N-(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)-3-hydroxy-4-[[2-methoxy-5-methyl-4-[(methylamino)sulphonyl]phenyl]azo]naphthalene-2-carboxamide

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From June 2012 to January 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD 407) performed under GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N-(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)-3-hydroxy-4-[[2-methoxy-5-[(phenylamino)carbonyl]phenyl]azo]naphthalene-2-carboxamide

EC Number:

235-425-2

EC Name:

N-(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)-3-hydroxy-4-[[2-methoxy-5-[(phenylamino)carbonyl]phenyl]azo]naphthalene-2-carboxamide

Cas Number:

12225-06-8

Molecular formula:

C32H24N6O5

IUPAC Name:

3-hydroxy-4-{[2-methoxy-5-(phenylcarbamoyl)phenyl]diazenyl}-N-(2-oxo-2,3-dihydro-1H-benzimidazol-5-yl)-2-naphthamide

Test material form:

solid: nanoform, no surface treatment

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Species/strain: Healthy Wistar rats, Crl: WI(Han) (Full Barrier)
- Source: Charles River, 97633 Sulzfeld, Germany
- Sex: male and female; the female animals were non-pregnant and nulliparous.
- Age at study initiation: males: 7-8 weeks old, females: 7-8 weeks old.
- Weight at study initiation: males: 144- 167 g, (mean: 156.10 g, ± 20% = 124.88 -187.32 g)
females: 119 -139 g, (mean: 128.73 g, ± 20% = 102.99- 154.48 g)
- Housing: Full barrier in an air-conditioned room; housed individually in IVC cages, type III, polysulphone cages on Altromin saw fibre bedding
- Diet: Altromin 1324 maintenance diet for rats and mice (lot no. 0715), ad libitum
- Water: tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals), ad libitum
- Certificates of food, water and bedding are filed at BSL BIOSERVICE
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-3
- Humidity (%): 55+/-10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

The animals of the main and recovery groups were treated with the test item or vehicle for 7 days per week for a period of 28 days. The test item formulation or vehicle was administered at a single dose per day to the animals by oral gavage. For each animal the individual dosing volume was calculated on the basis of the most recently measured body weight.
The application volume for all groups was 5 mL/kg body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For determination of the nominal concentration of test item in dosing formulations, samples were retained from all groups once weekly during the treatment period and analyzed on the same day within 6 hours (16 samples in total).
In the first week of treatment, samples for the testing of homogeneity were taken from the top, middle and bottom of the freshly prepared high, medium and low dose formulations and analyzed on the same day within 6 hours (9 samples in total).

All samples of dosing formulations were sent to analytic department of BSL BIOSERVICE Scientific Laboratories GmbH on particular day of sampling.
Formulation analysis was performed in accordance with GLP.

Duration of treatment / exposure:

28 days

Frequency of treatment:

once daily, 7 days per week for a period of 28 days.

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 100, 300, 1000 mg/kg Body weight
Basis:
actual ingested

No. of animals per sex per dose:

40 animals (20 males and 20 females) were included in the main study (5 male and 5 female animals per group). The main study included one control (C) and three dose groups (Low Dose = LD, Medium Dose = MD, High Dose = HD).
In addition, 20 animals (5 male and 5 female animals per group) were included in the control and high dose groups which were observed for a period of 14 days following the last administration. (Control Recovery = CR, High Dose Recovery = HDR).

Control animals:

yes, concurrent vehicle

Details on study design:

Preparation of the Animals:
Prior to the start of the treatment period, a detailed clinical observation outside the home cage was made. All animals were healthy and none of those showed pathological signs before the first administration. Before the first administration all animals used for the study were weighed and assigned to the experimental groups with achieving a most homogenous variation in body weight throughout the groups of males and females.

Dosage:
Following doses were selected in consultation with sponsor and on the basis of data from a BSL dose range finding study: 100, 300, 1000 mg/kg body weight.

The animals in the control group were handled in an identical manner to the test group subjects and received corn oil using the same volume as used for the treatment groups.

Administration of Doses:
The animals of the main and recovery groups were treated with the test item or vehicle on 7 days per week for a period of 28 days. The test item formulation or vehicle was administered at a single dose per day to the animals by oral gavage. For each animal the individual dosing volume was calculated on the basis of the most recently measured body weight.
The application volume for all groups was 5 mL/kg body weight.

Examinations

Observations and examinations performed and frequency:

Body Weight and Food Consumption:
The body weight was recorded once before the assignment to the experimental groups, on the first day of administration and weekly during the treatment and recovery period.
Food consumption was measured weekly during the treatment and recovery period.

