2-cyano-2-[2,3-dihydro-3-(tetrahydro-2,4,6-trioxo-5(2H)-pyrimidinylidene)-1H-isoindol-1-ylidene]-N-methylacetamide

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

acceptable restrictions: functional observation battery not included

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Functional observation battery missing

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Japanese Guidelines on Industrial Chemnicals (1986)

Deviations:

not specified

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2-cyano-2-[2,3-dihydro-3-(tetrahydro-2,4,6-trioxo-5(2H)-pyrimidinylidene)-1H-isoindol-1-ylidene]-N-methylacetamide

EC Number:

278-388-8

EC Name:

2-cyano-2-[2,3-dihydro-3-(tetrahydro-2,4,6-trioxo-5(2H)-pyrimidinylidene)-1H-isoindol-1-ylidene]-N-methylacetamide

Cas Number:

76199-85-4

Molecular formula:

C16H11N5O4

IUPAC Name:

2-cyano-N-methyl-2-[3-(2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene)-2,3-dihydro-1H-isoindol-1-ylidene]acetamide

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): C.I. Pigment Yellow 185
- Physical state: yellow powder
- Analytical purity: 99.3 %
- Lot/batch No.: 98-3107;
- Storage condition of test material: room temperature

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan, Inc.
- Age at study initiation: 5 weeks
- Weight at study initiation: males: 146-169 g, females 126-150 g
- Housing: Five rats of the same sex before grouping and two rats of the same sex after grouping were housed in each cage (polycarbonate cages (265W X 426D X 200H mm, Tokiwa Kagaku Kikai Co., Ltd.) with hard wood chip bedding (Beta chip, Charles River Japan, Inc.)).
- Diet: Pellet diet for experimental animals (Mi, Oriental Yeast Co., Ltd.), ad libitum
- Water: ordinary tap water ad libitum, renewed once a week, filtered through a 5 µm filter and disinfected with UV.
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 +/- 2
- Humidity (%): 55 +/- 15
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12h/12h

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.5 % CMC-Na aqueous solution mixed with 0.1% Tween 80

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The dosing solutions were prepared once a week, and were stored in a dark refrigerated place. They were used within 8 days. The dosing solutions were treated with ultrasonicator for about 10 minutes at the time of use and then were continuously stirred until administration.

VEHICLE
- Concentration in vehicle: 0.5%
- Amount of vehicle (if gavage): 10 mL/kg
- Lot/batch no. (if required): Tween 80: Tokyo Kasei Kogyo Co., Ltd., Lot No. 10546; CMC-Na: Iwai Chemicals Company, Lot No. 041210.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The homogeneity and stability of the test substance in the dosing solution for 8 days stored in a refrigerated place were confirmed at the dose of 0.4 and 100 mg/mL by HPLC method before the first administration. They were within ±10 for each indicated concentration.

Duration of treatment / exposure:

28 days

Frequency of treatment:

once a day in the morning

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

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

Doses / concentrations 5

Dose / conc.:

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

No. of animals per sex per dose:

6

Control animals:

yes

Details on study design:

- Dose selection rationale:
A dose-finding 7-day repeated dose toxicity study (the test substance was administered to three rats of both sexes at dose levels of 0, 100, 500, and 1000 mg/kg, respectively) was performed.
The study revealed yellowish stool which was considered to be related to defecation of the test substance in both sexes of all treatment groups, loose stool in males of the 500 mg/kg group, decreased MCV and MCH in females of the 1000 mg/kg group, an increased relative weight of the liver in males of the 100 and 1000 mg/kg groups.
There were no toxic changes considered to be related to the administration of the test substance in body weight or at necropsy. As there were no grave changes in the 1000 mg/kg group, the highest dose level was set at 1000 mg/kg; this is the upper limit as prescribed in the guideline.
- Post-exposure recovery period in satellite groups: Additionally 6 animals per sex in the control, 200, and 1000 mg/kg groups were subjected to a recovery period of 14 days.

Examinations

Observations and examinations performed and frequency:

OBSERVATIONS:
The day of the first administration was designated as Day 1, and the period from Day 1-7 was designated as Week 1. The period of Day 29 and after was designated as the recovery period.

CAGE SIDE OBSERVATIONS/Clinical Sign: Yes
- Time schedule: All rats were observed twice a day (before and after administration) during the treatment period and once a day on every other day in the morning.

