Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
other: subacute study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From9 December 1991 to 13 July 1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report Date:
1992

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Principles of method if other than guideline:
not applicable.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): P#620

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Japan, Inc.
- Age at study initiation: 5 weeks
- Weight at study initiation: males: 134 to 154 g; females: 115 to 134 g
- Fasting period before study: no data
- Housing: two rats of the same sex were housed in a single polycarbonate cage (265 W * 426 D * 200 H mm), with hard wood chips bedding on four-decker steel racks. The cages with bedding, feeders and watering bottles were autoclaved and changed every weeks.
- Diet (e.g. ad libitum): pellet diet, ad libitum
- Water (e.g. ad libitum): ordinary tap water, filtered through a 5µm filter and sterilized by UV irradiation, ad libitum.
- Acclimation period: during 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-25°C
- Humidity (%): 40 to 70%
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To: no data

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5% Tween 80 in 0.5% CMC-Na aqueous solution.
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: the test item was suspended in the vehicle using a ultra-high-speed homogeniser. The dosing solutions were prepared once a week and were kept in a refrigerator until just before dosing.

DIET PREPARATION
not applicable: gavage administration

VEHICLE
- Justification for use and choice of vehicle (if other than water): no data
- Concentration in vehicle: 0.5% Tween 80 in 0.5% CMC-Na aqueous solution
- Amount of vehicle (if gavage): 1 mL per 100 g bw
- Lot/batch no. (if required): no data
- Purity: no data
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
the low-dose and high-dose solutions were analysed by GC on 7 and 14 days after the preparation and the stability for 14 days of the dosing solutions in a refrigerator were confirmed (see table 7.5.1/1).
Duration of treatment / exposure:
28 days of exposure + 14 days of recovery period
Frequency of treatment:
Once a day in the morning.
Doses / concentrations
Remarks:
Doses / Concentrations:
0; 20; 140 and 1000 mg/kg bw/d
Basis:
actual ingested
No. of animals per sex per dose:
Male: 12 animals at 0 mg/kg bw/day (6 rats were autopsied on the day after the termination of treatment; 6 rats after the recovery period of 14 days)
Male: 6 animals at 20 mg/kg bw/day Male: 6 animals at 140 mg/kg bw/day Male: 12 animals at 1000 mg/kg bw/day (6 rats were autopsied on the day after the termination of treatment; 6 rats after the recovery period of 14 days)
Female: 12 animals at 0 mg/kg bw/day (6 rats were autopsied on the day after the termination of treatment; 6 rats after the recovery period of 14 days)
Female: 6 animals at 20 mg/kg bw/day Female: 6 animals at 140 mg/kg bw/day Female: 12 animals at 1000 mg/kg bw/day(6 rats were autopsied on the day after the termination of treatment; 6 rats after the recovery period of 14 days)
See Table 7.5.1/2
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: An eight-day preliminary study was performed at the doses of 0 and 1000 mg/kg bw/d, and a slight increase of the liver weight was observed, but no death was found. Based on these result, the high-dose was set at 1000 mg/kg bw/d, and the medium and lose doses were set at 140 and 20 mg/kg bw/d, respectively.
- Rationale for animal assignment (if not random): not applicable, randomly assignement
- Rationale for selecting satellite groups: no data
- Post-exposure recovery period in satellite groups: 14 days
- Section schedule rationale (if not random): no data
Positive control:
Not applicable.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: every day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule for examinations: all rats were palpated carefully once a week

BODY WEIGHT: Yes
- Time schedule for examinations: all rats were weighed on the first day of treatment, therefater once a week throughout the test period.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Not applicable

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / No data
Not applicable

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the schedulded sacrifice from all surviving animals
- Anaesthetic used for blood collection: Yes (Thiopental sodium)
- Animals fasted: No
- How many animals: all animals
- Parameters checked in table 7.5.1/3 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the schedulded sacrifice from all surviving animals
- Animals fasted: No
- How many animals: all animals
- Parameters checked in table 7.5.1/3 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: Fresh urine was collected 2 days before scheduled sacrifices and examined for pH, Occult blood, Protein, Glucose, Ketone, Bilirubin and Urobilinogen. Then urine was collected for 18 hours and also examined for Volume, Specific gravity, sodium, potassium and chloride
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters checked in table 7.5.1/3 were examined.