Clinical Observation:
All animals were observed for clinical signs during the entire treatment period of 28 days. The recovery animals were observed for an additional
period of 14 days following the last administration.
Twice daily all animals were observed for morbidity and mortality except for weekends and public holidays when observations were made once daily.

Ophthalmological examination, using an ophthalmoscope was made on all animals before the first administration and in the last week of the treatment period as well at the end of the recovery period in the recovery animals.

Functional Observation:
Once before the first exposure as well as once in the fourth week of exposure multiple detailed behavioural observations were made outside the home cage using a functional observational battery of tests . These tests were conducted in all animals.

Haematology:
One day after the last administration, blood was sampled from all surviving animals of the main study for a haematological evaluation. Blood from the recovery animals was sampled at the end of the recovery period.
Haematological parameters examined were: haematocrit value (Hct), haemoglobin content (Hb), red blood cell count (RBC), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), reticulocytes (Re), platelet count (PLT), white blood cells (WBC), neutrophils (Neu), lymphocytes (Lym), monocytes (Mono), eosinophils (Eos), basophils (Baso)

Blood Coagulation:
One day after the last administration, blood was sampled from all surviving animals of the main study for an evaluation of the coagulation parameters. Blood from the recovery animals was sampled at the end of the recovery period.
Blood Coagulation parameters examined were: prothrombin time (PT) and activated partial thromboplastin time (aPTT)

Clinical Biochemistry:
One day after the last administration, blood was sampled from all surviving animals of the main study for an evaluation of the clinical biochemistry. Blood from the recovery animals was sampled at the end of the recovery period.
Clinical biochemistry parameters examined were: alanine aminotransferase (ALAT), aspartate-aminotransferase (ASAT), alkaline phosphatase (AP), creatinine (Crea), total protein (TP), albumin (Alb), urea, total bilirubin (TBIL), total bile acids (TBA), total cholesterol (Chol), glucose (Gluc), sodium (Na), potassium (K)

Urinalysis:
Urinalysis was performed with samples collected from all animals prior to or as part of the sacrifice of the animals. The following parameters were measured using qualitative indicators (Heiland Urine Stripes URI 10SL).
Urine parameters examined were: urine colour/appearance, specific gravity, nitrite, ph-value (ph), protein, glucose, ketone bodies (ketones), urobilinogen (ubg), bilirubin, blood, leucocytes.

Sacrifice and pathology:

Pathology:

Gross necropsy:
All animals from the main and recovery groups were subjected to a detailed gross necropsy on day 29 for main group animals and day 43 for recovery group animals which included careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.
The wet weight of the organs (liver, kidneys, adrenals, testes, epididymides, prostate, seminal vesicles and coagulating glands, ovaries, uterus with cervix, thymus, thyroid/parathyroid glands, spleen, brain, pituitary gland and heart) of all sacrificed animals was recorded as soon as possible. Paired organs were weighed separately.
The following tissues from all animals were preserved in 10% neutral buffered formalin except eyes, testes and epididymides that were fixed in Modified Davidson’s fixative for approx. 24 hours before they were transferred to 10% neutral buffered formalin.
Preserved and Examined Tissues:
brain (cerebrum, cerebellum and pons), spinal cord, eye, liver, kidneys, adrenal glands, stomach, small and large intestines (including Peyer´s patches), thymus, thyroid, spleen, lung and trachea, mammary glands, skin, aorta, All gross lesions, heart, ovaries, uterus with cervix, vagina, testes, epididymides, prostate and seminal vesicles with coagulating glands as a whole, urinary bladder, lymphnodes (mesentric and axillary), peripheral nerve (e.g. sciatic nerve) with skeletal muscle, bone with bone marrow (sternum), pituitary gland, oesophagus, Salivary glands, pancreas.

Histopathology:
The preserved organs were examined histopathologically after preparation of paraffin sections and haematoxylin-eosin staining.
A histopathological evaluation was carried out on all animals of the control and high dose groups (of the main study) which were sacrificed at the end of the treatment period. Histopathological examinations were not extended to animals of the other dose groups and recovery animals, as no treatment related changes were observed in the high dose group.
Gross lesions macroscopically identified were examined microscopically.
The histological processing of tissues to microscope slides was performed at the GLP-certified contract laboratory Propath UK Ltd, Willow Court, Netherwood Road, Hereford HR2 6JU, Great Britain (test site for tissue processing). The histopathological evaluation was performed at the GLP-certified contract laboratory KALEIDIS – Consultancy in Histopathology (test site for histopathology), 6 rue du Gers, 68300 Saint-Louis, France. Blocking, embedding, cutting, H&E staining and scientific slide evaluation was performed according to the corresponding SOP’s of the test sites.

