specified
Executive summary:

The repeated dose 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.

Parameters evaluated included clinical signs, bodyweight changes, food consumption, urinanalysis, blood biochemistry, haematology, functional observational battery examination. Following sacrifice, organ weights were recorded gross pathology and histopathology examinations were carried out.

Under the conditions of the study no males or females were dead or moribund.  The given test material did not affect the general conditions, including the detailed observation in the FOB.  

During the dosing period, gains in the body weight were lower or tended to be lower in the males and females of the 100 mg/kg group.  However, the body weight gains were higher during the recovery period.  Therefore, the given test material showed a tendency to reduce the body weight gain and a tendency to recovery of the weight gain by the withdrawal was seen.

As an effect on the food consumption, the mean daily food consumption of female 100 mg/kg group was lower during the lactation period.  

In the haematological examination of the female 100 mg/kg group, haematocrit, haemoglobin and red blood cell count were higher and reticulocyte ratio tended to be lower on day 5 of lactation.  

In the blood chemical examination of the male 100 mg/kg group, triglyceride and total cholesterol were higher or tended to be higher. Moreover, total protein was lower in the female 100 mg/kg group on day 5 of lactation.  

In the serum protein electrophoresis of the female 100 mg/kg group on day 5 of lactation, the concentrations and ratios of albumin and gamma-globulin fractions and A/G ratio were lower, and the ratio of beta-globulin fraction was higher.  

Pathological examination revealed a macroscopical change as atrophy of testis and epididymis in the male 100 mg/kg group. Also, the weights of these organs were lower in the same group. These changes are considered to be caused by the dosing of test substance. As the histopathological findings, atrophy and vacuolation of seminiferous tubule, multinucleated giant cell formation and interstitial cell hyperplasia were observed in testis in the male 100 mg/kg group.  In addition, at the end of the recovery period, Sertoli cell-only syndrome (the finding which shows that most reproductive cells disappeared and only Sertoli cells remain in the seminiferous tubule) was found in the testis. At the end of the dosing period, decrease of sperm and cell debris in the lumen were found in the epididymis. At the end of the recovery period, decrease of sperm was found in the epididymis. In females, the spleen weight was lower in the 100 mg/kg group. However, no histopathological changes which were considered to be caused by the dosing of test material were found.  

Therefore, under the conditions of the study, the No Observed Adverse Effect Level was considered to be 20 mg/kg bw/day

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
30 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
This key study was part of a series of animal studies conducted to methods equivalent to modern regulatory standards.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

28-day repeated dosie toxicity (HRC, 1989)

The repeated dose toxicity of the test material was investigated in a GLP study which was conducted accoding to a methodology similar to the standardised guideline OECD 407.

During the study, groups of rats received DMI, formulated as a solution, at dose levels of 6, 30 and 150 mg/kg bw/day, daily, for a period of 28 consecutive days, by oral gavage. Clinical findings, bodyweight changes, food consumption, haematology parameters, biochemistry parameters and urinalayses were conducted throughout the study. Following treatment on day 28, the animals were sacrificed. Organs were weighed and macroscopic and microscopic pathology investigation ensued.

Under the conditions of the study, the No Observed Adverse Effect Level (NOAEL) of DMI for the males was judged to be 30 mg/kg/day each in the conditions of this study because of the effects on the testis and epididymis of the150 mg/kg group.

28-day repeated dose toxicity (Biosafety researc Centre, 2011)

The repeated dose 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.

Parameters evaluated included clinical signs, bodyweight changes, food consumption, urinanalysis, blood biochemistry, haematology, functional observational battery examination. Following sacrifice, organ weights were recorded gross pathology and histopathology examinations were carried out.

Under the conditions of the study no males or females were dead or moribund.  The given test material did not affect the general conditions, including the detailed observation in the FOB.  

During the dosing period, gains in the body weight were lower or tended to be lower in the males and females of the 100 mg/kg group.  However, the body weight gains were higher during the recovery period.  Therefore, the given test material showed a tendency to reduce the body weight gain and a tendency to recovery of the weight gain by the withdrawal was seen.

As an effect on the food consumption, the mean daily food consumption of female 100 mg/kg group was lower during the lactation period.  

In the haematological examination of the female 100 mg/kg group, haematocrit, haemoglobin and red blood cell count were higher and reticulocyte ratio tended to be lower on day 5 of lactation.  

In the blood chemical examination of the male 100 mg/kg group, triglyceride and total cholesterol were higher or tended to be higher. Moreover, total protein was lower in the female 100 mg/kg group on day 5 of lactation.  

In the serum protein electrophoresis of the female 100 mg/kg group on day 5 of lactation, the concentrations and ratios of albumin and gamma-globulin fractions and A/G ratio were lower, and the ratio of beta-globulin fraction was higher.  

Pathological examination revealed a macroscopical change as atrophy of testis and epididymis in the male 100 mg/kg group. Also, the weights of these organs were lower in the same group. These changes are considered to be caused by the dosing of test substance. As the histopathological findings, atrophy and vacuolation of seminiferous tubule, multinucleated giant cell formation and interstitial cell hyperplasia were observed in testis in the male 100 mg/kg group.  In addition, at the end of the recovery period, Sertoli cell-only syndrome (the finding which shows that most reproductive cells disappeared and only Sertoli cells remain in the seminiferous tubule) was found in the testis. At the end of the dosing period, decrease of sperm and cell debris in the lumen were found in the epididymis. At the end of the recovery period, decrease of sperm was found in the epididymis. In females, the spleen weight was lower in the 100 mg/kg group. However, no histopathological changes which were considered to be caused by the dosing of test material were found.  

Therefore, under the conditions of the study, the No Observed Adverse Effect Level was considered to be 20 mg/kg bw/day

Justification for classification or non-classification

The registered substance does not meet the criteria for classification for Specific Target Organ Toxicity - Repeated Exposure (STOT-RE) according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.