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Toxicological information

Repeated dose toxicity: oral

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Administrative data

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.
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report date:
2002

Materials and methods

Test guideline
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

Test material

Constituent 1
Reference substance name:
ferrous sulphate heptahydrate
IUPAC Name:
ferrous sulphate heptahydrate
Constituent 2
Chemical structure
Reference substance name:
iron(2+);sulfate;heptahydrate
Cas Number:
7782-63-0
Molecular formula:
FeH14O11S
IUPAC Name:
iron(2+);sulfate;heptahydrate
Details on test material:
- Name of test material (as cited in study report): Iron (II) sulfate heptahydrate
- Physical state: blue-green crystalline powder
- Analytical purity: 91.1 % (Lot No. 010628) and 90.6 % (Lot No. 010903)
- Impurities (identity and concentrations): Mg 0.3%, Mn 0.19%, Zn 75 ppm and Mg 0.28%, Mn 0.16%, Zn 84 ppm
- Storage condition of test material: room temperature under argon
- Stability under test conditions: The stability of test concentrations was confirmed for 6 hours in the dark at room temperature.

Test animals

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

Administration / exposure

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
Doses / concentrations
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

Examinations

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

Results and discussion

Results of examinations

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).

Effect levels

open allclose all
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

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

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

Applicant's summary and conclusion

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