Statistics:

A statistical assessment of the results of the body weight, food consumption, parameters of haematology, blood coagulation and clinical biochemistry and absolute and relative organ weights were performed for each gender by comparing values of dosed with control animals of the main groups using a one-way ANOVA and a post-hoc Dunnett Test. High dose and control values of the recovery groups were compared using a Student’s t-Test. These statistics were performed with GraphPad Prism 5.01 software (p<0.05 was considered as statistically significant).

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:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

Animal Survival:
No mortalities were observed in the study and all animals were sacrificed on day 29 (main group animals) and day 43 (recovery group animals).

Clinical Observations:
No test item related clinical signs were observed in any male and female animal during the entire study period. Few spontaneous clinical signs observed occasionally in male and female animals were alopecia from abdomen (1/5 in LD male), slight piloerection (2/5 in CR Males), moderate piloerection (1/5 in HDR male), slight salivation (1/5 in C female), moderate salivation (1/5 in LD female), aggressive behaviour (1/5 in HD female), moving the bedding, slightly increased spontaneous activity, aggressive behaviour, alopecia from abdomen and forepaws (1/5 in HDR female).
During the weekly detailed clinical observation, no significant changes or differences between the groups were observed.
There were no ophthalmoscopic findings in any of the animals of this study.

Functional Observation Battery:
No relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period. There were no biologically relevant differences in body temperature between the groups.

Body Weight Development:
In both males and females, no statistically significant effect was observed on body weight and body weight change in treatment groups during the entire study period except significant increase in mean daily weight change in HD males when compared with the controls. This significant effect is considered to be toxicologically irrelevant and non adverse. All group mean values for body weight and body weight change were comparable with respective controls and were within the normal range of variation for this strain.

Food Consumption:
Statistical analysis of food consumption data of male and female animals revealed no test item related effect on food consumption during the whole study period. In correlation to the body weight and body weight change, the food consumption in both males and females increased with the progress of the study in all groups.

Haematology and Blood Coagulation:
In males, at the end of the treatment period and recovery period, statistically significant increase in basophils in HD group and increase in large unstained cells and reticulocytes was observed in HDR group when compared to the controls. Since increase in basophils was within the normal range of variation and since difference in large unstained cells and reticulocytes was not observed at the end of treatment period in main study animals these statistically significant effects on few haematology parameters were considered to be non adverse and biologically irrelevant. Blood coagulation was not affected in males by the test item.
In females, at the end of the treatment period, no statistically significant difference was observed in any of the haematology parameters in treated groups when compared with the controls. However, statistically significant decrease in aPTT was observed in HDR group at the end of recovery period. Since the difference in aPTT was not observed at the end of treatment period in main study animals and since this difference was within the normal range of variation this effect was considered to be of no toxicological significance.
All remaining haematological parameters remained unaffected in male and females and were within the normal range of variation.

Clinical Biochemistry:
In males, at the end of the treatment period, no statistically significant difference was observed in any of the clinical biochemistry parameters in treated groups when compared with the controls. However, statistically significant increase in creatinine and decrease in TBA was observed in HDR group which was considered to be non adverse in the light of no such effect was observed in the main study animals sacrificed at the end of treatment period.
In females, at the end of the treatment period, statistically significant decrease in glucose in LD group and at the end of recovery period significant decrease in sodium was observed in HDR group when compared with the controls. Due to lack of dose dependency in effect on glucose and decrease in sodium was marginal, this effect on female clinical biochemistry parameters was not considered to be test item related.
All other clinical biochemistry parameters in male and females remained unaffected due to the treatment and were within the normal range of variation for this strain.

Urinalysis:
High protein and erythrocyte levels were found in the urine of few animals including control animals. All other urinary parameters were in the normal range of variation and no conspicuous differences between dose group and control group were observed.

Pathology:
At necropsy of male and females by using a high dose of Ketamine/Xylazine (2:1), macroscopic examination of the animals revealed few findings in males and females as follows:
Mandibular lymph nodes- discoloured red (1/5 in control male), Axillary lymph nodes- discoloured dark (1/5 in control and LD male), Thymus- discoloured dark (1/5 in control male), Epididymis (right)-yellow spot (1/5 in MD male), Uterus with oviduct and cervix- fluid distension (1/5 in control and LD female), Adrenal gland (Left) - red spot (1/5 in HD female) and Oesophagus- with black content (1/5 in control recovery female).
These gross pathological findings were spontaneous in nature and were assumed to be common background findings in this strain and as such not due to a systemic effect due to the test item administration.

