Reaction mass of Trisodium tris(D-gluconato(2-)-κO1,κO2)ferrate(3-) and Sodium chloride

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

no data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Well conducted non-GLP study. The study was performed according to methods similar to OECD407/408. Only male rats were used. 10 male animals were sacrificided at the beginning of the experiment in order to establish baseline levels of the analytical parameters to be measured. No other data were collected for these animals. No further control group was included.

Data source

Reference

Reference Type:

publication

Title:

Disposition, accumulation and toxicity of iron fed as iron (II) sulfate or as sodium iron EDTA in rats

Author:

Appel, M. J. , Kuper, C.F., Woutersen, R.A.

Year:

2001

Bibliographic source:

Food and Chemical Toxicology 39 261-269

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Principles of method if other than guideline:

- Only male rats were used.
- 31 or 61 days of exposure
- 10 male animals were sacrificed at the beginning of the experiment in order to establish baseline levels of the analytical parameters to be measured. No other data were collected for these animals. No further control group was included.

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

sodium iron EDTA

IUPAC Name:

sodium iron EDTA

Constituent 2

Reference substance name:

Sodium feredetate

EC Number:

239-802-2

EC Name:

Sodium feredetate

Cas Number:

15708-41-5

Molecular formula:

C10H12N2O8FeNa

IUPAC Name:

Sodium; 2-[2-(bis(carboxylatomethyl)amino)ethyl-(carboxylatomethyl)amino]acetate; iron(+3) cation

Details on test material:

Sodium iron EDTA (FeEDTA; approx. 13% iron) and iron (II) sulfate (FeSO4; approx. 20% iron) were obtained from Dr Paul Lohmann (Emmerthal, Germany).

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany (SPF colony)
- Age at study initiation: 5-6 weeks
- Weight at study initiation: no data
- Fasting period before study: not applicable
- Housing: individually under conventional laboratory conditions in one room, in suspended stainless-steel cages fitted with wire-mesh floor and front.
- Diet (e.g. ad libitum): Prior to the start of the experiment, a batch of modified AIN-93G (Reeves et al., 1993) diet was prepared freshly at
TNO and stored at or below 18C until use. Two batches of a commercially available low-iron AIN-93M diet ( < 5 mg iron/kg diet; lot nos 8315-6 and 8345-4) were obtained from Dyets Inc. (Bethlehem, PA, USA).
- Water (e.g. ad libitum): no data
- Acclimation period: ± 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): no data
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): no data

IN-LIFE DATES: From: To: no data

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): no data
- Mixing appropriate amounts with (Type of food): FeEDTA or FeSO4 were incorporated in the low-iron AIN-93M diet at constant concentrations by mixing in mechanical mixers at TNO.
- Storage temperature of food: Dietary iron was found to be stable after storage for 7 days at room temperature in an open container and after 7
days or 5 weeks in a refrigerator in a closed container and after 5 weeks in a freezer in a closed container.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The content, stability and homogeneous distribution of iron in the test diets was analyzed by means of atomic absorption spectrometry (AAS). The content of each of the six diets was measured three times during the study.

The iron content was close to the intended values in 17 of the 18 diet samples measured during the experiment. In the one case that the criteria for 'close to intended' were not met (10% of the intended concentration), a concentration of 61 mg Fe per kg diet was found, which was 13% lower than the intended 70 mg Fe/kg.

