Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information
A study was undertaken to investigate the effect of oxidative stress and chronic iron intoxication on the rat testes. The protective effect of vitamin E was also evaluated.
A study was undertaken to evaluate genome-related differences to iron overload between and within rodent species
Link to relevant study records
Reference
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.
Reason / purpose:
reference to same study
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
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
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.
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
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
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: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
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).
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.
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
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).
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.
Reproductive effects observed:
not specified

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

 

 

 

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).

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
A modern, guideline compliant screening study performed with a read-across substance is available.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

No adverse effects on reproductive performance or fertility were evident among rats dosed at 1000 mg/kg bw/day. Iron sulphate had no effects on oestrous cyclicity. Treatment of males and females prior to mating had no effect on reproductive performance or fertility or any of the reproduction performance indices investigated. Systemic toxicity was evident at the high dose level of 1000 mg/kg bw/day, but this had no effect on reproduction. Systemic effects included minor clinical signs (salivation) and the death of one male and female at 1000 mg/kg bw/day. Minor reductions in bodyweight were noted for both males and females, this was reflected in changes in relative organ weights, affecting adrenals, liver, pituitary, heart, brain, testes and uterus. Pathological changes were predominantly observed in the high dose group with sporadic cases of liver, spleen and renal changes observed. Generally these effects were not extended into the lower dose groups. Histopathological changes in the testes were considered incidental and not treatment related. The parental systemic toxicity NOAEL was 300 mg/kg bw/day.


Short description of key information:
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).

Justification for selection of Effect on fertility via oral route:
Only one study available for this endpoint

Effects on developmental toxicity

Description of key information
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).  There were no developmental toxicity effects apparent in the pups of the high dose group
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
No data
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.
Qualifier:
according to
Guideline:
other: OECD 422
Deviations:
no
Principles of method if other than guideline:
Refer to summary presented in section 7.8.1
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
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
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.
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 treatment and at the end of administration.
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
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
Duration of test:
49 days
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).
Maternal examinations:
Observation performed and frequency: General condition was observed twice a day (before and after treatment). 

Male body weights were  measured twice a week. Female body weights were measured twice a week  during the pre-mating and mating period and on days 0, 7, 14 and 21 of gestation and days 0 and 4 of lactation. 

Food consumption was determined twice a week during the pre- and post-mating period in males, and twice a week during the pre-mating period and on days 2, 9, 16 and 21 of gestation and day 4 of lactation in females. 

For 6 males and females per group, urinalysis was carried out before final administration. 

Blood samples were collected from the abdominal aorta on next day of the last  administration and hematological  and blood chemical examinations were carried out. 
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No
Fetal examinations:
- External examinations: Yes:
- Soft tissue examinations: No data
- Skeletal examinations: No data
- Head examinations: No data
Statistics:
Bartlett´s test, Dunnett´s test
Indices:
copulation index, fertility index, gestation index, delivery index, implantation index, live birth index and viability index
Historical control data:
No information
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
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).
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
In a well conducted OECD 422 study conducted to GLP (reliability score 1), the NOAEL for developmental 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).
Executive summary:

In a well conducted OECD 422 study conducted to GLP (reliability score 1), the NOAEL for developmental 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).

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
A modern, guideline compliant screening study performed with a read-across substance is available.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

In the screening test there were no adverse developmental effects observed at doses up to 1000 mg/kg bw/day. 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). The NOAEL for offspring was greater than 1000 mg/kg bw/day.


Justification for selection of Effect on developmental toxicity: via oral route:
Ony one study available for this endpoint

Toxicity to reproduction: other studies

Additional information

A screening study performed with the read-across substance iron sulphate does not show any developmental or reproductive toxicity at the limit dose of 1000 mg/kg bw/d. Parental toxicity was apparent at this dose level. Non-standard investigative studies indicate that iron overload may be associated with testicular pathology, however the results of these studies are of unclear relevance to the risk assessment. Further testing for developmental and reproductive toxicity is not proposed.

It is considered that based on the following points, a weight-of-evidence approach can be taken for reproductive and developmental endpoints for iron trinitrate, rather than proposing to conduct further, new experimental animal tests to fulfil the Annex requirements: the ubiquitous nature of iron in the environment; the fact that iron is an essential element; iron plays an important role in biological processes, with iron homeostasis being under strict control; iron has been given to pregnant women for many years without an effect on pregnancy outcome; available experimental animal data have not revealed any adverse effects on reproduction parameters and development. From the weight-of-evidence approach the following conclusion can be reached: Results from recent guideline oral screening studies performed on ferrous chloride and ferrous sulfate gave NOAELs for reproductive and developmental effects of ≥500 mg/kg body weight/day or ≥1000 mg/kg body weight/day (no adverse effects were observed), respectively. These findings are considered to be relevant to ferric as well as ferrous salts, as oxidation of ferrous to ferric occurs in the low pH of stomach before ingested iron is absorbed into the body. In humans, iron supplementation of about 5.8 to 11.7 mg/kg bw/d (for a 60kg individual) is routinely prescribed throughout pregnancy with no adverse effects on pregnancy outcome.

Justification for classification or non-classification

Based on the absence of reproductive or developmental toxicity effects at the limit dose of 1000 mg/kg bw/day in a sreening study performed with a read-across substance, there are no indications that iron trinatrate should be classified.