Iron

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well-documented, acceptable for assessment.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1996

Materials and methods

Test guideline

Qualifier:

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

Remarks:

Referance to GLP is not customary in publications.

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

carbonyl iron

IUPAC Name:

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

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- 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)


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

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- 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:

no

Duration of treatment / exposure:

12 weeks

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
35 (control), 350, 3500 and 20000 µg Fe/g diet.
Basis:
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:

no

Examinations

Observations and examinations performed and frequency:

NONHEME IRON
Determined in livers and hearts by bathophenanthroline reaction.

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

IMMUNOHISTOCHEMICAL STAINING
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:

HISTOPATHOLOGY
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.

Statistics:

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:

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:

NON-HEME IRON
-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.

LIPID PEROXIDATION (liver)
The measurements revealed significant increases in the livers of animals dosed with the two upper doses.

IMMUNOHISTOCHEMICAL STAINING (pancreas)
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)	9	350, 3500, 20000 µg Fe/g feed	3.15, 31.5, 180	71, 710, 4072
300 (after 12 weeks)	20	350, 3500, 20000 µg Fe/g feed	7,70, 400 mg Fe	26, 260, 1498

Applicant's summary and conclusion

Conclusions:

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.