Coupling reaction products of 3,3’-dichlorobiphenyl-4,4’-bis(diazonium) dichloride with N-(2,4-dialkylphenyl)-3-oxobutanamide and potassium 4-[(3-oxobutanoyl)amino]substituted benzene and their basic aluminium and earth alkali metal salts

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:

7 June 2007 - 9 January 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD guidelines and according to GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

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:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: "28-day Repeated Dose Toxicity Study in Mammalian Species" prescribed in "Concerning Testing Methods Relating to the New Chemical Substances" (Notification No. 1121002, MHLW, No. 2 (November 13, 2003), METI & No. 031121002, MOE (November 21, 2003))

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): S-500
- Substance type: yellow powder
- Physical state: solid
- Lot/batch No.: MF1456-2345
- Stability under test conditions: stable (confirmed by IR)
- Storage condition of test material: at room temperature

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan Inc. (Hino Breeding Center; 735, Shimokomatsuki, Hino-cho, Gamo-gun, Shiga 529-1633, Japan)
- Age at study initiation: 5 weeks old
- Weight at study initiation: 136.1-169.2 g (males) and 118.1-144.3 (females)
- Fasting period before study: not applicable
- Housing: stainless steel hanger cages with wire-mesh floor.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 10 days, including 6 days quarantine

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.1-23.9
- Humidity (%): 48.5-62.0
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: CMC-Na (carboxymethyl cellulose sodium salt)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was suspended with 0.5 w/v% CMC-Na solution in a mortar to 10.0 w/v% formulation (i.e. 100 mg/mL). The lower concentrations of 2.5 and 0.5 w/v% suspensions were diluted from 10.0 w/v% suspension. These were prepared once per 7 or 8 days and stored at the dark and cold place.

VEHICLE
- Justification for use and choice of vehicle (if other than water): when the preparation method of the dosing formulation method was investigated, the test substance was not dissolved in purified water but suspended with 0.5 w/v% CMC-Na solution (in purified water).
- Concentration in vehicle: 0.5% (w/v%) CMC-Na solution in purified water is used to prepare dose formulations: 0.5, 2.5 and 10 w/v% test substance, i.e. 5, 25 and 100 mg/ml.
- Amount of vehicle (if gavage): 10 mL/kg
- Lot/batch no. (if required): purified water: 070326A, CMC-Na: LT

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

At the start (first preparation) and the last (last preparation) of dosing, middle layers of 10.0, 2.5 and 0.5 w/v% formulations were taken (n=1) just after preparation, and quantitatively analyzed once for each by HPLC (n=1) after sample pretreatment.
The test substance in 10.0 and 0.5 w/v% formulations was stable for 8 days after preparation at cold and dark place and showed good homogeneity.
The concentration of test substance in 10.0, 2.5 and 0.5 w/v% dose formulations for subject study at the first and final preparation was acceptable level (ratio actual/nominal concentration: 99.6-102%).

Duration of treatment / exposure:

28 days exposure, followed by 14 days recovery period.

Frequency of treatment:

daily in the morning.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
50 mg/kg bw/d
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
250 mg/kg bw/d
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
1000 mg/kg bw/d
Basis:
actual ingested

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: A 14-day repeated oral dose toxicity study was performed at 4 doses: 25, 250, 500 and 1000 mg/kg bw/d. As a result, no adverse effects attributable to the test substance were noted in males and females of all dose groups.
- Rationale for animal assignment (if not random): ensuring homogeneity of mean body weights using body weight-stratified randomization
- Rationale for selecting satellite groups: not indicated
- Post-exposure recovery period in satellite groups: control and 1000 mg/kg bw/d dose groups for 14 days.
- Section schedule rationale (if not random): not indicated

Positive control:

not applicable

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily during dosing period: before, during and after dosing and in the afternoon. Daily during the recovery period: in the morning and in the afternoon.
- Cage side observations: general condition: mortality, stool, fur, scab formation

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before dosing in all animals. Thereafter, on a blind test basis once weekly during the dosing and recovery periods.

BODY WEIGHT: Yes
- Time schedule for examinations: on day before dosing, and on days 1, 3, 8, 12, 17, 21, 26 and 28 during dosing period, and on days 1, 5, 10 and 14 during recovery period. And immediately before necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No, as g food/rat/day.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of dosing period or recovery period
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes, overnight for 16-20 h
- How many animals: all
- Parameters examined: red blood cell count, white blood cell count, hemoglobin conc., hematocrit value, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, reticulocyte count, prothrombin time, activated partial thromboplastin time, differentiation of leucocytes (neutrophils, eosinophils, basophils, lymphocytes, monocytes, large unstained cells)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of dosing period or recovery period
- Animals fasted: Yes, overnight for 16-20 h
- How many animals: all
- Parameters examined: asparate aminotransferase, alanine aminotransferase, alkaline phosphatase, cholinesterase, gamma-glutamyl transpeptidase, total cholesterol, triglyceride, glucose, total protein, albumin, A/G ratio, blood urea nitrogen, creatinine, total bilirubin, calcium, inorganic phosphorus, sodium, potassium, chloride

URINALYSIS: Yes
- Time schedule for collection of urine: end of dosing period or recovery period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, but drinking water ad libitum
- Parameters examined: urine volume, colour, turbidity, urine specific gravity, pH, protein, glucose, occult blood, urinary sediments.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: week 4 of dosing
- Dose groups that were examined: all animals
- Battery of functions tested: reflex test / locomotor activity / grip strength

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Body surface, all orifices, subcutaneous tissue, cranial, thoracic, abdominal and pelvic cavities, and contents.

