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Diss Factsheets

Administrative data

Description of key information

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
No data
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: 1d The study was well documented and meets generally accepted scientific principles, and conducted in compliance with GLP.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc., Yokohama, Japan
- Age at study initiation: 8 weeks
- Weight at study initiation: male: 341 -383 g; female: 222 -255 g
- Housing: stainless steel cage
- Diet (ad libitum): CRF-1 from Oriental Yeast Co., Ltd.
- Water (ad libitum): tap water
- Acclimation period: 12 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 26
- Humidity (%): 40 - 70
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12 / 12
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Test substance was prepared with water for injection purposes. Gavage solutions were prepared freshly everytime and used within 6 hours.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of test concentrations was confirmed for 6 hours in the dark at room temperature.
The concentrations were measured in samples used for males at the first teatment and at the end of administration.
Duration of treatment / exposure:
Males: Total of 49 days beginning 14 days before mating, Females: Total of 42-47 days from 14 days before mating to day 5 of lactation
Frequency of treatment:
once daily
Remarks:
Doses / Concentrations:
30, 100, 300, 1000 mg/kg bw/day
Basis:
other: nominal conc.
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
Post-exposure period: none
- Dose selection rationale: Doses selected for the main studies were based on gross pathology finging observed in the 14-days preliminary studies (Study No. 100520P).
- Rationale for animal assignment (if not random): No data
- Rationale for selecting satellite groups: To study repeated dose toxicity in non-pregnant females
- Post-exposure recovery period in satellite groups: No post-exposure period
- Section schedule rationale (if not random): No data
Positive control:
None
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations: general condition and mortality

BODY WEIGHT: Yes
- Time schedule for examinations: male: twice weekly; female: twice weekly, during pregnancy on days 0, 7 14 and 21, during lactation on days 0 and 4

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on day after last application
- Anaesthetic used for blood collection: Yes (identity) Pentobarbital-Na
- Animals fasted: Yes
- How many animals: 6 of each group
- Parameters checked: RBC, Hemoglobin, Hematocrit, MCV, MCH, MCHC, Platelet, Reticulocyte, PT, APTT, Fibrinogen, WBC, Differential leukocyte: Lymphocyte, Neutrophil, Eosinophil, Basophil, Monocyte

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on day after last application
- Animals fasted: Yes
- How many animals: 6 of each group
- Parameters checked: AST, ALT, ALP, gamma-GTP, T-potein, Albumin, A/G, T-bilirubin, BUN, Creatinine, Glucose, T-cholesterol, Triglyceride, Na, K, Cl, Ca, Inorganic-p, Fe

URINALYSIS: Yes
- Time schedule for collection of urine: before end of exposure
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: Color, pH, Protein, Glucose, Ketone body, Bilirubin, Occult blood, Urobilinogen, Urinary sediments, Epithelial cells, Erythrocytes, Leukocytes, Casts, Crystals

- Reproductive behavior od parental animals: see section 7.8.1

- Development of F1 generation: see section 7.8.1
Sacrifice and pathology:
GROSS PATHOLOGY: Yes: males: brain, pituitary, thyroids, thymus, heart, liver, spleen, kidneys, adrenals, testes, epididymides; females: brain, pituitary, thyroids, thymus, heart, liver, spleen, kidneys, adrenals, ovaries, uterus
HISTOPATHOLOGY: Yes: males: eyeball, thymus, heart, lung, stomach, liver, pancreas, spleen, kidney, urinary bladder, testis, epididymis, prostate,bone marrow; females: heart, lung, liver, spleen, kidney, urinary bladder, pituitary,
Other examinations:
Estrus cycle, reproductive performance, observation of pups
Statistics:
Bartlett's test, Dunnett's and Chi² test
Clinical signs:
no effects observed
Description (incidence and severity):
No adverse systemic effects
Mortality:
no mortality observed
Description (incidence):
No adverse systemic effects
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No significant effects
Food efficiency:
not examined
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:
effects observed, treatment-related
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
male: 1000 mg/kg bw/day: 1 died on day 27 and salivation, 300 mg/kg bw/day: salivation; female: 1000 mg/kg bw/day: 1 died on day 19 and salivation, 300 mg/kg bw/day: salivation