Duration of treatment / exposure:

31 or 61 days

Frequency of treatment:

daily via the diet

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
FeSO4 or FeEDTA were mixed with these diets to obtain diets containing 35, 70 or 140 mg Fe/kg diet. ± FeEDTA = FENaEDTA.3H2O 264, 528, 1056 mg/kg diet (mol weight Fe 55.847, mol weight EDTA-FeNa 3H2O 421.096)
Basis:
nominal in diet

Doses / concentrations 2

Remarks:

Doses / Concentrations:
The mean intake of iron was 2.81, 5.67, 11.19 mg/kg bw/day for the -low, -mid and high-dose, respectively. ± FeEDTA = FENaEDTA.3H2O 21, 43, 84 mg/kg bw/day (mol weight Fe 55.847, mol weight EDTA-FeNa 3H2O 421.096)
Basis:
actual ingested

No. of animals per sex per dose:

40 male rats, 20 were sacrificed after 31 days and the remaining were sacrificed after 61 days

Control animals:

other: - 10 male animals were sacrificided at the beginning of the experiment in order to establish baseline levels of the analytical parameters to be measured. No other data were collected for these animals. No further control group was included.

Details on study design:

- Dose selection rationale: no data
- Rationale for animal assignment (if not random): randomized by computer
- Rationale for selecting satellite groups: not applicable
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rationale (if not random): no data

Positive control:

Not applicable

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: No

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Each animal was observed daily and all abnormalities, signs of ill-health or reactions to treatment were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each animal was recorded once during the acclimatization period, at initiation of treatment, and once weekly thereafter. In addition, the animals were weighed on the day of scheduled autopsy in order to determine their correct organ to body weight ratios.

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: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No
Food consumption was measured per animal twice weekly, over periods of 3 or 4 days, by weighing the feeders. The results were expressed in g per animal per day.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes
The efficiency of food utilization was calculated and expressed in g weight gain per g food consumed.

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At autopsy
- Anaesthetic used for blood collection: Yes; No data on identity
- Animals fasted: Yes
- How many animals: All
- Parameters examined: hemoglobin, packed cell volume, red blood cell count, total white blood cell count, differential white blood cell count, prothrombin time and thrombocyte count. The following parameters were calculated: mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At autopsy
- Animals fasted: Yes
- How many animals: all
- Parameters examined: alkaline phosphatase activity (ALP), aspartate aminotransferase activity (ASAT), alanine arninotransferase activity (ALAT), gamma glutamyl transferase activity (GGT), total protein, albumin, ratio albumin to globulin, urea, creatinine, glucose, bilirubin (total), cholesterol (total), triglycerides, phospholipids, calcium (Ca), sodium (Na), potassium (K), chloride (Cl) and inorganic phosphate

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:
- Intake of iron from FeEDTA or FeSO4
- Determination of iron and total iron binding capacity (TIBC) in blood plasma

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
After completion of the treatment periods, the animals were killed on several successive working days, in such a sequence that the average time of killing was approximately the same for each group. The animals were killed by exsanguination from the abdominal aorta under ether anesthesia
and then examined macroscopically for pathological changes.
The adrenals, brain, caecum, colon, heart, kidneys, liver, oesophagus, rectum, small intestines (duodenum, ileum, jejunum), spleen, stomach, testes and thymus, were excised, examined for gross lesions and preserved in a neutral aqueous phosphate buffered 4% solution of formaldehyde (10% solution of formalin). The organs that are italicised were weighed prior to preservation.
HISTOPATHOLOGY: Yes
Samples of liver, spleen and all gross lesions were embedded in paraffin wax, sectioned at 5 µm and stained with haematoxylin and eosin and with Perl's Prussian blue for iron. The stained sections, except the Perl's Prussian blue stained sections of gross lesions, were examined by light microscopy by an experienced pathologist.
Fresh samples of the liver, spleen and kidneys of all animals were analyzed for non-heme iron content by the bathophenanthroline reaction and the results were expressed as µg Fe/g tissue (wet weight). The analysis was performed according to the method described by Whittaker et al. (1997). Before analysis of study samples, the method was validated for the different tissues under investigation.