BODY WEIGHT: Yes
- Time schedule for examinations: once a week

FOOD CONSUMPTION: once a week

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 29 and 43 at the scheduled sacrifices
- Anaesthetic used for blood collection: Yes, blood samples were obtained from the inferior vena cava of the rat without fasting, under intraperitoneal thiopental sodium anesthesia.
- Parameters examined and the corresponding method:
(1) Erythrocyte count (RBC): Sheath flow DC impedance detection method
(2) Hemoglobin concentration (Hb): SLS hemoglobin method
(3) Hematocrit (Ht): Cumulative pulse height detection method
(4) Mean corpuscular volume (MCV): Calculated from (1) and (3)
(5) Mean corpuscular hemoglobin (MCH): Calculated from (1) and (2)
(6) Mean corpuscular hemoglobin concentration (MCHC): Calculated from (2) and (3)
(7) Reticulocyte count: Flow cytometry by argon laser method
(8) Platelet count (PLT): Sheath flow DC impedance detection method
(9) Prothrombin time: Modified Quick one method
(10) Activated partial thromboplastin time (APTT): Activated cephaloplastin method
(11) Leukocyte count (WBC): RF/DC impedance detection method
(12) Leukocyte count-differential: Counting after Wright's stain

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day 29 and 43 at the scheduled sacrifices
- Animals fasted: No
- Parameters examined and the corresponding method:
(1) ASAT (GOT): UV-rate method (modified JSCC method)
(2) ALAT (GPT): UV-rate method (modified JSCC method)
(3) γGT: γ-glutamyl-p-nitroanilide substrate-method (modified SSCC method)
(4) Alkaline phosphatase (ALP) p-nitrophenyl-phosphate substrate-method (modified JSCC method)
(5) Total bilirubin: Enzymatic method (BOD method)
(6) Urea nitrogen: Enzymatic-UV method (Urease-GLDH method)
(7) Creatinine: Jaffé method
(8) Glucose: Enzymatic-UV method (GlcK-G6PDH method)
(9) Total cholesterol: Enzymatic method (CES-CO-POD method)
(10) Triglycerides: Enzymatic method (LPL-GK-G3PO-POD method)
(11) Total protein: Biuret method
(12) Albumin: BCG method
(13) A/G ratio: Calculated from (11) and (12)
(14) Calcium: OCPC method
(15) Inorganic phosphorus: Enzymatic method (PNP-XOD-POD method)
(16) Sodium (Na): Ion-selective electrode method
(17) Potassium (K): Ion-selective electrode method
(18) Chloride (Cl): Ion-selective eleotrode method

URINALYSIS: Yes
- Time schedule for collection of urine:
Fresh urine was collected from six males and females of each group on Day 23, and examined. As the result, urinalysis during the recovery period was not performed, since no changes suspected to be the effect of administration of the test substance were found.
- Parameters examined and the corresponding method:
(1) pH Paper tests (Multistix, Bayer Medical Ltd.)
(2) Protein Paper tests (Multistix, Bayer Medical Ltd.)
(3) Glucose Paper tests (Multistix, Bayer Medical Ltd.)
(4) Ketone bodies Paper tests (Multistix, Bayer Medical Ltd.)
(5) Bilirubin Paper tests (Multistix, Bayer Medical Ltd.)
(6) Occult blood Paper tests (Multistix, Bayer Medical Ltd.)
(7) Urobilinogen Paper tests (Multistix, Bayer Medical Ltd.)

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Organ weight of the brain, liver, kidneys, adrenals, thymus, spleen, testes, and ovaries

HISTOPATHOLOGY: Yes
- Histological examination was performed on following organs and tissues of the control and 1000 mg/kg groups at the end of the treatment period, along with all gross lesions found in any group: heart, liver, spieen, kidney, and adrenal.
Histological specimens were stained with H.E. stain in accordance with routine methods and examined. Additionally, all testes in any group at the end of the treatment period were also examined histologically since small of bilateral testes was observed with one male of the 1000 mg/kg group at necropsy at the end of the treatment period.