BODY WEIGHT
male: 1000 mg/kg bw/day: reduced between days 11 and 49; female: 1000 mg/kg bw/day: reduced during pregnancy on day 21 (not significant)
FOOD CONSUMPTION
male/female: 1000 mg/kg bw/day: reduced on day 3

HAEMATOLOGY
male: 1000 mg/kg bw/day: RBC and APTT reduced; MCV, MCH and reticulocytes increased, increase at 300 mg/kg bw/day of MCH is considered to be not toxicologically relevant, because no change in RBC is observed ; female: 30 and 1000 mg/kg bw/day: MCV and MCH increased; 1000 mg/kg bw/day: hemoglobin increased, increase at 30 and 1000 mg/kg bw/day of MCH, MCV and hemoglobin is considered to be not toxicologically relevant, because no change in RBC is observed .

CLINICAL CHEMISTRY
male: 1000 mg/kg bw/day: total-protein, albumin, calcium decreased; ALT, gamma-GTP and A/G increased; T-bilirubin was increased at 30 and 100 mg/kg bw/day, but no changes at 300 and 1000 mg/kg bw/day; female: 300 mg/kg bw/day: inorganic-p increased; 1000 mg/kg bw/day: gamma-GTP and inorganic-p increased; ALP decreased at 30, 100 and 300 mg/kg bw/day, but no changes at 1000 mg/kg bw/day.

URINALYSIS
male: 1000 mg/kg bw/day: volume increased and specific gravity decreased

ORGAN WEIGHTS
male: 1000 mg/kg bw/day: absolute and relative weight of adrenals, relative weight of liver increased; absolute testes weight increased at 300 mg/kg bw/day, but not at 1000 mg/kg bw/day; absolute weights of pituitary and heart were decreased; relative weight of brain and testes increased: these changes are considered to be due to the significant body weight loss
females: 1000 mg/kg bw/day: absolute and relative weight of liver increased relative weight of uterus increased at 1000 mg/kg bw/day, but this was considered to be due to the significant body weight loss.

GROSS PATHOLOGY and HISTOPATHOLOGY: NON-NEOPLASTIC
male survivors:
1000 mg/kg bw/day: thymus: atrophy in 2 animals; stomach inflammation and ulcers in glandular stomach (1 animal); bleeding (1 case); inflammatory cell infiltration in submucosal glandular stomach (2 cases); vacuolization of the forestomach epithelium (1 case); liver: yellow-brown pigmentation of periportal hepatocytes (6 cases); pigmentation of periportal Kupffer cells (3 cases): probably due to iron; spleen: extramedullary hematopoiesis (4 cases); yellow-brown pigmentation in red pulp (6 cases); kidney: basophilic changes in tubular epithelium (4 cases); bone marrow: hematopoiesis in the femur (1 case)
300 mg/kg bw/day: spleen: yellow-brown pigmentation in the red pulp (6 cases); extramedullary hematopoiesis (5 cases)

100 mg/kg bw/day: spleen: yellow-brown pigmentation in the red pulp (6 cases); extramedullary hematopoiesis (2 cases); kidney: basophilic changes in tubular epithelium (1 case)

30 mg/kg bw/day: spleen: yellow-brown pigmentation in the red pulp (6 cases); extramedullary hematopoiesis (1 cases); kidney: basophilic changes in tubular epithelium (2 cases)
Control: yellow-brown pigmentation in the red pulp (6 cases); extramedullary hematopoiesis (2 cases)

dead males:
1000 mg/kg bw/day: mineral deposits in heart, lung congestion, pigmentation of periportal hepatocytes
The adrenal glands showed abnormalities (abnormal growth) in the autopsy.

