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:
other: Guideline study and GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
Batch.No.: Kn-Gi 9328-168
SAT: 07006

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
90 days
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 60, 100, 140 mg/kg bw/day
Basis:

No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:
Clinical sign of transient lethargy was occasionally observed in both male and female rats from second week.
The severity of clinical signs viz. salivation, lethargy and tremors was observed in dose dependent manner but these signs were
transient within the study period. Other clinical signs were found incidental.

Sacrifice and pathology:
Gross pathological examinations of the animals of either sex belonging to the control as well as various
treated groups revealed lesions in liver, lungs, heart, spleen, (female) uterus (female), watery fluid in thoracic
cavity and abdominal cavity.
Minor histological changes, except some seen in liver, heart and kidney, found in organs of all treated groups
compared well with the control and recovery control groups and were at par with our historical control data
(Prasad, et al., 2001). Hence, these changes were considered to be spontaneous/incidental and adaptive/
physiological in nature.
Hepatic, nephritic and cardiac changes were often seen not strictly dose-related and/or even found in control
animals. Nevertheless centrilobular hepatocyte degeneration in combination with MNC infiltration found in
G4 animals seems to be a treatment related effect. Similar effects were found in kidneys of G3 and G4
animals (predominately in males) where regenerative tubules were in combination of MNC infiltration. The
hearts of these dosed animals (G3, G4) showed myocardial degeneration/necrosis/fibrosis and ventricle
dilation which did not recovered during the treatment free period of 28 days may be treatment related.
Other examinations:
Haematology
ln males, significant , in females, significantly increased platelets, MCHC and WBC (all the test substance treated goups but not in G6) were
observed as compared to the control group (Gl). Above alterations were inconsistent and/or within biological range, therefore considered as incidental findings.
Clinical Chemistry
ln males, significantly increased AST and calcium (G2 and G3), phosphorus (G2, G3,G4 and G6), BUN and urea (G6) and significantly reduced glucose (G2), urea (G2), cholesterol (G3) and chloride (G2 and G3) were observed as compared to the confol
group Gl.
ln females, were observed as compared to the control group (Gt). Significant alteration were obseryed in AST (G2 and G3 either sex), ALT (G2 and G3 in females and phosphorus (either sex of all treated groups). Except of the increased phosphorus, all other clinical chemistry data
were inconsistent and/or with in biological limit hence considered as incidental and not related to test substance toxicity.
Significant decrease in terminal body weight in G3 and G4 males was suggestive of toxic effects of test substance at higher dose levels (i.e. mid and high). Alteration observed in hepatic weight was considered treatment related, whereas, changes observed in other organs are considered incidental as they are within normal range/due to experimental stress/absence of significance pathological
lesions. The dose-related increase in relative liver weight is considered to be a physiological adaptation to the enforced xenobiotic metabolisrn
in the liver and without toxicologieal concern.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
effects observed, treatment-related
Ophthalmological findings:
effects observed, treatment-related
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
effects observed, treatment-related

Effect levels

Dose descriptor:
NOAEL
Effect level:
>= 60 other: mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the result of present study, it is concluded that the NOAEL of Kn 172 in Wistar rats over a period of 90 days is 60 mg/kg bw/day.



Executive summary:

This study was conducted to assess the toxic effects of Kn 172 (N-(4-amino-3-methylphenyl)-N-[3-(1Himidazol-

1yl)-propyl]amine x 3 HCl) in SPF Wistar rats when administered by oral gavage for a period of 90 consecutive days. The methods followed were as per the guidelines of OECD N° 408 (September 21, 1998).

60 male and 60 female rats of Wistar strains were randomly divided into six groups (G1 to G6). Each group

comprised of 10 rats per sex per group. The animals were treated with Kn 172 in distilled water by oral gavage at dose levels of 60 (G2 - low dose), 100 (G3 - mid dose) and 140 mg/kg bw/day (G4 - high dose) for a period of 90 consecutive days. Concurrent control group (G1 - 0 mg/kg bw/day) animals were treated with vehicle (distilled water). In addition, high dose recovery (G6 - 140 mg/kg bw/day) and control recovery group (G5 - 0 mg/kg bw/day) were treated for 90 days and observed further for a period of 28 days to investigate the persistence, recovery or delayed effect of Kn 172, if any.

The dose formulations were prepared by dissolving Kn 172 in distilled water. The dose formulations were tested for homogeneity of mixing and active ingredient concentration by validated analytical method. Stability test results revealed 95.23, 95.83 and 100.84 % mean recovery of active ingredient at 6, 10 and 14 mg/mL concentrations, respectively. The dose formulations were prepared fresh everyday prior to dosing. Triplicate sample analysis of test substance in distilled water revealed homogenous mixing of test substance in the vehicle.

