Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Feb-June 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to OECD and GLP guidelines.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
other: Concerning testing methods relating to new chemical substances (METI, 2011 and rev 2012)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): X-22-1622
- Physical state: colourless clear liquid
- Lot No.: 311030

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Labs Japan
- Age at study initiation: 5 weeks
- Weight at study initiation: 124-144 g for males and 114-132 g for females
- Fasting period before study: no
- Housing: stainless steel cages with mesh-floor; metabolic cage during urine collection
- Diet (ad libitum): pelleted diet (MF, lot 131004 and 131209, Oriental Yeast Co., Ltd, Japan)
- Water (ad libitum): chlorinated (maintained at 3-5 ppm chlorine)
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.2-23.4
- Humidity (%): 48.7-62.6
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 25 Feb - 8 April 2014

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: test chemical was weighed and dissolved in corn oil to prepare 10.0 w/v% formulation. Formulations of 3.00 and 1.00 w/v% were prepared by diluting with corn oil. Dosing formulations were administered within 11 days of preparation.

VEHICLE
- Justification for use and choice of vehicle (if other than water): corn oil had been used for a similar substance and the test substance dissolved at 10 w/v% in corn oil; historical control data of corn oil were available
- Concentration in vehicle: 1, 3, or 10% w/v
- Amount of vehicle (if gavage): 10 ml/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
CERI, Stability analysis of X-22-1622, stability and concentration analyses of the formulation of X-22-1622, study no. X18-1058, 18 June 2014.
Stability during 13 days under the storage conditions was determined for 10.0 and 0.100 w/v% formulations. Concentrations of the test chemical in the 10.0, 3.00 and 1.00 w/v% formulations was determined with gas chromatography.
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg bw/d; incl. recovery for vehicle control and high dose
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: no adverse effect on clinical observations, body weight, necropsy and organ weights was observed in a 7-day range-finding study administering rats (3/sex) 0, 30, 100, 300 or 1000 mg/kg bw/d (study no. P11-1058).
- Rationale for selecting satellite groups: no reason given
- Post-exposure recovery period in satellite groups: 14 days for 5/sex
Positive control:
no

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 3 times daily during the dosing period

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the dosing period and once a week during dosing and recovery periods

BODY WEIGHT: Yes
- Time schedule for examinations: day 1, 3, 8, 12, 17, 21, 26 and 28 of dosing period and day 1, 5, 10 and 14 of recovery period

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, on day 1, 3, 8, 15, 22 and 28 of dosing period and day 1, 4, 8 and 14 of recovery period

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No, but included in clinical observations; no further details

HAEMATOLOGY: Yes, parameters according to OECD 407 (2008)
- Time schedule for collection of blood: after urine collection on the day of necropsy
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes, 16-20 hours
- How many animals: all animals

CLINICAL CHEMISTRY: Yes, parameters according to OECD 407 (2008), including bile acids, bilirubin and triglycerides
- Time schedule for collection of blood: after urine collection on the day of necropsy
- Animals fasted: Yes, 16-20 hours
- How many animals: all animals

URINALYSIS: Yes, parameters according to OECD 407 (2008); urine sediments only in control and high dose group
- Time schedule for collection of urine: last night of the dosing period or recovery period (16 hours)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: week 4 of dosing period; since a significant chang in grip strength of the hindlimbs was found in males at 1000 mg/kg bw/d, it was also examined in males in week 2 of recovery.
- Dose groups that were examined: all groups
- Battery of functions tested: reflexes and grip strength and locomotor activity
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, with organs weighed according to OECD 407 (2008) and additional estrous cycle determination
HISTOPATHOLOGY: Yes, according to OECD 407 (2008) for control and high dose; liver for all females; testes, epididymides also for recovery males; ovaries, vagina and uterus also for recovery females
Other examinations:
Since vacuolization of the periportal hepatocytes was suspected to be fatty accumulation in females of the 1000 mg/kg bw/d group, staining by oil red O was performed for frozen sections of the liver from one female at 1000 mg/kg bw/d.
Statistics:
Main group: Bartlett's test for homogeneity of variances and when variances were homogeneous at a significance of 5% Dunnett's test was conducted. When variances were not homogeneous, nonparametric Dunnett's test was conducted. Nonparametric Dunnett's test for defecation frequency and urination.
Recovery group: F-test for variance ratio followed by Student's t-test if no significant difference (5% level) or Aspin-Welch t-test if significant difference. Mann-Whitney U-test for defecation frequency and urination.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY: no treatment-related effects observed

BODY WEIGHT AND WEIGHT GAIN: no treatment-related effects observed on final body weight and body weight gain

FOOD CONSUMPTION: no treatment-related effects observed

HAEMATOLOGY: Prothrombin time and activated partial thromboplastin time were significantly prolonged in males at 1000 mg/kg bw/d (120 and 129% of control, resp). However, control values and values at 1000 mg/kg bw/d were within the historical control data and disappeared after recovery and, therefore, were considered to be not toxicologically relevant. The ratio of large unstained cells was significantly increased and hemoglobin concentration and hematocrit value were significantly decreased (both 96% of control), but within historical control data, in females at 1000 mg/kg bw/d; these effects were not considered toxicologically relevant.
At the end of the recovery period MCHC and large unstained cells were increased in males at 1000 mg/kg bw/d, but still within the historical control data. Therefore, these isolated parameters were considered to be not toxicologically relevant. In females, only reticulocyte count was increased, but within historical control data and not considered toxicologically relevant.