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:
Food consumption was calculated as the total amount of food consumed in each cage divided by the number of rats living in a cage.
Water consumptionj: an aincrease of water consumption was noted in the clinical observation at 2 weeks after commencement of treatment. So, water consumption was calculated as the total amount of water consumed in each cage divided by the number of rats living in a cage from 3 weeks after commencement of treatment.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 7.5.1/4)
HISTOPATHOLOGY: Yes (see table 7.5.1/4)
Other examinations:
no other examination
Statistics:
Bartlett's test, ANOVA, Dunnett's test, Scheffes's test, Kruskal-Wallis 's test and Che2 Test.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
only salivation
Mortality:
mortality observed, treatment-related
Description (incidence):
only salivation
Body weight and weight changes:
no effects observed
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:
effects observed, treatment-related
Description (incidence and severity):
increase in platelet counts, decrease in hemoglobin concentration, MCHC, prothrombin time, increase in the rate of lymphoid cells.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Increase of gamma-GTP, total cholesterol, calcium of total protein and albumin.; decrease of GOT and A/G ratio, of glucose and chloride.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
decrease of urobilinogen and specific gravity, ketone, increase of urine volume, potassium.
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Liver and Kidney
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
brownish change of the Liver, renal discoloration, focal hemorrhage of the lungs, hepatodiaphragmatic nodule, hemorrhage of the eye left ball, pyelectasis.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Diffuse hypertrophy of hepatocytes (+ bile pigment), hyaline droplets in the tubular epithelium of kidneys, basophilic change of renal tubular epithelium
Histopathological findings: neoplastic:
no effects observed
Details on results:
Clinical observations: No death occured. Salivation was observed in all male and female rats treated with 1000 mg/Kg/day 5 days after treatment began and thereafter until dosing ceased. Salivation was also observed in one male and one female rat treated with 140 mg/kg/day after 22 and 24 days treatment respectively and thereafter until dosing ceased. Salivation occurred before or after dosing and disappeared within 40 min. Salivation was not observed during the recovery period.

Body weight: no effect. The body weights of all dose groups for both sexes increased at the same rate as that of the control group, during both the treatment and recovery periods.

Food Consumption:
not applicable

Water Consumption:
not applicable

Laboratory findings:

Haematological Effects: increase in platelet counts in males of the high-dose group, and decrease in hemoglobin concentration, MCHC, prothrombin time, and an increase in the rate of lymphoid cells in females of the high-dose group. Examination at the end of the recovery period revealed increases in reticulocyte counts and platelet counts in males of the high-dose group and decreases in hemoglobin concentration and leukocytes counts in females of the high-dose group.

Blood chemical Analysis: Examination at the end of the treatment period revealed increases in ç-GTP, total cholesterol, and calcium in the both sexes of the high dose-group, decrease of GOT and A/G ratio in males of the high-dose group, decrease of glucose and increase of total protein in females of the high-dose group, and increase of albumin and decrease of chloride in females of the low-and high-dose groups. Examination at the end of the recovery period revealed that alkaline phosphatase and A/G ratio were decreased and total choloesterol was increased in females of the high-dose group.

Urinalysis: Examination at the end of the treatment period revealed that urobilinogen and specific gravity were decreased and the urine volume was increased in both sexes dosed at 1000 mg/kg bw/day. In males dosed at 1000 mg/kg bw/day there was also a decrease in ketone levels. In female rats dosed at both 1000 mg/kg bw/day and 140 mg/kg bw/day potassium levels were increased.
Examination at the end of the recovery period revealed acidification of urine and a decrease in protein in males dosed at 1000 mg/kg bw/day.

Effects in organs:

Organ weight: Examination at the end of the treatment period revealed that both absolute and relative liver weights were increased in both sexes dosed at 1000 mg/kg bw/day. Examination at the end of the recovery period revealed that the relative liver weight was increased in both sexes dosed at 1000 mg/kg bw/day. Furthermore, both absolute and relative kidney weights were increased in males dosed at 1000 mg/kg bw/ day.

Gross pathology: Autopsies at the end of the treatment showed brownish change of the liver in 6 male rats and 4 female rats doses at 1000 mg/kg bw/day. Renal discolouration was seen in two males dosed at 140 mg/kg bw/day and six males dosed at 1000 mg/kg bw/day.
Terminal examination at the end of the recovery at the end of the recovery period showed renal discolouration in one male dosed at 1000 mg/kg bw/day.

Microscopic examination: Examination at the end of the treatment period revealed diffuse hypertrophy of hepatocytes in animals dosed at 1000 mg/kg bw/day. The cytoplasm of the affected hepatocytes appeared eosinophilic and granular. This change was not observed in animals examined after the recovery period.
Bile pigments in hepatocytes and connective tissues, bile plugs in the intralobular bile duct and cholangitis which consisted mainly of lymphocytic infiltration were found in males dosed at 1000 mg/kg bw/day.
Bile pigments in connective tissues, bile plugs in the intralobular bile duct and cholangitis were found at the end of the recovery period, but there was a tendency toward recovery.
In the kidneys, hyaline droplets in the tubular epithelium was observed in all the control males and treated males. However, in males examined at the end of the treatment period and dosed at 140 and 1000 mg/kg bw/day, the findings were classified as moderate; cytoplasm of the tubular epithelium was filled with droplets of hyaline of various sizes and the lesion was seen to be widespread. Only one male dosed at 1000 mg/kg bw/day was found to have this change after the recovery period.
A basophilic change of the renal tubular epithelium was found in one male dosed at 140 mg/kg/day and two males dosed at 1000 mg/kg/day at the end of the treatment period. In this lesion, the basophilic focus of the renal tubule was seen in the cortex, occasional mitoses were encountered. The
same change was also noted in four males dosed at 1000 mg/kg bw/day at the end of the recovery period.

