Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Purity: 100 w/w% (as a reaction product)

Test animals

Species:
rat
Strain:
other: Crj:CD (SD) IGS
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at receipt: Not reported
- Age a study initiation: 5 weeks
- Weight at study initiation: 123.1-144.5 g (males); 106.9-126.4 g (females)
- Housing: Individually in wire net floor cages made of stainless steel
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: Not reported

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2°C
- Humidity (%): 55 ± 10%
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light
- Air Changes: 10-15 times/hr

Administration / exposure

Route of administration:
oral: gavage
Details on oral exposure:
PREPARATION OF TEST SUBSTANCE FORMULATION:
-Solvent used: 0.5 w/v% aqueous carboxymethyl cellulose sodium (CMC-Na) solution
-Preparation frequency: More than once for 7 days
-Preparation details: A 0.5 w/v% solution of CMC-Na was prepared with purified water. The test substance was precisely weighed, finely pulverized in a mortar, and suspended while dropping 0.5 w/v% aqueous CMC-Na solution to prepare 10.0, 2.0, and 0.4 w/v%. The preparation was carried out under lighting of a sodium lamp.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Method of Analysis: HPLC

Homogeneity (Relative standard deviation: 5% or less of Target)
Conducted on prepared solutions: 10.0 and 0.5 (w/v%)
Results:
Nominal conc. 10.0 (w/v%)
Actual conc. (w/v%)
 Top layer of measurement: 10.0
 Middle layer: 10.7
 Bottom layer: 10.3
Mean actual conc. (w/v%): 10.3
Relative standard deviation: 3.4%

Nominal conc. 10.05 (w/v%)
Actual conc. (w/v%)
 Top layer of measurement: 0.0516
 Middle layer: 0.0504
 Bottom layer: 0.0508
Mean actual conc. (w/v%): 0.0509%
Relative standard deviation: 1.2%

Stability of the test substance:
There was no change in the infrared absorption spectrum provided by the sponsor, and that measured prior to the dosage period. Also, no change could be seen after the dosage period. The calibration curve showed a correlation coefficient of R=0.999 and good linearity.

Stability in prepared solutions (100 ± 10% of Time Zero)
Conducted on prepared solutions: 10.0 and 0.5 (w/v%)
Results:
Nominal conc. 10.0 (w/v%)
 Immediately after preparation: mean conc. 10.3 (w/v%)
 3 days after preparation: mean conc. 1.04 (w/v%)
 7 days after preparation: mean conc. 9.96 (w/v%)
Rate to the conc. (7 days) measured immediately after preparation: 96.7%

Nominal conc. 0.05 (w/v%)
 Immediately after preparation: mean conc. 0.0509 (w/v%)
 3 days after preparation: mean conc. 0.0505 (w/v%)
 7 days after preparation: mean conc. 0.0487 (w/v%)
Rate to the conc. (7 days) measured immediately after preparation: 95.7%
Duration of treatment / exposure:
28 days
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
40 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Control and high dose groups consisted of 12 animals/sex, for which 6 animals/sex were used in a 14 day recovery group. Low and middle dose groups consisted of 6 animals/sex.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on no observed effects in a 14-day pretest at 50, 250, and 1000 mg/kg/day.
Positive control:
None

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: note than once a day for mortality and moribundity.

DETAILED CLINICAL OBSERVATIONS: Yes/No
- Time schedule:

BODY WEIGHT: Yes
- Time schedule for examinations: The day prior to the start of dosing; days 1, 3, 8, 12, 17, 21, 26, and 28 of the dosing period; and on days 1, 5, 10, and 14 of the recovery period. Also once immediately before necropsy.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Once prior to dosing on days 3, 8, 15, and 28 for the dosing period; and on days 4, 8, and 14 for the recovery period.

FOOD EFFICIENCY: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: End of dosing and recovery periods.
- Anaesthetic used for blood collection: ether
- Animals fasted: Yes, overnight (16-20 hrs)
- How many animals: All animals
- Parameters checked in table No. 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: End of dosing and recovery periods.
- Animals fasted: Yes, overnight (16-20 hrs)
- How many animals: All animals
- Parameters checked in table No. 2 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: 16-hr urine sample: day 28 of the dosing period and day 14 of the recovery period. .
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Not reported.
- Parameters checked in table No. 2 were examined.

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 3)
HISTOPATHOLOGY: Yes (see table 3)
Statistics:
For weight, the amount of feed intake, the hematological parameters, blood parameters, urine volume, and organ weight, an equi-variance test of the Bartlett method was performed, and if the equi-variance was recognized at a 5% meaningful level, a one-dimensional arrangement variance analysis was performed. If a meaningful difference was recognized in the variance analysis, a test of the Dunnett method was performed between the medium control group and each dosage group.
If the equi-variance was not recognized, a test of Kruskal-Wallis was performed, and if a meaningful difference was recognized, a nonparametric test of the Dunnett method was performed between the medium control group and each dosage group.