Organ Weight:
In males, at the end of treatment period, statistically significant increase in absolute and relative (to brain and body weight) prostate weight in MD group, increase in absolute pituitary weight in HD group and increase in relative (to brain weight) pituitary weight in MD and HD group were observed when compared with controls. Statistical analysis of data from animals sacrificed at the end of recovery period revealed decrease in relative (to body and brain weight) total kidney weights in HDR group when compared to the corresponding control.
In females, statistically significant increase in absolute and relative (to brain and body weight) total adrenal weights in LD and MD group and decrease in absolute and relative (to brain and body weight) thyroid/parathyroid weights in MD group were observed when compared with controls. Absolute and relative (to brain and body weight) organ weight data from females sacrificed at the end of recovery period remained unaffected due to the treatment and all group mean and individual values from treated groups were comparable with the controls.
Due to lack of dose dependency and in the light of absence of histopathological findings in the few organs with statistical significance, this effect on organ weight data was considered to be non adverse and biologically irrelevant.

Histopathology:
No test item-related macroscopic organ changes and no toxicologically relevant histopathological findings were observed in this study.
As a conclusion, based on the pathological evaluation, the dose of 1000 mg/kg/day is considered to be the NOAEL (No Observed Adverse Effect Level) for pathology under the conditions of this study.

Dose Formulation Analysis:
Concentration analysis of formulation samples was determined in study week 1, 2, 3 and 4 for all dose groups. The mean recoveries observed in LD, MD and HD groups were 90.8%, 101.0%, and 79.7% of the nominal concentration, respectively.
Homogeneity of formulation samples was determined in study week 1 for all dose groups. The mean recoveries observed for LD group was 89.2%, for MD group 93.9% and for HD group 81.2% of the nominal value. The coefficients of variation of the different sampling locations (top, middle, bottom) was in LD group 1.4%, in MD group 1.9% and in HD group 2.8%.


Stability in the vehicle:
> 72 h at a concentration of 0.1 mg/mL and 50 mg/mL at room temperature (analysed by the sponsor)

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

other: content C.I.

Sex:

male/female

Basis for effect level:

other: No relevant signs of toxicity or mortality was observed up to the highest dose tested (i.e. 1000 mg/kg body weight) in both male and females.

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Under the conditions of the present study, the repeated oral administration of test material to male and female Wistar rats at doses of 100, 300 and 1000 mg/kg body weight for 28 days was associated with no relevant signs of toxicity or mortality.
Based on the data generated from this study, the NOAEL (No Observed Adverse Effect Level) of the teat material is considered to be 1000 mg/kg body weight/day (i.h. highest dose tested) for the 28-day repeated dose oral toxicity study in male and female rats.

Executive summary:

The aim of this study was to assess the possible health hazards which could arise from repeated exposure of the test material via oral administration to rats over a period of 28 days. In order to allow a detection of possible delayed occurrence or persistence of or recovery from toxic effects, the animals in the recovery groups were observed for a period of 14 days following the last administration.

The test item was administered daily in graduated doses to 3 groups of test animals, one dose level per group for a treatment period of 28 days. Animals of an additional control group were handled identically as the dose groups but received corn oil, the vehicle used in this study. The 4 groups comprised of 5 male and 5 female Wistar rats Crl: WI(Han). In addition, 20 recovery-animals (5 males and 5 females per group) were included in the control and high dose groups.

The doses evaluated were 0, 100, 300, 1000 mg/kg body weight.

Dose concentrations were based on the purity/content of the test item (97.3 %).

The test item formulation was prepared freshly on each day of administration. The test item was dissolved in corn oil and administered daily during a 28-day treatment period to male and female animals by oral gavage. Dose volumes were adjusted individually based on a twice-weekly body weight measurement.The application volume for all groups was 5 mL/kg body weight.

During the period of dose administration, the animals were observed precisely each day for signs of toxicity. Body weight was measured twice a week and food consumption was measured weekly. At the end of the treatment period, all main study animals and at the end of recovery period, all recovery animals were sacrificed and subjected to necropsy. The wet weight of a subset of tissues was determined and a set of organs/tissues was preserved in 10% neutral buffered formalin. Full histopathological evaluation of the tissues was performed on high dose and control animals.Organs showing gross abnormalities were also examined histopathologically.