Each rat was observed for mortality and visible signs of reaction to treatment twice a day throughout the studyperiod. Body weight and food consumption were determined weekly. Detailed clinical examination/neurobehavioural observations were conducted on each animal once prior to initiation of treatment and at weekly intervals thereafter. Ophthalmological examination was made on all animals prior to the initiation of treatment. Prior to terminal and recovery sacrifice, ophthalmologic examination was performed in all surviving animals. Functional observational battery (FOB) test was performed on all surviving animals during 12th week of treatment (G1 to G4) and 4th week of recovery period (G5 and G6).

Haematological and biochemical analysis were performed on blood samples of all surviving animals at the end of the treatment and the recovery periods. Urine samples were collected from all surviving rats at the end of the treatment and the recovery period for urine analysis.

All the rats were sacrificed by carbon dioxide asphyxiation and subjected to a gross pathological examination at the end of the treatment and the recovery periods. Absolute organ weights were recorded and relative organ weights were calculated for the organs viz., adrenals, brain, ovaries/testes, uterus/epididymides, heart, kidneys, liver, spleen and thymus in all rats. The required organs were collected from all the animals at necropsy. Histopathological examination was made on all preserved organs from the control and the high dose group animals and liver, kidneys, heart, lung and other organs showing gross lesions from rest all the

groups viz., low, mid and recovery groups (control and high dose).

The findings from the present study were:

Total two males and two females of G4 (high dose) and two males and one female of G6 (high dose recovery)

died during the study.

Clinical signs of mild to severe salivation, piloerection, lethargy and tremors occurred shortly after gavages

application of Kn 172. All these clinical signs were transient within the study period and persisted typically

for approximately 30 minutes post dosing.

Ophthalmological examinations performed prior to sacrifice did not reveal any treatment related abnormality. No treatment related changes were observed in home cage and handling observations of Neurobehavioural/detailed clinical observations. In open field observation there was significant reduction in rearing count in all Kn 172 treated groups in male and female. Motor activity was significantly decreased in G3 and G4 females. There was no significant finding in hindlimb foot splay, grip strength and motor activity. In sensory reactivity, approach and tail pinch response seems to be slightly affected.

Treatment related significant reduction in mean body weight (male rats from G3, G4 and G6) and percent body weight change (either sex from G3, G4 and G6) was observed when compared with the respective control groups, which was mostly recovered during the recovery period of 28 days (G6 - male and female).

Mean food consumption was reduced in G4 and G6 males for a certain period and intermittently in G3 male and high dose females (G4 and G6). By the end of the study, this parameter was not different in treated animals compared to their control groups. This temporary alteration is considered as an adaptation but without toxicological relevance.

No treatment related changes were observed in any of the hematological parameter in any groups of either sex whereas evaluation of clinical chemistry parameters revealed treatment related alteration in AST, ALT and phosphorus.

Significant decrease in terminal body weight in G3 and G4 males was suggestive of toxic effects of test substance at higher dose levels (i.e. mid and high). Alteration observed in hepatic weight was considered treatment related, whereas, changes observed in other organs are considered incidental as they are within normal range/due to experimental stress/absence of significance pathological lesions.

Gross pathological examinations of the animals of either sex belonging to the control as well as various treated groups revealed lesions in liver, lungs, heart, spleen, (female) uterus (female), watery fluid in thoracic cavity and abdominal cavity.

Minor histological changes, except some seen in liver, heart and kidney, found in organs of all treated groups compared well with the control and recovery control groups and were at par with our historical control data (Prasad, et al., 2001). Hence, these changes were considered to be spontaneous/incidental and adaptive/ physiological in nature.

Hepatic, nephritic and cardiac changes were often seen not strictly dose-related and/or even found in control animals. Nevertheless centrilobular hepatocyte degeneration in combination with MNC infiltration found in G4 animals seems to be a treatment related effect. Similar effects were found in kidneys of G3 and G4 animals (predominately in males) where regenerative tubules were in combination of MNC infiltration. The hearts of these dosed animals (G3, G4) showed myocardial degeneration/necrosis/fibrosis and ventricledilation which did not recovered during the treatment free period of 28 days may be treatment related.

Due to the effects found in the liver, kidneys and hearts of G3 and G4 dosed animals the test substance was considered to be systemic toxic at concentrations of 100 mg/kg bw/day or exceeding concentrations under the used test conditions. At G2 level (60 mg/kg bw/day) animals were considered to be unaffected in regard to any relevant toxicological effects for either sex.