Other examinations:

None

Statistics:

Body weights were analyses by one-way analysis of covariance (covariate: body weight on day 0) followed by Dunnett's multiple comparison tests, food consumption and food conversion efficiency were analyzed by one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison tests, red blood cell and coagulation variables, total white blood cell counts, absolute differential white blood cell counts, clinical chemistry values, plasma iron, TIBC and organ weights were analyzed by one-way ANOVA followed by Dunnett's multiple comparison tests, relative differential white blood cell counts were analyzed by Kruskal-Wallis nonparametric ANOVA followed by Mann-Whitney U-tests, histopathological changes were analyzed by Fisher's exact probability test and non-heme iron analyses in tissue were analyzed by two-way ANOVA followed by Student's t-tests. All tests were two-sided. Probability values of P < 0.05 were considered significant.

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:

no effects observed

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

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:

Hematology
After 31 days of feeding, the mean corpuscular hemoglobin was statistically significantly higher in groups receiving the high dose of FeSO4 and the mid and high dose of FeEDTA in comparison to the group receiving the low dose of FeSO4. The mean corpuscular hemoglobin concentration was statistically significantly higher in the group receiving the high dose of FeEDTA in comparison to the group receiving the low dose of FeS04. No other statistically significant differences in red blood cell or coagulation variables were observed after 31 days of feeding.
After 61 days of feeding, no statistically significant differences in red blood cell or coagulation variables were observed. After 31 days of feeding, the absolute number of eosinophils was statistically significantly higher in the high-dose FeEDTA group in comparison to the lowand mid-dose FeEDTA groups and the mid-dose FeSO4 group. The percentage of eosinophils was statistic all significantly higher in the high-dose FeEDTA group in comparison to the low-dose FeEDTA and the mid-dose FeSO4 groups. No other statistically significant differences in white blood cell counts were observed.
After 61 days of feeding, no statistically significant differences in white blood cell counts were observed.

Clinical chemistry values in plasma
The following statistically significant differences were observed:

At day 32
Alkaline phosphatase activity was decreased in the mid-dose FeEDTA group in comparison to the low-, mid- and high-dose FeSO4-groups. At day 62, this difference was no longer present. The concentration of total bilirubin was higher in the mid-dose FeSO4 group in comparison to the low-dose FeEDTA and FeSO4 - groups. At day 62, this difference was no longer present. Sodium and chloride concentrations decreased with
increasing dietary levels of iron (from both sources). At day 62, the difference in chloride concentrations was no longer present.

At day 62
Total protein concentrations were lower in the FeEDTA groups than in the FeSO4 groups and albumin concentrations were lower in the mid- and high-dose FeEDTA groups than in the low-dose FeEDTA group and the low- and mid-dose FeSO4 groups. The calcium concentration was lower in the high-dose FeEDTA group than in the low-dose FeSO4 and FeEDTA groups and the mid-dose FeSO4 group. Sodium concentrations were lower in the high-dose FeEDTA and FeSO4groups than in the low-dose FeSO4 group.

Total iron binding capacity in blood plasma
After 31 days of feeding, no statistically significant differences were observed in TIBC along the groups.
After 61 days of feeding, a statistically significantly higher TIBC was observed in the low-dose FeSO4 group compared to the other groups. Furthermore, the TIBC was statistically significantly lower in the high dose FeEDTA group than in the low-dose FeEDTA and the mid-dose FeSO4 groups.

Iron disposition: non-heme iron in liver, spleen and kidney
A statistically significant increase in non-heme iron concentration in the liver with increasing dietary iron concentrations after 31 and 61 days of feeding was found. Independently from the dose level, feeding FeEDTA resulted in statistically significantly lower non-heme iron concentrations in the liver compared to FeSO4 after 31 and 61 days of feeding. After 31 days of feeding, the non-heme iron concentrations in the spleen had increased statistically significantly with increasing dietary iron concentrations. Independently from the dose level, feeding FeEDTA resulted in statistically significantly lower non-heme iron concentrations in the spleen compared to feeding FeSO4 for 3 1 days. After 61 days of feeding, the effect of the dose level had disappeared, but feeding FeEDTA still resulted in statistically significantly lower non-heme iron concentrations in the spleen, compared to FeSO4. After 31 and 61 days of feeding, the non-heme iron concentrations in the kidneys had increased statistically
significantly with increasing dietary iron concentrations, but no statistically significant differences in non-heme iron concentrations between FeEDTA or FeSO4 feeding were observed.