female survivors:
1000 mg/kg bw/day: liver: yellow-brown pigmentation of periportal hepatocytes (6 cases) probably due to iron; spleen: yellow-brown pigmentation in the red pulp (6 cases); extramedullary hematopoiesis (6 cases)

300 mg/kg bw/day: spleen: yellow-brown pigmentation in the red pulp (6 cases); extramedullary hematopoiesis (6 cases)

100 mg/kg bw/day: spleen: yellow-brown pigmentation in the red pulp (6 cases); extramedullary hematopoiesis (6 cases)

30 mg/kg bw/day: spleen: yellow-brown pigmentation in the red pulp (5 cases); extramedullary hematopoiesis (6 cases)

Control: spleen: yellow-brown pigmentation in the red pulp (6 cases); extramedullary hematopoiesis (6 cases)

dead females:
Lung congestion and edema, mineral deposits in liver
Abnormalities in pituitary (mass), adrenal (enlargement) and thymus (atrophy) were found at autopsy.

OTHER FINDINGS
Parent animal reproduction:
Reproductive performance displayed no significant changes between treatment groups and controls (number of estrous cases, copulation index, number of days before copulation, fertility index, gestation length, gestation index, delivery conditions, nursing conditions, number of corpora lutea, number of implantation sites, or implantation rate).

Pups:
No significant changes between treatment groups and controls were observed (number, number of stillbriths, number of live pups on day 0 of lactation, sex ratio, delivery index, birth index, live birth index, general signs, number of live pups on day 4 of lactation, viability index on day 4 of lactation, external observation, body weight change, necropsy findings).
Dose descriptor:
NOAEL
Remarks:
repeated dose toxicity
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat. (total fraction)
Remarks:
FeSO4.7H2O
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat. (total fraction)
Remarks:
FeSO4.7H2O
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat. (total fraction)
Remarks:
FeSO4.7H2O
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Dose descriptor:
NOAEL
Remarks:
repeated dose toxicity
Effect level:
20 mg/kg bw/day (actual dose received)
Based on:
element (total fraction)
Remarks:
Fe
Sex:
male/female
Basis for effect level:
other: recalculated value from the FeSO4 level
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
>= 200 mg/kg bw/day (actual dose received)
Based on:
element (total fraction)
Remarks:
Fe
Sex:
male/female
Basis for effect level:
other: recalculated value from the FeSO4 level
Remarks on result:
not determinable due to absence of adverse toxic effects
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Effect level:
>= 200 mg/kg bw/day (actual dose received)
Based on:
element (total fraction)
Remarks:
Fe
Sex:
male/female
Basis for effect level:
other: recalculated value from the FeSO4 level
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
not specified

Table 1: Urinalysis (urine volume and specific gravity) results for males

 Dose (mg/kg bw/day)

 0

30

 100

300 

1000 

 Number of animals

 6

 6

 6

 6

 6

 Urine volume (mL) mean

 13.4

 13.2

14.6 

 17.6

 28.7**

 SD

 3.7

 3.4

 2.6

 6.7

 8.0

 Specific gravity mean

 1.055

 1.056

 1.052

 1.051

 1.027**

 SD

0.013

 0.012

 0.007

 0.014

 0.004

Note:**, p<0.01

Table 2: Haematology results for males

 Dose (mg/kg bw/day)

 0

 30

 100

300 

 1000

 Number of animals

 6

 6

 6

 6

 6

 RBC (10e+4/uL) mean

 839

 826

 807

 798

689**

 SD

 25

 29

 47

 38

 113

 ATPP (sec.) mean

 31.6

32.5 

 30.7

 30.2

 25.6**

 SD

 1.9

 2.0

 2.0

 3.8

 2.3

 MCV (fL) mean

 53.9

 56.1

 55.3

 56.3

 60.0**

 SD

 1.4

 1.7

 0.5

 1.8

 5.5

MCH (pg) mean

 18.3

 19.1

 18.8

 19.3*

 20.5**

 SD

 0.3

 0.5

 0.3

 0.5

 1.4

 Reticulocyte (0/00) mean

 26

 28

 27

 29

 65*

SD 

 4

 5

 3

 4

 68

Note:*, p<0.05; **, p<0.01

Table 3: Blood chemistry for males

 Dose (mg/kg bw/day)

