5-[(2,3-dihydro-6-methyl-2-oxo-1H-benzimidazol-5-yl)azo]barbituric acid

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:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

limit dose study

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

5-[(2,3-dihydro-6-methyl-2-oxo-1H-benzimidazol-5-yl)azo]barbituric acid

EC Number:

276-344-2

EC Name:

5-[(2,3-dihydro-6-methyl-2-oxo-1H-benzimidazol-5-yl)azo]barbituric acid

Cas Number:

72102-84-2

Molecular formula:

C12H10N6O4

IUPAC Name:

5-[(1E)-2-(6-methyl-2-oxo-2,3-dihydro-1H-1,3-benzodiazol-5-yl)diazen-1-yl]-1,3-diazinane-2,4,6-trione

Test material form:

solid

Details on test material:

CAS No.: 72102-84-2

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Reason for the selection: The rat is a frequently used laboratory animal, and there is comprehensive experience with this animal species. Moreover, the rat has been proposed as a suitable animal species by the OECD and the EPA for this type of study.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services GmbH, Sulzfeld, Germany
- Age at study initiation: 33 +/- 1 days
- Fasting period before study: over night
- Housing: 5 per cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

The test substance was applied as a suspension. To prepare this suspension, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, drinking water was filled up to the desired volume, subsequently mixed by a magnetic stirrer. During administration of the test substance, preparations were kept homogeneous by stirring with a magnetic stirrer. The test-substance preparations were produced once a week.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of Cromophtal Orange K 2960 in drinking water for a period of 7 days at room temperature was proven before the start of the administration period. Homogeneity was verified in 3 samples (was used as a concentration control at the same time) at the beginning of the study. In addition, samples for homogeneity and concentration control analyses were taken weekly. These samples were stored deep-frozen and only analyzed, if the results from the analysis at the beginning were outside of the expected range of 90-110%.

Duration of treatment / exposure:

28d

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

10.0 g/100 ml concentration

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Positive control:

not applicable

Examinations

Observations and examinations performed and frequency:

Mortality
A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsy

Clinical observations
The animals were examined for evident signs of toxicity or mortality twice a day (in the morning and in the late afternoon) from Mondays to Fridays and once a day (in the morning) on Saturdays, Sundays and public holidays. Abnormalities and changes were documented for each animal.

Detailed clinical observations
Detailed clinical observations (DCO) were performed in all animals prior to the administration period and thereafter at weekly intervals. The findings were ranked according to the degree of severity, if applicable. The animals were transferred to a standard arena (50 × 37.5 cm with sides of 25 cm height). The following parameters were examined:

1. Abnormal behavior when handled
2. Fur
3. Skin
4. Posture
5. Salivation
6. Respiration
7. Activity/ arousal level
8. Tremors
9. Convulsions
10. Abnormal movements
11. Gait abnormalities
12. Lacrimation
13. Palpebral closure
14. Exophthalmos
15. Assessment of the feces discharged during the examination (appearance/ consistency)
16. Assessment of the urine discharged during the examination
17. Pupil size

Food consumption
Food consumption was determined weekly and calculated as mean food consumption in grams per animal and day.

Water consumption
Drinking water consumption was observed by daily visual inspection of the water bottles for any overt changes in volume.

Body weight data
Body weight was determined before the start of the administration period in order to randomize the animals. During the administration period the body weight was determined on day 0 (start of the administration period) and thereafter at weekly intervals. The difference between the body weight on the respective day of weighing and the body weight on day 0 was calculated as body weight change.

CLINICAL PATHOLOGY
All animals were subjected to plasma concentration analysis for the parent compound. According to time schedule (see section 2.4.), EDTA blood samples (about 100 µL) were collected from non-fasted animals by puncturing the retro-bulbar venous plexus under isoflurane anesthesia. After plasma preparation, the samples were transferred to the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, 67056 Ludwigshafen, Germany, and frozen at about -80°C until analysis

Sacrifice and pathology:

Necropsy
The animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology.

Organ weights
The following weights were determined in all animals sacrificed on schedule:

1. Anesthetized animals
2. Kidneys
3. Liver
4. Spleen

Organ/tissue fixation
The following organs or tissues were fixed in 4% neutral-buffered formaldehyde solution:

5. All gross lesions
6. Kidneys
7. Liver
8. Spleen

From the liver, each one slice of the Lobus dexter medialis and the Lobus sinister lateralis was fixed in Carnoy’s solution and embedded in paraplast.

