Registration Dossier

Administrative data

short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
GLP compliance:
Limit test:

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Test material form:
solid: particulate/powder
Details on test material:
Pigment orange 16 (hereinafter abbreviated as PO16) was used as a test substance. It is also called “Disazo orange” and its name in English is C. I. Pigment Orange 16 having CAS No. 6505-28-8, molecular weight 620.66 and molecular formula C34H32N6O6.
Purity: 99 wt% or higher
The provided test substance contained a water-soluble compound (NaCl) 0.18% as impurity

For this specific material, a particle size characterization is not available.
Specific details on test material used for the study:
- Purity: 99 wt% or higher (impurity: NaCl 0.18 %)
- Name of test material (as cited in study report): Pigment orange 16 (C.I. Pigment Orange 16), Disazo orange

Test animals

other: Sprague-Dawley [Crj:CD (SD) IGS, SPF]
Details on test animals or test system and environmental conditions:
- Source: Tsukuba Breeding Center, Charles River Laboratories Japan, Inc.)
- Age at study initiation: Four week
- Weight at study initiation: Males: 160 g- 187.5 g (mean: 176.6 g); Females: 135.5 - 164.4 g (mean: 149.7 g)
- Housing: Through the whole breeding period, the animals were housed and bred individually in metal cages with wire-mesh floor (220 w x 270 d x 190 h mm)
- Diet: Solid feed (CE-2, Clea Japan, Inc.), ad libitum
- Water: Tap water, ad libitum
- Acclimation period: 9 days

- Temperature: 24 +/- 1 °C
- Humidity: 50 - 65 %
- Air changes: 15 times of ventilation/hour
- Photoperiod: 12-hour light cycle

Administration / exposure

Route of administration:
oral: gavage
corn oil
Details on oral exposure:
Dosing volume: 10 mL/kg
Analytical verification of doses or concentrations:
Duration of treatment / exposure:
28 days
Frequency of treatment:
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Control group and 100 mg/kg dosing group: 10 animal per sex
Other groups: 5 animals per sex
Control animals:
yes, concurrent vehicle
Details on study design:
recovery period of 14 days


Observations and examinations performed and frequency:
- Time schedule for examinations: The body weight was measured on day 1 immediately before dosing and on day 5 of week 1 of administration period, and at a frequency of twice a week from week 2 of administration period onward during the administration period and the recovery test period. In addition, the body weight was also measured on the day of end of administration period, on the day of end of recovery test period and on the necropsy day

- Time schedule for collection of urine: Urine was collected 4 hours after dosing or fresh urine
- Parameters examined: Color tone, turbidity, pH, occult blood, protein, sugar, and ketone body, Urobilinogen and bilirubin

- Time schedule for collection of blood: End of the test
- Animals fasted: Yes, 18 – 24 hours before blood collection (before autopsy).
- How many animals: All the animals which were autopsied at the end of administration period or at the end of recovery test period
- Parameters examined: Red blood cell count (RBC), White blood cell count (WBC), Hemoglobin content (Hb), Mean corpuscular volume (MCV), Platelet count, Hematocrit (Ht), Mean corpuscular hemoglobin(MCH), Mean corpuscular hemoglobin concentration (MCHC), Differential white blood count, Reticulocyte count, Prothrombin time (PT), Activated partial thromboplastin time (APTT).

Sacrifice and pathology:

Organ weights determined:
brain, heart, liver, kidneys, thymus, spleen, adrenals, testes, epididymis, ovaries

Organ tissues fixed/preserved were
brain, hypophysis, spinal cord, eye ball, thyroid gland, parathyroid gland, heart*, trachea, bronchus, lungs, liver*, kidneys*, thymus, spleen*, adrenal*, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, testis*, epididymis*, prostate, seminal vesicle, ovary*, uterus, vagina, mammary gland, urinary bladder, mandibular lymph node, mesenteric lymph node, skeletal muscle (leg region), sciatic nerve, femur bone marrow, pancreas, submaxillary gland, sublingual gland, tongue, esophagus, aorta, Harderian gland, skin, and gross lesion.
For body weight, food consumption, hematological and blood biochemical values and the respective measured values of organ weights, the average value and standard deviation were obtained for each group.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
no effects observed
Haematological findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Details on results:
FOOD CONSUMPTION: For females in the 100 mg/kg dosing group, food consumptions at week 4 of administration significantly decreased compared with the control group. In this regard, it is assumed that one animal of the said group showed decreased food consumption due to the false dosing in the same period.

HAEMATOLOGY: For the result of hematological examination at the end of administration period, no significant differences in every examination item were found between the control group and the test substance dosing groups. On the other hand, females in the test substance dosing groups showed an increase in red blood cell count at the end of the recovery test period.

For the examination at the end of administration period, a significantly decreased value of A/G ratio was found in males of the 300 mg/kg dosing group, and a significantly decreased value of potassium concentration was found in females of the 1000 mg/kg dosing group. For the examination at the end of the recovery test period, a significantly decreased value of total cholesterol was found in females of the test substance dosing groups, but there were no significant differences in other test items between the control group and the test substance dosing groups.

The relative weights of ovaries in the 100 mg/kg dosing group autopsied at the end of administration period showed a significant increase. Except for said case, no significant differences were found in other items including the organ weights of the cases autopsied at the end of administration period.

HISTOPATHOLOGY: For the control group and the 1000 mg/kg dosing group autopsied at the end of administration period, the findings were seen in the lever, kidneys and spleen of both males and females and in the heart of males. However, any differences in the occurrence frequency and extent of the findings were not found among the groups. Furthermore, in 1 female of the control group in which alopecia was macroscopically seen was also found crust as a corresponding change and accessory spleen as well. In 1 female of the 300 mg/kg dosing group were no changes seen except for slight extramedullary hematopoiesis and deposit of brown pigment.

Effect levels

Dose descriptor:
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: No adverse effects were observed at the limit dose.

Target system / organ toxicity

Critical effects observed:

Applicant's summary and conclusion

No adverse effects were observed upon subacute gavage dosing of rats at doses of up to 1000 mg/kg bw.