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Diss Factsheets

Administrative data

acute toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study.

Data source

Reference Type:
study report

Materials and methods

Test guideline
according to guideline
OECD Guideline 402 (Acute Dermal Toxicity)
GLP compliance:
Test type:
standard acute method
Limit test:

Test material

Constituent 1
Chemical structure
Reference substance name:
Ammonium iron(3+) hexakis(cyano-C)ferrate(4-)
EC Number:
EC Name:
Ammonium iron(3+) hexakis(cyano-C)ferrate(4-)
Cas Number:
Molecular formula:
ammonium iron(3+) hexakis(cyano-C)ferrate(4-)
Test material form:
solid: particulate/powder
migrated information: powder
Details on test material:
- Name: Ammonium iron (3+) hexakis (cyano-C) ferrate (4-) (abbreviation: Milori Blue 905)
- Lot number: K73-4766
- Purity: 93.6% (Non-GLP)
- Description: Blue powder (Non-GLP)
- Storage conditions and location: Room temperature (actual range: 18.9º to 21.3ºC, permissible range: 1°C to 30°C) in an air-tight container (storage cabinet in test substance storage room A046)
- Expiration date: March 24, 2015

Test animals

Details on test animals or test system and environmental conditions:
Charles River Laboratories Japan, Inc. (Hino Breeding Center)

At receipt: 6-week-old males and 9-week-old females
At administration: 7-week-old males and 10-week-old females

Body weight range at receipt and administration
(1) At the receipt
actual range: 170.9 to 181.6 g, permissible range: 150 to 220 g (males)
actual range: 193.4 to 208.3 g, permissible range: 180 to 270 g (females)
(2) At the administration (permissible range: 200 to 300 g)
actual range: 229.1 to 239.8 g (males)
actual range: 220.8 to 228.7 g (females)

Quarantine and acclimatization
At animal receipt, species, strain, age, number, and sex were checked, and observation of clinical signs, external appearance and body weight measurement were performed. The period of quarantine and acclimatization was set at 5 days for males and females. During this period, all animals were examined for clinical signs once daily and body weights (on the day of receipt and 5 days after receipt) to confirm their health status. Since all animals were healthy, they were all used for the group assignment.

Identification of animals during the quarantine and acclimatization periods
At the receipt, animals were identified by marking a unique code on the tail with a red oily felt-tip pen and by a cage card bearing the study number, quarantine animal number, and sex attached to the front of each cage.

Group assignment
The animals were assigned to groups by the total randomization method on the day before dosing based on the body weights recorded on the same day. Animals weighing 200 to 300 g and within ± 20% of the mean body weight (calculated for each sex) were used for this study. The remaining animals will be excluded from this study, and then euthanized by exsanguination under sodium pentobarbital anesthesia.

Identification of animals after group assignment
Animals were identified by marking animal number on the tail with a black oily felt-tip pen, and by a cage card bearing the study number, animal number, test substance name, dose level, and sex attached to the front of each cage.

Environmental conditions
23.4º to 24.3ºC (acceptable range: 21º to 27ºC)

Relative humidity
46.8% to 60.9% (acceptable range: 35% to 75%)

10 to 20 air-changes per hour

Twelve hour light cycle: 7:00 light on and 19:00 light off

Breeding materials
Stainless-steel cages ( 226 × 346 × 198 mm)

Cage racks and feeder
Made by stainless-steel

Number of animals per cage
One animal per cage

Exchange frequency of housing equipment
Cage, cage rack, and feeder: once at group assignment
Sanitary tray: 2 or 3 times a week

Cleaning and sanitation
All housing equipments were sterilized by autoclaving after washing with water. The floor of the animal room was cleaned and wiped every day with a disinfectant-soaked mop. NaClO was used as disinfectant every day.

Type of diet: Pellet diet (CRF-1, Oriental Yeast Co., Ltd.), ad libitum
Type of water: Well water
Method of sanitization: Admixed with NaClO (about 2 ppm)
Method of water supply: ad libitum (automatic watering system)

Administration / exposure

Type of coverage:
Details on dermal exposure:
(1) The hair on the dorsal skin (areas of about 5  6 cm) were clipped with electric clippers (Speedik DSC-08, Blade: 0.1 mm, Shimizu E.C.) and an electric shaver (BS5884, Braun), taking care not to injure the skin, at about 24 hours before administration.
(2) The animals were fitted with a collar after the hair clipping.
(3) The test substance application area was prepared to fall within a range accounting for about 10% of the total body surface (about 4 x 5 cm).
(4) The required amount of the test substance was applied uniformly on a lint cloth (about 4 × 5 cm) lined with an impermeable sheet (BlendermTM, 3M Health Care). The lint cloth was moistened with an appropriate amount (0.5 mL) of water for injection (lot No. K9L78, Otsuka Pharmaceutical Factory, Inc.).
(5) The lint cloth loaded with the test substance was applied to the skin, and the application area was covered with an elastic adhesive bandage (SILKYTEX, ALCARE Co. Ltd.) for about 24 hours.
(6) After about 24-hour exposure, the test substance, lint cloth, and elastic adhesive bandage were removed.
(7) The application area was wiped with a gauze sheet immersed in lukewarm water and water was removed with dry tissue paper. The animals were fitted with the collar during the administration and recovery periods (Day 1 to 15).
Duration of exposure:
24 h
2000 mg/kg bw
No. of animals per sex per dose:
7 animals per sex per dose
Control animals:
Details on study design:
6.7 Observations and measurements
The following observations and measurements were performed. The day of the initial administration was designated as Day 1.
Clinical observation
All animals were observed for their mortality and clinical signs before the dosing and at 30 minutes, 1, 3, and 5 hours postdose on the day of the administration and once daily for 14 days thereafter.

Body weight
All animals were subjected to the body weight measurement just before the administration (Day 1) and on Days 3, 8, and 15. Moreover, the body weight gain between each measurement day was calculated.

Pathological examination (Necropsy)
At the end of the 14-day observation period, all animals were euthanized by exsanguination under intraperitoneal sodium pentobarbital (30 mg/kg) anesthesia and dissected immediately, and all organs and tissues were examined macroscopically.

Results and discussion

Effect levels
Dose descriptor:
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
No deaths occured suring the study period.
Clinical signs:
other: No abnormality was seen in either sex .
Gross pathology:
No abnormality was seen in either sex.

Applicant's summary and conclusion

Interpretation of results:
not classified
Migrated information Criteria used for interpretation of results: EU
CLP: not classified
DSD: not classified