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Repeated dose toxicity: oral

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sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well-documented, acceptable for assessment.
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference Type:

Materials and methods

Test guideline
no guideline followed
Principles of method if other than guideline:
Feeding diets containing 35, 350, 3500 and 20000 µg Fe/g were fed to 11, 10, 10 and 18 male Sprague-Dawley rats, respectively, for 12 weeks. The effects on the histopathology of liver, pancreas, spleen and heart were examined.
GLP compliance:
not specified
Referance to GLP is not customary in publications.
Limit test:

Test material

Constituent 1
Reference substance name:
carbonyl iron
carbonyl iron
Details on test material:
Name of test material (as cited in study report): carbonyl iron (ISP Technologies, Inc., Wayne, NJ)
- Physical state: solid (spherical particles, with average particle size between 7-9 µm)
- Analytical purity: 99%
- Other: carbonyl iron is an extremely pure form of iron. It is produced after treatment of Fe with CO, that first results into iron pentacarbolyl. Thereafter, the latter is decomposed, yielding CO and pure iron powder.

Test animals

Details on test animals or test system and environmental conditions:
- Source: Bruce Spruce Farms, Inc., Altamont, NY
- Age at study initiation: weanling

- Housing: stainless stell cages, individually housed
- Diet: ad libitum
- Water: ad libitum (destilled, deionized)

- Temperature (°C): controlled
- Photoperiod (hrs dark / hrs light):controlled

Administration / exposure

Route of administration:
oral: feed
unchanged (no vehicle)
Details on oral exposure:
- Mixing appropriate amounts with (Type of food): AIN-76A diet (Dyets, Inc., Bethlehem, PA) which contained 200 g/kg casein, 3 g/kg DL-methionine, 150 g/kg cornstarch, 500 g/kg glucose, 50 g/kg fiber Celufil, 35 g/kg AIN-76 mineral mix, and 10 g/kg AIN-76 vitamin mix with 50 mg menadione/kg and 2 g choline bitartrate/kg.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
Duration of treatment / exposure:
12 weeks
Frequency of treatment:
Doses / concentrations
Doses / Concentrations:
35 (control), 350, 3500 and 20000 µg Fe/g diet.
nominal in diet
No. of animals per sex per dose:
11, 10, 10, 18 respectively (doses mentioned above)
Control animals:
yes, plain diet
Details on study design:
The animals were fed with the carbonyl Fe diet for 12 weeks. At end of the 12-week the animals were fasted for approximately 15 h, anesthetized by intramuscular injection of 5 mg of ketamine hydrocloride/ 100 g bw and decapitated.
Positive control:


Observations and examinations performed and frequency:
Determined in livers and hearts by bathophenanthroline reaction.

Liver lipid conjugated dienes were determined by a modified method of Watson et al., (1984).

Formalin-fixed, paraffin-embedded pancreas sections were also processed for in situ end-labelling of 3'-OH DNA strand breaks localized in apoptotic bodies using the ApopTag Detection Kit.

Sacrifice and pathology:
A complete necropsy was performed on each animal and tissues were fixed in 10 % neutral buffered formalin.
One set of tissues were stained with hematoxylin and eosin and another set of tisssues with Perl's Prussian blue for Fe. The tissues examined were the following: liver, heart, spleen and pancreas.
1-way ANOVA, Scheffe multiple-comparison method, Pearson's product for determination of correlation coefficients.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No mortality was observed in the control and lowest dose group. For the two highest dose groups mortality rates were 20 % and 28 %, respectively.
mortality observed, treatment-related
Description (incidence):
No mortality was observed in the control and lowest dose group. For the two highest dose groups mortality rates were 20 % and 28 %, respectively.
Body weight and weight changes:
not specified
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
effects observed, treatment-related
Details on results:
-Dose-related increase in liver in the rats fed with diets containing 3500 and 20000 µg Fe/g diet.
-Dose-related increase in the hearts of all groups.

The measurements revealed significant increases in the livers of animals dosed with the two upper doses.

Nuclei and nuclear fragments, characteristic of apoptosis, were observed interspersed among the pancreatic tissues.

LIVER: hepatocellular hypertrophy in animals receiving the highest dose. Dose-related accumulation of cytoplasmic Fe-positive material within hepatocytes or intrasinusoidal cells.
HEART: the incidence of severity of cardiomyopathy increased with higher dietary concentrations of Fe (marked increase at the highest dose) and was characterized by a spectrum of lesions.
SPLEEN: hemosiderin was present in the sinusoidal macrophages of all animals, treated and untreated, but it increased with increasing dose of Fe (Prussian blue reaction). Splenic atrophy at the two upper doses (white pulp), characterized by a loss of cells (Table II, attachment).
PANCREAS: presence of apoptotic cells in animals fed with 350 µg Fe/g; pancreatic atrophy observed in animals fed with 3500 and 20000 µg Fe/g diets, associated with extensive loss of both endocrine and exocrine tissue.Difuse replacement of pancreatic tissue by adipose tissue with infiltration of the fibrovascular stroma by polymorphonuclear cells, macrophages and a small number of lymphocytes.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1: Dose calculated by the submitter based on the species-specific allometric equation for food consumption of US EPA (1988).

Average weight (g)

Food consumption (g/day)

Fe in diet


mg Fe/day

Dose (mg/kg bw)

44 (weanling)


350, 3500, 20000 µg Fe/g feed

3.15, 31.5, 180

71, 710, 4072

300 (after 12 weeks)


350, 3500, 20000 µg Fe/g feed

7,70, 400 mg Fe

26, 260, 1498

Applicant's summary and conclusion

The study provides information on the mechanism of toxic action of iron, in cases of overload.
Executive summary:

In a subchronic toxicity study feeding diets containing 35, 350, 3500 and 20000 µg Fe/g were fed to 11, 10, 10 and 18 male Sprague-Dawley rats, respectively. The treatment lasted 12 weeks. The findings revealed a direct correlation between increased liver nonheme Fe and lipid peroxidation measured by the lipid-conjugated diene assay. Histopathological examinations revealed hepatocellular hemosiderosis, myocardial degeneration and necrosis, splenic lymphoid atrophy and Fe deposition in the sinuisoidal macrophages, and pancreatic atrophy. The toxic effects include cellular apoptosis or necrosis in heart, spleen and pancreas and when coupled with the findings on lipid peroxidation, suggests that oxidative stress is involved in pathogenesis of lesions.