 0

 30

 100

 300

 1000

 Number of animals

 6

 6

 6

 6

 ALT (IU/L) mean

 30.4

 34.2

 33.9

 29.8

 57.0*

 SD

 4.7

 7.9

 7.8

 4.3

 27.0

 Gamma-GTP (IU/L) mean

 0.42

 0.49

 0.40

 0.36

 0.71*

 SD

 0.14

 0.14

 0.16

 0.07

 0.29

 T-protein (g/dL) mean

 5.6

 5.5

 5.5

 5.6

 4.5**

 SD

 0.3

 0.2

 0.2

 0.2

 0.3

 Albumin (g/dL) mean

 2.78

 2.75

 2.81

 2.78

 2.35**

 SD

 0.08

 0.16

 0.15

 0.12

 0.17

 A/G mean

 0.99

 0.99

 1.03

 1.00

 1.10*

 SD

0.06

 0.05

 0.07

 0.05

 0.08

Note:*, p<0.05; **, p<0.01

Table 4: Blood chemistry for females

 Dose (mg/kg bw/day)

 0

 30

 100

 300

 1000

Number of animals

 6

 6

 6

 6

 6

 Gamma-GTP (IU/L) mean

 0.57

 0.70

 0.60

 0.70

 1.31*

 SD

 0.17

 0.16

 0.12

 0.18

 0.85

 Inorganic-p (mg/dL) mean

 8.9

 9.0

 9.1

 10.1*

 10.3**

 SD

 0.9

 0.7

 0.8

 0.4

 0.4

Note :*, p<0.05; **, p<0.01

Table 5: Organ weights for males (only statistically significant changes included)

 Dose (mg/kg bw/day)

 0

 30

 100

 300

 1000

 Number of animals

 6

 6

 6

 6

 6

 Body weight (g) mean

485

 483

 474

 478

 426**

SD

32

 38

 31

 32

 47

 Brain relative (g%) mean

0.44 

 0.43

 0.44

 0.44

 0.50**

 SD

 0.03

 0.03

 0.02

 0.03

 0.05

 Pituitary absolute (g) mean

14.9 

 16.4

 15.2

 14.0

 13.0*

 SD

1.7 

 2.1

 1.7

 1.6

 1.6

 Heart absolute (g) mean

 1.40

 1.41

 1.46

 1.41

 1.27*

 SD

 0.12

 0.12

 0.08

 0.12

 0.14

 Liver relative (g%) mean

2.53 

 2.47

 2.56

 2.65

 2.96**

 SD

0.12 

 0.19

 0.21

 0.16

 0.33

 Adrenals absolute (mg) mean

 53.3

 57.3

 53.4

 55.4

 66.6**

 SD

 7.0

 9.0

 7.0

 6.5

 9.3

 Adrenals relative (mg%) mean

 11.0

 11.9

 11.3

 11.6

 15.9**

 SD

 1.5

 2.0

 1.6

 1.6

 3.3

Note: :*, p<0.05; **, p<0.01

Table 6: Organ weights for females (only statistically significant changes included)

 Dose (mg/kg bw/day)

 0

 30

 100

 300

 1000

 Number of animals

 6

 6

 6

 6

 Body weight (g) mean

 302

 306

 302

 301

 293

 SD

 21

 14

 17

 17

 14

Liver absolute (g) mean

 10.62

 10.76

 10.76

 10.63

 11.89*

 SD

 1.14

 0.65

 1.23

 0.83

 1.20

 Liver relative (g%) mean

 3.52

 3.52

 3.56

 3.54

 4.06**

 SD

 0.21

 0.19

 0.29

 0.23

 0.37

Uterus relative(g%) mean

 0.19

 0.20

 0.18

 0.20

 0.22*

 SD

 0.02

 0.03

 0.02

 0.03

 0.04

Note:*, p<0.05; **, p<0.01

Conclusions:
In a good quality OECD 422 study conducted to GLP (reliability score 1) the NOAEL for repeated dose toxicity of iron sulphate heptahydrate was 100 mg/kg bw/day (equivalent to 20 mg Fe/kg bw/day for both sexes) based on the extramedullary hematopoiesis of the spleen in males and increased levels of inorganic phosphate in females at 300 mg/kg bw/day in rats.
Executive summary:

In a good quality OECD 422 study conducted to GLP (reliability score 1) the NOAEL for repeated dose toxicity of iron sulphate heptahydrate was 100 mg/kg bw/day (equivalent to 20 mg Fe/kg bw/day for both sexes) based on the extramedullary hematopoiesis of the spleen in males and increased levels of inorganic phosphate in females at 300 mg/kg bw/d

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Qualifier:
no guideline followed
Principles of method if other than guideline:
The present feeding study was performed to provide data on the disposition, accumulation and toxicity of sodium iron EDTA in comparison with iron (II) sulphate in rats on administration via the diet for 31 and 61.
GLP compliance:
not specified
Limit test:
no
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
- Age at study initiation: 5-6 weeks
- Weight at study initiation: no data
- Fasting period before study: no data
- Housing: individually
- Diet (e.g. ad libitum): AIN-93G for at least 14 days prior to start experiment
- Water (e.g. ad libitum): no data
- Acclimation period: at least 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): no data
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): no data
Route of administration:
oral: feed
Vehicle:
other: FeSO4 incorporated in diet
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Mixing appropriate amounts with (Type of food): low-iron AIN-93M diet
- Storage temperature of food: at or below 18 °C
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The content, stability and homogenous distribution of iron in the test diets was analyzed by means of atomic absorption spectrometry (AAS). The content of each concentration of diet was measured three times during the study.
Duration of treatment / exposure:
31 or 61 days
Frequency of treatment:
daily in feed
Remarks:
Doses / Concentrations:
0, 35, 70, and 140 mg Fe/kg in feed giving a maximum dose of 11.5 mg Fe/kg body weight, equivalent to 31.2 mg ferrous sulphate/kg bw
Basis:
nominal in diet
No. of animals per sex per dose:
40
Control animals:
other: FeSO4 group served as control group for FeEDTA
Details on study design:
Rationale for animal assignment (if not random): random
Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: once during the aclimatization 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 was measured per animal twice weekly, over periods of 3 or 4 days, by weighing the feeders. The results were expressed in g / animal / day/day: Yes
- The intake of iron from FeSO4 / kg bw / day was calculated from the nominal dietary level of iron, the food intake, and the body weight (mean of the body weights measured at the start and the end of each week): Yes

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at autopsy
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes
- How many animals: 20/group

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at autopsy
- Animals fasted: Yes
- How many animals: 20/group
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Other examinations:
Non-heme iron analysis in tissue
Statistics:
Body weights were analysed 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, 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 non-parametric ANOVA followed by Mann-Whitney U-tests, histopathological changes were analyzed by Fisher's exact probability test and non-heme iron analysesin 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.
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
Haematological findings:
no effects observed
Description (incidence and severity):
in red and white blood cell or coagulation variables
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Dose descriptor:
NOAEL
Effect level:
31.2 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks:
FeSO4
Sex:
male
Basis for effect level:
other: no iron excess in tissue or any other toxicologically significant effects
Dose descriptor:
NOAEL
Effect level:
11.5 mg/kg bw/day (nominal)
Based on:
element
Remarks:
Fe
Sex:
male
Critical effects observed:
not specified

Iron analyses of the diet indicated that the test material was homogenously distributed and in 17/18 diet samples the Fe content was within 10 % of the intended value (in one case 13 % lower).

Clinical observations

None considered treatment related.