CLINICAL CHEMISTRY: Alinine aminotransferase was increased (183% of control) and chloride was decreased (99% of control, but within historical control data) in females at 1000 mg/kg bw/d; the effects were no longer present at the end of the recovery period. Total protein and calcium were increased at the end of the recovery period (within historical control data). No effects were found in males.

URINALYSIS: An increased amount of protein was observed in one male at 300 and 1000 mg/kg bw/d during the dosing period, but not during recovery; no changes observed in females. In the absence of a dose-related response, the change in males is not considered to be treatment-related.

NEUROBEHAVIOUR: significant increase in grip strength of hindlimb was found in males at 1000 mg/kg bw/d, which had disappeared in the recovery group.

ORGAN WEIGHTS: Relative liver weight was increased (116% of control) in females at 1000 mg/kg bw/d. Absolute and relative thyroid weight was increased at 1000 mg/kg bw; at the end of the recovery period absolute and relative thyroid weight was decreased relative to control.
In the recovery group, absolute epididymides weight was decreased in males. Females still showed an increase in relative liver weight (109% of control). Females also showed increased absolute and relative uterus weight during administration period.

GROSS PATHOLOGY: A grayish region of the lungs was observed in one male, of which the body weight and food consumption were low during the administration period, at 1000 mg/kg bw/d. After recovery pelvic dilatation of the right kidney was seen in one male at 1000 mg/kg bw/d. No changes were observed in females.

HISTOPATHOLOGY: The male with grayish lungs at 1000 mg/kg bw/d showed a moderate focal inflammation of the lung. Microgranuloma in the liver and lymphocyte infiltration in the ventral prostate were observed each in one male at 1000 mg/kg bw/d, the latter was considered incidental. Very slight or slight fatty (confirmed by Oil red O) changes of periportal hepatocytes in 4/5 females, slight or moderate microgranuloma in the liver of 3 females was seen at 1000 mg/kg bw/d. Very slight mineralization in the cortico-medullary junction of the kidney in 2 females (vs 1 control female with slight mineralization) was observed at 1000 mg/kg bw/d and not considered toxicologically relevant. Very slight fatty changes of periportal hepatocytes in 2/5 females, and slight microgranuloma in one female were observed at 300 mg/kg bw/d. Very slight fatty changes of periportal hepatocytes was also observed in one female at 100 mg/kg bw/d.
After recovery, one male at 1000 mg/kg bw/d showed unilateral pelvic dilatation of the kidney (also gross observation). Although unilateral focal atrophy of seminiferous tubules in the testis was observed in 1/5 males at 1000 mg/kg bw/d, no abnormalities were observed in the epididymides whose weight had significantly decreased. No abnormalities were seen in the liver of females whose relative weight had increased, nor in the uterus of females whose absolute weight had increased at 1000 mg/kg bw/d. No abnormalities were seen in the ovary or vagina. therefore, the effect on uterus weight was considered not treatment-related. No fatty changes or microgranuloma in the liver were observed.

HISTORICAL CONTROL DATA:
During administration period (no data during recovery):
PT: 10.55-22.75 sec in 99 males
APTT: 16.02-27.22 sec in 99 males

ESTROUS CYCLE: no effect on length of estrous cycle was shown

Effect levels

Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: fatty changes of periportal hepatocytes and microgranuloma in the liver at 300 mg/kg bw/d and higher in females, which had disappeared after recovery

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Dosing formulations were confirmed to be stable under the storage conditions, since the concentration of the test chemical in the formulations after storage were within 100 +/-10% of those analysed immediately after preparation (99.4% for 10.0 w/v% and 100% for 0.100 w/v%). The concentration of the test chemical in the 10.0, 3.00 and 1.00 w/v% formulations were within 100 +/-10% of each nominal concentration (99.3% for the 10.0 w/v%, 98.7% for the 3.00 w/v% and 96.6% for the 1.00 w/v% formulation.

Applicant's summary and conclusion

Conclusions:
The NOAEL was determined to be 100 mg/kg bw/d based on fatty changes of periportal hepatocytes and microgranuloma in the liver at 300 mg/kg bw/d and higher in females.
Executive summary:

Rats were exposed to 0, 100, 300 or 1000 mg/kg bw/d X-22-1622 in corn oil by gavage for 28 days according to OECD 407 and GLP. For the control group and the 1000 mg/kg bw/d group a recovery period of 14 days was included.

No (toxicologically relevant) effects were observed on mortality, clinical signs, functional behaviour, body weight, food consumption, haematology and urinalysis. Absolute and relative liver weight were increased in females at 1000 mg/kg bw/d, and accompanied by very slight or slight fatty changes of periportal hepatocytes in 4/5 females and slight or moderate microgranuloma in the liver in 3/5 females. Microgranuloma was also seen in one male at 1000 mg/kg bw/d. At 300 mg/kg bw/d very slight fatty changes of periportal hepatocytes were seen in 2/5 females and slight microgranuloma in 1/5 female. Liver effects had disappeared after recovery.

Based on the above results, the NOAEL was determined to be 100 mg/kg bw/d.