Statistics:

The numerical data were analyzed by multiple comparison tests. They were first analyzed by Bartlett's test. If the group variance was determined to be homogeneous, all groups were compared by a one-way analysis of variance. If Bartlett's test indicated heterogeneous variance, the Kruskal-Wallis test was employed, and a Dunnett's test or Dunnett type multiple comparison was used when there was a significant difference between the groups. The categorical data were analyzed by R x C Chi-square test, and when there was significance, Armitage's Chi-square test was used to compare the difference between the control group and each treatment group.
Statistical analysis was performed on items listed below. The analysis was not performed on cinical sign, necropsy, or histological findings.
Multiple comparison test: body weight, food consumption, hematology, blood chemistry, and organ weight.
Chi-square test: urinalysis.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No abnormalities were observed in any group.
Yellowish stool was observed in all cages of the 200 and 1000 mg/kg groups during the treatment period. However, this change was considered to be related to the color of the test substance, and was not a biological reaction.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The body weight of the treatment groups progressed in the same manner as that of the control group.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The food consumption of the treatment groups changed in the same manner as that of the control group.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No changes related to the administration of the test substance were observed at the end of the treatment or recovery periods.

Decreased MCV (Mean Corpuscular Volume) and MCH (Mean Corpuscular hemoglobin) were observed in females of the 200 mg/kg group and a decreased MCHC (Mean Corpuscular hemoglobin concentration) was observed in males of the 8 and 40 mg/kg groups at the end of the treatment period; however, these changes were considered unrelated to the administration of the test substance, since they were slight and not observed in the 1000 mg/kg group.

An increased ratio of monocyte in leukocyte count-differential was observed in females of the 200 and 1000 mg/kg groups at the end of the recovery period; however, this change was considered unrelated to the administration of the test substance since this change was within normal range and not observed at the end of the treatment period.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No changes related to the administration of the test substance were observed at the end of the treatment or recovery periods.
Decreased sodium was observed in females of the 200 mg/kg group at the end of the recovery period; however, this change was considered unrelated to the administration of the test substance, since this change was slight and not observed in the 1000 mg/kg group and at the end of the treatment period.

Endocrine findings:

not examined

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

No changes related to the administration of the test substance were observed during the treatment period.
Decreased ketone bodies were observed in females of the 8 mg/kg group; however, this change was considered unrelated to the administration of the test substance, since this change was not observed in the 40 mg/kg or above groups.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No changes were observed at the end of the treatment and recovery periods.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No changes related to the administration of the test substance were observed at the end of the treatment or recovery periods.
Although thymic remnant in neck in the thymus, nodule in the spleen, hemorrhage in the lungs, hepatodiaphragmatic nodule in the liver, cyst in the kidneys, and bilateral small in the testes were observed in the treatment groups at the end of the treatment or recovery periods, all of them were considered unrelated to the administration of the test substance, because of their circumstances of occurrence.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No changes related to the administration of the test substance were observed.
Although focal inflammatory cell infiltration in the heart, nodular lymphoid hyperplasia in the spleen, focal hemorrhage in the lungs, microgranuloma in the liver, basophilic tubule, cyst, hyaline droplet in the proximal tubular epithelium, and focal interstitial lymphocytic infiltration in the kidneys were observed in the treatment groups at the end of the treatment or recovery periods, all of them were considered unrelated to the administration of the test substance, because of their circumstances of occurrence.
They were spontaneous changes frequently seen in this strain of rat.
And additionally, testes of all animals in all groups at the end of the treatment period were examined histologically since small of bilateral testes was observed with one male of the 1000 mg/kg group at the necropsy of the end of the treatment period. As the result, bilateral diffuse atrophy of seminiferous tubule was observed in the animals with small testes; however, no histological changes were observed in the testes of the other animals. According to the result, the change observed in the testes was considered to be a spontaneous change.

Effect levels

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed up to and including the highest tested dose

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Under the conditions of this test the no-observed-effect-level (NOEL) of the test substance was determined to be 1000 mg/kg bw in both sexes after oral application for 28 days.

Executive summary:

The 28 day repeated dose study with rats was performed according to the OECD guideline 407 following GLP requirements. The study deviated from the guideline in that a functional observation battery was not included. The test substance (purity: 99.3 weight-%) was administered by gavage to male and female SD rats for 28 days at doses of 0, 8, 40, 200 and 1000 mg/kg bw to investigate its toxicity and, if necessary possible reversibility. A control group was dosed with a vehicle (0.5 % CMC-Na aqueous solution mixed with 0.1% Tween 80) only.

No changes attributable to the administration of the test substance were observed in any observations or measurements, namely, clinical sign, body weight, food consumption, hematology, blood chemistry, urinalysis, organ weight, necropsy or histology. Therefore, under the conditions of this test the NOEL of the test substance was determined to be 1000 mg/kg bw in both sexes.