Registration Dossier

Toxicological information

Toxicity to reproduction

Currently viewing:

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was well documented and meets generally accepted scientific principles, and conducted in compliance with GLP.
Cross-reference
Reason / purpose:
reference to same study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid: crystalline
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 (Lot No. 010628) and Mg 0.28%, Mn 0.16%, Zn 84 ppm (Lot No. 010903)
- 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 and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc., Yokohama, Japan
- Age at study initiation: 10 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 exposure:
PREPARATION OF DOSING SOLUTIONS: Test substance was prepared with water for injection purposes. Gavage solutions were prepared freshly each time and used within 6 hours.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: up to 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
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 beginning 14 days before mating .
Frequency of treatment:
Once daily
Details on study schedule:
Not applicable as only screening study.
Doses / concentrations
Remarks:
Doses / Concentrations:
30, 100, 300, 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses selected for the main studies were based on gross pathology findings observed in the 14-days preliminary studies (Study No. 100520P).
Positive control:
None

Examinations

Parental animals: Observations and examinations:
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, 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, ?-GTP, T-protein, 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.

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.
Oestrous cyclicity (parental animals):
Number of times in estrus before and during administration till mating confirmed, number of corpora lutea
Sperm parameters (parental animals):
Parameters examined in P male parental generations:
testis weight and abnormality, epididymis weight and abnormality, abnormality of prostate
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals, one day after the last administration (day 50);
- Maternal animals: All surviving animals, 25 days after mating

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS: yes

Organs examined at necropsy.  Organ weight: Brain, liver, kidney, spleen, heart, adrenal, pituitary, thyroids, thymus, testis, epididymis, ovary, uterus. Microscopic examination: Brain, pituitary, thymus, thyroid, adrenal, spleen, heart, esophagus, stomach, liver, pancreas, duodenum, jejunum, ileum, 
cecum, colon, rectum, trachea, lung, kidney, urinary bladder, testis, epididymis, prostate, seminal vesicle, ovary, uterus, vagina, eye, harderian gland, mammary gland, sternum, femur, spinal cord, lymph node, mandibular lymph node, salivary grand, parathyroid grand, sciatic nerve, 
bone marrow.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring was sacrificed at 4 days of age.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
Statistics:
Bartlett´s test, Dunnett´s test and ?2 test
Reproductive indices:
copulation index, fertility index, gestation index, delivery index
Offspring viability indices:
implantation index, live birth index and viability index

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
no systemic effects
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
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
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
males: 1000 mg/kg bw/day: 1 died on day 27 and salivation; 300 mg/kg bw/day: salivation
females: 1000 mg/kg bw/day: 1 died on day 19 and salivation; 300 mg/kg bw/day: salivation

BODY WEIGHT
males: 1000 mg/kg bw/day: reduced between days 11 and 49;
females: 1000 mg/kg bw/day: reduced during pregnancy on day 21 (not significant)

FOOD CONSUMPTION
males/females: 1000 mg/kg bw/day: reduced on day 3

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
no effect on estrous cycle


REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- All 12 pairs of each dose group copulated successfully (copulation index 100%).
- All females except one female became pregnant (fertility index: 100).
-The gestation index was 100% for all dose groups

ORGAN WEIGHTS (PARENTAL ANIMALS)
- males: 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.

- Significant higher absolute testis weight observed in males exposure to the 300 mg/kg bw/day, but not in males treated with 1000 mg/kg bw/day. This increase is not considered to be treatment related. The statistically significant higher relative testis weights (1000 mg/kg bw/d) may be due to reduced body weights. No effect on epididymis weights, no abnormalities in testes, epididymides and prostates observed.


GROSS PATHOLOGY (PARENTAL ANIMALS)
- males:
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.

- females:
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.

HISTOPATHOLOGY (PARENTAL ANIMALS)
males testis:
-mild atrophy of seminiferous tubules: 1/7 animals at 1000 mg/kg bw/d
-moderate atrophy of seminiferous tubules: 1/7 animals at 1000 mg/kg bw/d
-mild hyperplasia of Leydig’s cell: 1/6 animals at 1000 mg/kg bw/d
-mild degeneration of seminiferous tubules: 1/6 animals at control and 1/7 at 1000 mg/kg bw/d
-moderate degeneration of seminiferous tubules: 1/6 animals at control and 1/7 at 1000 mg/kg bw/d
-moderate exfoliated round spermatids of seminiferous tubules: 1/6 animals in the control group and 1/7 at 1000 mg/kg bw/d

males epididymis:
-marked emptiness: 1/7 animals 1/7 at 1000 mg/kg bw/d

males prostate:
-mild lymphoid cell infiltration: 3/6 animals in the control groups
-moderate lymphoid cell infiltration: 3/6 animals at 1000 mg/kg bw/d

The effects observed in histopathology in male reproductive organs occured spontaneous and were not considered compound-related.

females: histopathological examinations of the ovary was not carried out.

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).

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: For parental repeated dose toxicity.
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: For reproductive toxicity.

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

VIABILITY (OFFSPRING): No adverse findings. 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 (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: For reproductive toxicity.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Number of times in estrus and conceiving days of females (P)

Group [mg/kg bw/d]

Control

iron(II)sulfate heptahydrate

0

30

100

300

1000

Number of females

12

12

12

12

12

Number of estrous cases before mating (14 days)

3.3 ± 0.9

3.4 ± 0.5

3.4 ± 0.7

3.8 ± 0.5

3.4 ± 0.5

Number of conceiving days 

2.3 ± 1.1

2.6 ± 1.2

2.6 ± 1.2

2.5 ± 0.8

3.1 ± 1.0

Organ weight of male rats (P)

 

Group [mg/kg bw/d]

Control

iron(II)sulfate heptahydrate

0

30

100

300

1000

Number of animals

12

12

12

12

12

Absolute body weight [g]

485 ± 32

483 ± 38

474 ± 31

478 ± 32

426 ± 47**

Absolute testes weight [g]

3.16 ± 0.17

3.30 ± 17

3.30 ± 0.18

3.45 ± 0.26*

3.26 ± 0.41

Relative testes weight [g]

0.65 ± 0.05

0.69 ± 0.07

0.70 ± 0.05

0.72 ± 0.06

0.77 ± 0.12**

Absolute epididymides weight [g]

1.28 ± 0.07

1.29 ± 0.14

1.25 ± 0.10

1.30 ± 0.08

1.21 ± 0.14

Relative epididymides weight [g]

0.26 ± 0.02

0.27 ± 0.04

0.27 ± 0.03

0.270 ± 0.03

0.29 ± 0.04

*: P < 0.05

**: P < 0.01

 

 

 

Applicant's summary and conclusion

Conclusions:
The NOAEL for reproductive toxicity established in a screening study was at least 1000 mg/kg bw/day iron sulphate heptahydrate (equivalent to 200 mg/kg bw/day iron) in rats. The NOAEL for parental toxicity was 100 mg/kg bw/day (equivalent to 20 mg/kg bw/day iron).
Executive summary:

In an OECD 422 screening study the NOAEL for reproductive toxicity was at least 1000 mg/kg bw/day iron sulphate heptahydrate (equivalent to 200 mg/kg bw/day iron) in rats. The NOAEL for parental toxicity was 100 mg/kg bw/day (equivalent to 20 mg/kg bw/day iron).