Body/organ weights

No statistically significant differences in terminal body weights or absolute and relative organ weights.

Food consumption and Food conversion efficiency (FCE)

Overall food consumption for the test groups ranged from 23.6 to 24.5 g/rat/day. Sporadic statistically significant changes were not dose related and were considered chance findings. FCE showed statistically significant differences among groups in weeks 1, 3 and 4, in most cases the FCE was significantly lower in the high dose ferrous sulphate group.

Iron intake

Mean intakes for the ferrous sulphate groups were 2.84, 5.69 and 11.54 mg/kg/bw for the low, mid and high dose groups respectively.

Haematology

At 61 days there were no statistically significant effects in any of the parameters investigated (haemoglobin, packed cell volume, RBC, total or differential white cell count, prothrombin time, thromobcyte count, MCV, MCH or MCHC). Statistically significant changes observed in some parameters after 31 days were not considered of toxicological significance.

Clinical chemistry

Sporadic changes were not dose related and not considered of toxicological significance.

Iron levels

There were no statistically significant changes in plasma iron concentrations in the ferrous sulphate treated groups. After 61 days total iron binding capacity (TIBC) was increased in the low-dose ferrous sulphate group compared to other groups (TIBC lower at higher dose levels). TIBC ferrous sulphate low dose 99.6, mid dose 90.9 and high dose 90.3 mmol/L. Irrespective of the dose, treatment with ferrous sulphate generally resulted in higher TIBC values than Fe EDTA.

Iron deposition

A statistically significant increase in non-haem iron concentration in the liver with increasing dietary iron concentrations was observed after 31 and 61 days. In the kidney non-haem iron also increased with increasing dietary concentrations. In the spleen this dose related increase was observed following 31 days exposure to ferrous sulphate but this significance was lost by 61 days. In general iron derived from ferrous sulphate was accumulated more efficiently than Fe EDTA.

Results for ferrous sulphate were Liver baseline iron content 62.2 mg/g wet tissue; at 31 days low 67.2, mid 89, high dose 116.1; at 61 days low 105.6, mid 120.2, high 126.4 mg/g wet tissue.

Spleen baseline iron content 26, at 31days low dose 171, mid dose 213, high 285.6 mg/g wet tissue, spleen after 61 days low 1018.5, mid 1107.2, high dose 1126.4 mg/g wet tissue.

Kidney baseline iron content 17.1 mg/g wet wt, at 31days low dose 29, mid 30, high 31.5; at 61 days low dose 43.5, mid 49, high 48 mg/g wet tissue.

Pathology

No remarkable macroscopic changes. In those tissues examined no histopathological changes related to dose or type of iron administered. After 31 days exposure to ferrous sulphate there was no Prussian blue staining in the liver however after 61 days there was positive staining for iron predominantly in the Kupffer cells. The incidence was low dose 11/20, mid and high dose 9/20 with a higher incidence of Kupffer cell staining in the mid and high doses. In the spleen Prussian blue staining was observed at 31 days mostly in the red pulp and the degree (intensity and number of positive cells) was rather higher in the high dose group. After 61 days the degree of staining was further increased and extended form the red pulp to the red pulp and marginal sinus. The degree of staining increased from very slight/slight to moderate in the high dose ferrous sulphate group.

Conclusions:
Dietary exposure to ferrous sulphate did not result in toxicologically relevant changes in clinical signs, body or organ weights, food consumption red and white cell or coagulation variables or in pathology.

It is concluded that, under the conditions of the present study, iron is accumulated from the diet in liver, spleen and kidneys in a dose-dependant manner. Feeding iron (ferrous sulphate) up to 11.5 mgFe/kg bw/day did not result in tissue iron excess nor in any other toxicologically significant effects.
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 mg/kg bw/day, derived from FeSO4 did not result in tissue iron excess nor in any other toxicologically significant effects.

Endpoint conclusion
Species:
rat

Additional information

Justification for classification or non-classification

In relation to the information available the UVCB substance does not show Repeated dose toxicity