Haematological and clinical biochemistry examinations were made on blood samples obtained from the overnight fasted main and recovery animals at the terminal sacrifice.

Summary of Results:

Animal Survival:

No mortalities were observed in the study and all animals were sacrificed on day 29 (main group animals) and day 43 (recovery group animals).

Clinical Observations:

No test item related clinical signs were observed in any male and female animal during the entire study period except spontaneous clinical signs observed occasionally in few animals.

During the weekly detailed clinical observation, no significant changes or differences between the groups were observed.

There were no ophthalmoscopic findings in any of the animals of this study.

Functional Observation Battery:

No relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period.

Body Weight Development:

In both males and females, no test item related effect on body weight and body weight change was observed in treatment groups during the entire study period.

Food Consumption:

Statistical analysis of food consumption data of male and female animals revealed no test item related effect on food consumption during the whole study period.

Haematology and Blood Coagulation:

In males, at the end of the treatment period and recovery period, statistically significant increase in basophils in HD group and increase in large unstained cells and reticulocytes was observed in HDR group when compared to the controls. Since increase in basophils was within the normal range of variation and since difference in large unstained cells and reticulocytes was not observed at the end of treatment period in main study animals, these effects were considered to be non adverse and biologically irrelevant.

In females, at the end of the treatment period, no statistically significant difference was observed in any of the haematology parameters in treated groups when compared with the controls. However, statistically significant decrease in aPTT was observed in HDR group at the end of recovery period. Since the difference in aPTT was not observed at the end of treatment period in main study animals and this difference was within the normal range of variation and therefore this effect was considered to be of no toxicological significance.

Clinical Biochemistry:

In males, at the end of the treatment period, no statistically significant difference was observed in any of the clinical biochemistry parameters in treated groups when compared with the controls. However, statistically significant increase in creatinine and decrease in TBA was observed in HDR group. In females, at the end of the treatment period, statistically significant decrease in glucose in LD group and at the end of recovery period significant decrease in sodium was observed in HDR group when compared with the controls.

Urinalysis:

High protein and erythrocyte levels were found in the urine of few animals including control animals. All other urinary parameters were in the normal range of variation and no conspicuous differences between dose group and control group were observed.

Pathology:

At necropsy of male and females, macroscopic examination of the animals revealed few spontaneous and common background findings and as such not due to a systemic effect due to the test item administration.

Organ Weight:

In males, at the end of treatment period, statistically significant increase in absolute and relative (to brain and body weight) prostate weight in MD group, increase in absolute pituitary weight in HD group and increase in relative (to brain weight) pituitary weight in MD and HD group were observed when compared with controls. Statistical analysis of data from animals sacrificed at the end of recovery period revealed decrease in relative (to body and brain weight) total kidney weights in HDR group when compared to the corresponding control.

In females, statistically significant increase in absolute and relative (to brain and body weight) total adrenal weights in LD and MD group and decrease in absolute and relative (to brain and body weight) thyroid/parathyroid weights in MD group were observed when compared with controls. Absolute and relative (to brain and body weight) organ weight data from females sacrificed at the end of recovery period remained unaffected due to the treatment and all group mean and individual values from treated groups were comparable with the controls. Due to lack of dose dependency and in the light of absence of histopathological findings in the few organs with statistical significance, these effects were considered to be non adverse and biologically irrelevant.

Histopathology:

No test item-related macroscopic organ changes and no toxicologically relevant histopathological findings were observed in this study.

As a conclusion, based on the pathological evaluation, the dose of 1000 mg/kg/day is considered to be the NOAEL (No Observed Adverse Effect Level) for pathology under the conditions of this study.

Dose Formulation Analysis:

Concentration analysis of formulation samples was determined in study week 1, 2, 3 and 4 for all dose groups. The mean recoveries observed in LD, MD and HD groups were 90.8%, 101.0%, and 79.7% of the nominal concentration, respectively.

Homogeneity of formulation samples was determined in study week 1 for all dose groups. The mean recoveries observed for LD group was 89.2%, for MD group 93.9% and for HD group 81.2% of the nominal value. The coefficients of variation of the different sampling locations (top, middle, bottom) was in LD group 1.4%, in MD group 1.9% and in HD group 2.8%. Stability in the vehicle: > 72 h at a concentration of 0.1 mg/mL and 50 mg/mL at room temperature (analysed by the sponsor)