Microscopic examination of Prussian blue-stained sections
After 31 days of feeding, no positive Prussian blue reaction was observed in the liver.
After 61 days of feeding, a positive Prussian blue reaction in the liver was observed in 11, nine and nine animals of the low-, mid and high-dose FeEDTA groups, respectively, and in six, 15 and 14 animals of to the low-, mid- and high-dose FeSO4 groups, respectively. In animals with a positive reaction, the blue staining was found predominantly in the Kupffer cells. The incidences of blue staining in hepatocytes and Kupffer cells of livers of the FeEDTA exposed animals was not influenced by the dose. In the mid- and high-dose FeSO4 groups, the incidences of blue staining in the Kupffer cells were higher than those in the low-dose FeSO4 group.
After 31 days of feeding, the spleen of all animals, low Fe-group animals included, demonstrated a positive Prussian blue reaction. Blue staining was mainly confined to the red pulp, and the degree (intensity of the staining and the number of positive cells) varied from
very slight to slight. The incidence of slight blue staining tended to be somewhat higher in the high-dose FeSO4 and FeEDTA groups compared to the low Fe-groups. No differences were observed between the FeSO4 and FeEDTA groups maintained on diets with comparable iron content.
After 61 days of feeding, a positive reaction in the spleen was observed in all animals. Compared to animals exposed for 31 days, the degree of staining in the spleen of animals exposed for 61 days was somewhat increased, and the staining was extended from red pulp only to red pulp
and marginal sinus, especially in animals with moderate blue staining. A distinct shift from (very) slight to moderate degree of blue staining was observed in the high-dose FeSO4 group when compared to the low-dose FeSO4 group, and in the mid- and high-dose FeEDTA groups
when compared to the low-dose FeEDTA group.

Effect levels

Dose descriptor:

NOAEL

Effect level:

> 11.2 mg/kg bw/day (actual dose received)

Based on:

element

Sex:

male

Basis for effect level:

other: No test item / dose response related toxicological effects observed; 11.2 mg Fe per kg bw corresponds with 421/56 x 11.2 = 84 mg EDTA-FeNa.3H2O per kg bw.

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

- It was concluded that, with respect to iron parameters in blood plasma, feeding FeSO4 or FeEDTA up to dietary concentrations of 140 mg Fe/kg, did not result in excessive iron loading.
- In the present study, the dietary exposure to iron from FeSO4 or FeEDTA did not result in toxicologically relevant changes in clinical signs, body or organ weights, food consumption, red and white blood cell or coagulation variables or cliincal chemistry or in pathology.

Executive summary:

A study was performed to provide data on the disposition, accumulation and toxicity of sodium iron EDTA in comparison with iron (II) sulfate in rats on administration via the diet for 31 and 61 days. Clinical signs, body weights, food consumption, food conversion efficiency, hematology, clinical chemistry and pathology of selected organs were used as criteria for disclosing possible harmful effects. Determination of iron and total iron binding capacity in blood plasma and non-heme iron analysis in liver, spleen and kidneys were used to assess the disposition and accumulation of iron originating from sodium iron EDTA or iron (II) sulfate.

It was concluded that, under the conditions of the present study, iron is accumulated from the diet in liver, spleen and kidneys in a dose-dependent manner, and iron derived from FeEDTA is taken up and/or accumulated less efficiently in liver and spleen than iron from FeSO4.

Moreover, feeding iron up to 11.5 and 11.2 mg/kg body weight/day, derived from FeSO4 and FeEDTA, respectively, did not result in tissue iron excess nor in any other toxicologically significant effects. The latter value corresponds to 84 mg EDTA-FeNa.3H2O per kg bw.