From all animals, fresh liver (Lobus caudatus, processus papillaris about 0.5 g]), left kidney and spleen (half of the organ) samples were taken during necropsy and immediately frozen in dried ice. The frozen tissue samples were transferred to the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, 67056 Ludwigshafen, Germany, and stored at about -80°C until analysis.

Histopathology
Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings according to the table below:
1. All gross lesions, Hematoxylin and Eosin (H&E) stain in all animals affected
2. Kidney, right, Hematoxylin and Eosin (H&E) stain, in all animals
3. Liver, Hematoxylin and Eosin (H&E) stain, in all animals
4. Spleen, Hematoxylin and Eosin (H&E) stain, in all animals

Other examinations:

BIOAVAILABILITY
The plasma samples taken on study day 21 as well as all fresh tissue samples (liver, kidney, spleen) taken on the day of necropsy were transferred to the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, 67056 Ludwigshafen, Germany, and frozen at about -80°C until analysis for potential analytical determination of the parent compound in these tissued.
The analyses were carried out as a separate study at Analytical Biochemical Laboratory (ABL), Assen, The Netherlands, under the responsibility of a Study Director of this test facility.

Statistics:

Body weight, body weight change: A comparison of each group with the control group was performed using DUNNETT's test (two-sided) for the hypothesis of equal means, * for p ≤ 0.05 ** for p ≤ 0.01

Weight parameters: A pairwise comparison of the test group with the control group was performed using the WILCOXON-test (two-sided). * for p ≤ 0.05 ** for p ≤ 0.01

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

Orange discolored feces were observed in all animals of test group 1 (1000 mg/kg bw/d) from study day 1 onwards until the end of the administration period. No other findings occurred in animals of test groups 1 (1000 mg/kg bw/d).

Mortality:

no mortality observed

Description (incidence):

No animal died prematurely in the present study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

With regard to the mean body weights as well as mean body weight change values, no significant changes to the control values were observed in all animals of test groups 1 (1000 mg/kg bw/d).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No test substance-related, adverse findings were observed. All recorded values were within the biological range typical for this strain of animals.

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

No test substance-related, adverse changes with regard to water consumption were observed.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not specified

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Absolute and relative organ weights
When compared to control group 0 (set to 100%), no significant absolute and relative weight changes were noted.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopically, an orange discoloration of contents was observed in the glandular stomach (6 of 10 animals), the jejunum (8 of 10 animals) and the cecum (all animals) of treated animals.

All other findings occurred either individually or were biologically equally distributed over control and treatment group. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

The kidneys of male animals of test group 1 (1000 mg/kg bw/d) showed an increased incidence and marginally increased severity of eosinophilic droplets in proximal tubular epithelial cells (see table below). Since the grading and severity of eosinophilic droplets in treated animals were within the range of historical control data, and since there were no correlating weight changes of the kidneys in these animals, the finding was regarded not to be treatment-related.

Histopathological findings: neoplastic:

not examined

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no effects up to the limit dose

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

Histopathology: eosinophilic droplets in proximal tubular epithelial cells in male animals

	male animals
Test group (mg/kg bw/d)	0	1000
No. of animals	5	5
Eosinophilic droplets in tubular epithelial cells	3	6
Grade 1	2	2
Grade 2	1	4

Bioavailability

The test item and internal standard were found to be stable during sample collection, sample handling, sample processing, and after repeated freezing and thawing. In addition, the method shows no analytical carry-over.

 

No residues of the applied test substance could be detected in rat plasma samples. However, small amounts could be detected in

·        liver tissue of 4 individual animals ranging from 0.94 to 11.1 ng/mg tissue,

·        spleen tissue of 3 individual animals ranging from 0.98 to 7.91 ng/mg tissue,

·        kidney tissue of 6 individual animals ranging from 1.23 to 15.3 ng/mg tissue.

 

Thus, it could be concluded that the test item is bioavailable.

Applicant's summary and conclusion

Conclusions:

The oral administration of the test item by gavage to male Wistar rats for 4 weeks did not result in any findings of toxicological concern even at a dose level of 1000 mg/kg bw/d. Therefore, under the conditions of the present study the no observed adverse effect level (NOAEL) was below 1000 mg/kg bw/d in male Wistar rats.
However, the analytical study results revealed bioavailability of the test item after oral administration at limit dose.