Effect levels

open allclose all
Dose descriptor:
NOEL
Effect level:
20 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effect
Dose descriptor:
NOAEL
Effect level:
ca. 140 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The NOAEL is determined at 140 mg/kg bw/d based only on salivation. No cholangitis associated with cholestasis was observed.
Dose descriptor:
LOAEL
Effect level:
ca. 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Food consumption by all dose groups of both sexes increased in the same fashion as the control group during the treatment and recovery periods. Water consumption by the high-dose group increased in the treatment period, but was the same as that of the control group during the recovery periods.

Applicant's summary and conclusion

Conclusions:
In conclusion, under the test conditions, the LOAEL is considered to be at 1000 mg/kg bw/d in a worst-case taking into account systemic adverse effects of the substance. P#620 is not classified for damage to organs through prolonged oral dose repeated exposure according to the criteria of the Annex VI of the Regulation (EC) No 1272/2008 (CLP) and of the Directive 67/548/EEC.
Executive summary:

In a subacute oral toxicity exposure study, performed similarly to OECD guideline No. 407 and in compliance with the GLP, P#620 (purity > 99%) diluted in an 0.5% CMC-Na aqueous solution with 0.5% Tween 80 was administered by gavage to male and female Crj: CD(SD) rats (6/sex/conditions) for 28 days at three dose levels. A recovery period was set for rats of the control and high-dose groups. Control rats were given the vehicle alone. An eight-day preliminary study was performed at the doses of 0 and 1000 mg/kg bw/d, and a slight increase of the liver weight was observed, but no death was found. Based on these results, the high-dose was set at 1000 mg/kg bw, and the medium and lose doses were set at 140 and 20 mg/kg bw/d, respectively.

Clinical signs were observed each day whereas body-weight was measured once a week. Before the scheduled sacrifice, urine were collected and further analyzed. At the end of the treatment period (for all groups) or after the recovery period (for the control and high-dose group), the animals were sacrificed and further observed for hematology, blood chemistry, gross macroscopy, organ weight and histopathology.

P#620 induced increase in platelet counts in males of the high-dose group, and decrease in hemoglobin concentration, MCHC, prothrombin time, and an increase in the rate of lymphoid cells in females of the high-dose group but these effects were considered as not adverse. The test substance induced also absolute and relative increase of liver weight accompanied with brownish change of the liver and hypertrophy of hepatocytes in the high-dose groups of both sexes.

It is however well known that these changes occur as an induction of the microsomal drug metabolizing enzyme systems caused by the treatment of several compounds, and are considered to be cellular adaptation phenomena. Furthermore, these changes had tendency to recover after withdrawal. Bile pigments in hepatocyte and connective tissues, bile plugs in interlobular bile duct and cholangitis (lymphocytic infiltration) were observed in the high-dose males.

It is assumed that the observed centrilobular hepatocyte enlargement occurs as an induction of the microsomal drug metabolizing enzyme systems caused by the treatment and is considered as cellular adaptation phenomena in the absence of associated inflammatory or degenerative changes. However, the adaptation response to the treatment with Amber core was very important at the highest dose (1000 mg/kg bw/d) in the 28-day repeated oral dose toxicity study as described in this dossier (see § 7.5.1). In fact, the observation of cholangitis (inflammatory) associated to cholestasis in both sexes at the highest dose of Amber core in the sub-acute repeated oral toxicity study showed that the substance induced adverse systemic toxicity. Therefore, even if this effect had tendency to recover after withdrawal (2-week recovery period), the highest dose of 1000 mg/kg bw/d was considered as a LOAEL in a worst-case.

Hyaline droplets of the renal tubular epithelium and basophilic changes were observed in the mid and high-dose male group. This lesion is known to be spontaneous in male rats only and is not observed in other species. Therefore, this effect is specific to male rat, and is not relevant for the risk assessment in human. Therefore, a NOAEL of 140 mg/kg bw/d can be considered based only on the excess of salivation in both sexes. Finally the 20 mg/kg bw/d is considered as a NOEL.

In conclusion, under the test conditions, the highest dose (1000 mg/kg bw/d) is considered as a LOAEL in a worst-case taking into account a potential adverse systemic effect of the substance. Therefore, P#620 is not classified for damage to organs through prolonged oral dose repeated exposure according to the criteria of the Annex VI of the Regulation (EC) No 1272/2008 (CLP) and of the Directive 67/548/EEC.

This study is considered as acceptable and satisfies the requirement for repeated dose toxicty endpoint.