ORGAN WEIGHTS: Yes
Liver, kidneys, heart, testes, epididymides, ovaries, brain , spleen, thymus and adrenals.

HISTOPATHOLOGY: Yes
Trachea, lungs, forestomach, glandular stomach, duodenum, jejunum, ileum, cecum, colon, rectum, liver, heart, kidneys, urinary bladder, testes, pididymides, prostate, seminal vesicles, ovaries, uterus, vagina, cerebrum, cerebellum, pons, spinal cord, sciatic nerve, bone marrow, axillar and mesenteric lymph nodes, spleen, thymus, pituitary gland, thyroids, parathyroids, adrenals, eye balls.

Other examinations:

Not applicable.

Statistics:

Bartlett's test for homogeneity of variance.
Dunnett's test for the difference between vehicle and exposed group (if variances were homogenous at 5% significance level)
Nonparametric Dunnett's test for the difference between vehicle and exposed group (if variances were not homogenous)

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:

not examined

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:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
Yellow stool was continuously observed in both sexes of 250 and 1000 mg/kg groups. Yellow stool was not observed two days after termination of dosing in both sexes of 1000 mg/kg recovery group.

HAEMATOLOGY
Hemoglobin concentration was decreased statistically in males of 1000 mg/kg group and ratio of large unstained cells was increased statistically in females of the 1000 mg/kg group at termination of the dosing period. These changes (after dosing period) were however within the historical control data.

CLINICAL CHEMISTRY
Sodium was increased statistically in males of the 1000 mg/kg group after dosing period. This change was however within the historical control data.

ORGAN WEIGHTS
Increases in relative liver and spleen weights in males of the 1000 mg/kg group after recovery period were noted. There were however no histopathological changes and these changes were within the historical control data.

GROSS PATHOLOGY
Incidental changes occurred, in small numbers, and often in normal animals.
After dosing period: cyst pituitary gland (1 male, 1000 mg/kg), diverticulum of jejunum (1 female, 1000 mg/kg), smaller left lobe of thyroid (1 female, 250 mg/kg), small testis (1 male, control).
After recovery period: recessed region of kidney (1 female, 1000 mg/kg), pelvic dilatation of kidney (1 male, control), diverticulum of jejunum (1 female, control)

HISTOPATHOLOGY: NON-NEOPLASTIC
Incidental changes occurred, in small numbers, and often in normal animals.
After dosing period:
- cyst formation in pars nervosa of pituitary gland (1 male, 1000 mg/kg) (same animal as mentioned under GROSS PATHOLOGY)
- diverticulum jejunum (1 female, 1000 mg/kg) (same animal as mentioned under GROSS PATHOLOGY)
- hypolasia of left lobe in thyroid (1 female, 250 mg/kg) (same animal as mentioned under GROSS PATHOLOGY)
- subcapsular solitary cyst in medulla of kidney (1 male, 1000 mg/kg)
- solitary cyst in medulla of kidney (1 male, 1000 mg/kg)
- increased follicular cysts of ovary and mineralization in cortico-medullary junction of kidney (1 female, 1000 mg/kg)
- mineralization in cortico-medullary junction of kidney (1 female, 1000 mg/kg and 1 female, control)
- degeneration of spermatocytes or inhibited spermiation and deep retention of spermatids of the testis and decreased spermatozoa in lumen or germ cell debris in lumen of the epididymis (1 male, control) (same animal as mentioned under GROSS PATHOLOGY)
- karyomegaly of tubular epithelium in corticomedullary junction of kidney (1 male, control)
- ectopic ossification of adrenal (1 male, control)
- ultimobranchial rest of thyroid (1 female, control)
- ectopic striated muscle of lung (1 female, control)

After recovery period:
- focal atrophy of nephrons with lymphocyte infiltration and fibrosis in kidney (1 female, 1000 mg/kg) (same animal as mentioned under GROSS PATHOLOGY)
- microgranuloma of liver (1 male, 1000 mg/kg)
- pelvic dilation in kidney (1 male, control) (same animal as mentioned under GROSS PATHOLOGY)
- focal diverticulum in jejunum (1 female, control) (same animal as mentioned under GROSS PATHOLOGY)

Effect levels

Dose descriptor:

NOEL

Effect level:

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

Sex:

male/female

Basis for effect level:

other: No abnormalities caused by the test substance were noted in the test. Yellow stool (at 250 and 1000 mg/kg bw dose groups) were an effect from the yellow colour of the test substance.

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

S-500 was orally administered by gavage to Crl:CD(SD) rats at doses of 50, 250 and 1000 mg/kg bw/day to perform a 28-day toxicity study followed by a 14-day recovery study.
Yellow stool was noted in males and females of the groups of 250 mg/kg and more. This change was not considered to be of toxicological significance since the effect is due to the colour of the test substance.
No abnormalities attributable to the test substance were observed in detailed clinical observations, sensorimotor functions, body weights, food intakes, hematological examinations, blood chemical examinations, urinalyses or pathological examinations. No abnormalities were also noted in the recovery test.
From the above mentioned results, the NOEL of S-500 in rats under the present study conditions was estimated to be 1000 mg/kg/day.