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
Description (incidence and severity):
During the recovery period, the amount of feed intake was significantly increased from vehicle control at p<0.05 for males on day 8 and day 14 in the 1000 mg/kg group. This was not considered test substance related as no similar change was seen at the end of the dosing period.
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 males, at the end of the dosing period, the extension of the thromboplastin of the activated part was observed only in the 40 mg/kg group (p<0.05). This was not considered test substance related. At the end of the recovery period, no abnormalities were observed in males. In females, reduction of the average red blood cell volume, the average amount of red blood cell hemoglobin, and the extension of the thromboplastin of the activated part were observed in the 1000 mg/kg group only at the end of the recovery period. These were significantly different from vehicle control at p<0.05. These changes were not considered test substance related as no similar change was seen at the end of the dosing period.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In males, at the end of the dosing period, GOT was reduced (p<0.01) and blood sugar was increased (p<0.05) in the 40 mg/kg group, and GOT was reduced in the 200 mg/kg group (p<0.01). These changes were not considered test substance related. In females, at the end of the dosing period, γ-GTP was increased (p<0.01) in the 1000 mg/kg group and GOT was reduced in the 40 mg/kg group (p<0.05). The test substance influence on the increase of γ-GTP was considered very slight without an accompanying tissue change. The change in GOT was not considered test substance related. In males, at the end of the recovery period, choline esterase (p<0.05) and total cholesterol (p<0.01) were increased in the 1000 mg/kg group. In females, at the end or the recovery period, GPT was reduced (p<0.01) in the 1000 mg/kg group. These changes were not considered test substance related.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
In males, at the end of the recovery period, the absolute brain weight was increased (p<0.05) in the 1000 mg/kg group. In females, at the end of the recovery period the absolute liver (p<0.05), kidneys (P<0.05), and adrenal (p<0.01) weights were decreased in the 1000 mg/kg group. These were not considered test substance related as no similar change was seen at the end of the dosing period.
Gross pathological findings:
no effects observed
Description (incidence and severity):
In females, at the end of the dosing period, the black part (1/6) of the mucous membrane of the glandular stomach was observed in the 40 mg/kg group. This was not considered test substance related. In males, at the end of the recovery period, the swelling increase (1/6) of the spleen was observed in the 1000 mg/kg group. This was not considered test substance related as no similar change was seen at the end of the dosing period.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
In males, at the end of the dosing period, an increase was observed in hyaline droplets in the kidneys (+, 2/6) in the medium control group. In females, at the end of the dosing period, a mineral precipitation (++, 1/6) of the skin and marrow boundary of the kidneys was observed in the medium control group, necrosis (+, 1/1) of the mucous membrane of the fundus gland of the glandular stomach was observed in the 40 mg/kg group, and mineral precipitation (+, 1/6) at the skin and marrow boundary of the kidneys was seen in the 1000 mg/kg group. The mineral precipitation was considered not test substance related as similar changes were observed in the control group. In males, at the end of the recovery period, local necrosis (+, 1/6) of the spleen was seen in the 1000 mg/kg group. This was not considered test substance related as no similar change was seen at the end of the dosing period.
Histopathological findings: neoplastic:
not examined

Effect levels

Dose descriptor:
NOEL
Effect level:
200 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
clinical biochemistry

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
NOEL = 200 mg/kg/day
Executive summary:

A 28-day repeated oral gavage study with a 14-day recovery period was conducted using groups of 6 male and 6 female Crj:CD (SD) IGS rats per dose level. Rats were 5 weeks of age at study start. Dose concentrations were 0, 40, 200, and 1000 mg/kg/day for the test groups and 0 and 1000 mg/kg for the recovery groups. All rats survived and there were no test substance related effects on clinical signs, body weight and weight gain, feed consumption, hematology at the end of the dosing period, urinalysis, organ weight, and gross pathology.

At the end of the dosing period, γ-GTP was increased in females in the 1000 mg/kg/group. The test substance influence on the increase of γ-GTP was considered very slight without an accompanying tissue change. At the end of the dosing period, mineral precipitation of the skin an marrow boundary of the kidneys was observed in females in the 1000 mg/kg group; however since a similar change was also observed in the control group, it was not considered not test substance related.

In the 1000 mg/kg male recovery animals, increased choline esterase, increased total cholesterol, swelling and local necrosis of the spleen were observed. In females recovery animals, increased feed intake, reduction in the average red blood cell volume and the average amount of red blood cell hemoglobin, extension of the thromboplastin of the activated part, reduction of GPT, and reduced liver, kidney, and adrenal weight were observed on day 8 and day 14. Since no similar changes were observed at the end of the dosing period, these changes were not considered test substance related.

Based on the increased γ-GTP in females in the 1000 mg/kg/group, the NOEL in rats was estimated as 200 mg/kg/day.