hydronium;iron(3+);hexahydroxide;disulfate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

12 April 2004 to 29 June 2004

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Justification for type of information:

The Reporting Format for the Chemical Category According to ECHA (2008) Guidance R.6.2.6.2 can be found in the Endpoint Summary of Toxicokinetics, metabolism and distribution.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

developmental toxicity

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

12 April 2004 to 29 June 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Justification for type of information:

The Reporting Format for the Chemical Category According to ECHA (2008) Guidance R.6.2.6.2 can be found in the Endpoint Summary of Toxicokinetics, metabolism and distribution.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

other: 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

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Sam: Strain of TacN(SD), Samtako Bio Korea, 77-1 Seorang-dong, Osan-si, Gyeonggido.

- Age at study initiation: 8 weeks old.

- Weight at study initiation: Male: 269.23 - 302.18 g., Female: 191.34 - 221.60 g.

- Fasting period before study: The day before the necropsy.

- Housing:
Facility used:Stainless steel wired cage (260W X 350L X 210H (mm), Daejong Lab), polycarbonate cage (260W X 420L X 180H (mm), MJLTD).
Pre-mating period: 1 - 2 individuals (separate female and male rats), in stainless steel wire cage.
Mating period: One female and one male in one cage, in stainless steel wire cage.
Gestation and nursing period (females): Females individually housed in polycarbonate cage.
Post-mating period (males): 1 - 2 individuals, in stainless steel wire cage.
recovery group: 1 - 2 individuals, in stainless steel wire cage.

- Diet : The lab rat feed 5057 (Agri Purina Korea, 627 Jangdang-dong, Pyeongtaek, Gyeonggido), ad libitum.Analysis of the diet met the criteria of the protocol.

- Water (e.g. ad libitum): Mains water, filtred and sterilised, ad libitum. Analysis of the water met the criteria of the drinking water quality (Ministry of Environment).

- Acclimation period: 7 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.7 - 23.0ºC.
- Humidity (%): 40.8 ± 69.1%.
- Air changes (per hr): 10 - 15 changes per hour, front ventilation.
- Photoperiod (hrs dark / hrs light): 12 hrs light (7am - 7pm) / 12 hrs dark. 150 - 300 lux was used.

IN-LIFE DATES:
- Males: From Day 0 To: Day 43.
- Female: From: Day 0 Up To: Day 5 post partum, (female mated but did not show gestation signs) Up To: gestation Day 27.
(Recovery group)
- Males: From: Day 0 To: Day 57.
- Females. From: Day 0 To: Day 69.

Route of administration:

oral: gavage

Type of inhalation exposure (if applicable):

other: not applicable

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The substance was suspended in the water for injection by dose and formulated just before the dosing.

VEHICLE (Water for injection)
- Amount of vehicle : 10 ml/kg/bw.
- Lot/batch no. : AAW4AB, Choongwae Pharma Corporation.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

Not applicable.

Details on mating procedure:

- Impregnation procedure: cohoused.

(If cohoused)
- M/F ratio per cage: 1:1.

- Length of cohabitation: 2 weeks.

- Proof of pregnancy: Not defined. The copulation was confirmed in the morning and afternoon by the fall of vaginal plug. The date when copulation was confirmed was set to gestation Day 0.

- Others: Gestation diagnosis and calcuation of delivery date: Individuals which did not deliver up to gestation Day 26 and showed the signs of gestation such as inflated mammary glands and abdominal enlargement were regarded as not pregnant. The final gestation was decided by the implantation signs in the uterus in the necropsy. Delivery signs were observed every day. When the delivery was confirmed up to the end of observation (5pm), the day was set on post partum Day 0. When delivery was made after the observation was over, the day after delivery day was set on post partum Day 0.

Duration of treatment / exposure:

(Males) from Day 0 to Day 42.
(Female) from Day 0 to Day 4 post-partum; females mated but not showing gestation signs up to gestation Day 26.

Frequency of treatment:

Once daily.

Duration of test:

IN-LIFE DATES:
- Males: From Day 0 To: Day 43.
- Female: From: Day 0 Up To: Day 5 post partum, (female mated but did not show gestation signs) Up To: gestation Day 27.
(Recovery group)
- Males: From: Day 0 To: Day 57.
- Females. From: Day 0 To: Day 69.

Dose / conc.:

125 mg/kg bw/day (nominal)

Dose / conc.:

250 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

No. of animals per sex per dose:

15 animals per sex per dose and the control group.
(Recovery group; 5 animals per sex at 500 mg/kg/day and control group.)

Control animals:

yes, concurrent vehicle

Maternal examinations:

- Dose selection rationale: Based on the results in the preliminary test (mortality took place at the dose level of 1000 mg/kg).

- Rationale for animal assignment (if not random): Based on the body weights measured at the end of the quarantine and purification period, rats were deployed as G1 - G2 - G3 - G4 - G4 - G3 - G2 - G1 - G1 - G1 - G3 - G4, where G1 of control, G2 125 mg/kg, G3 of 250 mg/kg and G4 of 500 mg/kg.

- Post-exposure recovery period in satellite groups: For two weeks.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No.
- Number of corpora lutea: Yes.
- Number of implantations: Yes.
- Number of early resorptions: No.
- Number of late resorptions: No.

Fetal examinations:

Birth rate, surviving rate, mortality on Day 0 and 4 post partum, body weight Day 0 and 4 post partum, external appearance and sex ratio were examined.

- External examinations: Yes.
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No

Statistics:

Levene's test. If significant in the one-way ANOVA due to homogeneous distribution, Dunnett's t-test was conducted. If the distribution was not homogeneous, the proper data transformation was conducted and the the Levene's test was repeated. If the distribution was homogeneous, the one-way ANOVA was conducted. In case of significance, Dunnett's t-test was repeated.

Indices:

- Mating

Copulation rate (%) = (number of female and male rats confirmed for copulation ÷ number of male rats used for mating)x100

Male impregnation rate (%) = (number of pregnant female rats ÷ number of female rats used for mating)x100

Female impregnation rate (%) = (number of pregnant female rats ÷ number of female rats confirmed for copulation)x100

Delivery rate (%) = (number of female rats that delivered live neonatas ÷ number of female rats confirmed for copulation)x100

- Measurement of corpora lutea and implantation

% pre – implantation loss = [1 – (number of implantation ÷ number of corpora lutea)] x 100

% post – implantation loss = [1 - (number of neonates – number of corpora lutea)] x 100

- Observation of neonates

Birth rate (%) = (Number of live neonates during delivery ÷ total number of neonates) x 100

Survival rate on Day 0post partum(
%) = (Number of live neonates on delivery date ÷Number of live neonates during delivery) x 100

Survival rate on Day 4post partum(%) = (Number of live neonates on Day 4post partum÷Number of live neonates on Day 0post partum) x 100

Sex ratio on Day 0post partum=(Number of survived male neonates on Day 0post partum÷ number of survived female neonates on Day 0post partum) x 100

Sex ratio on Day 4post partum=(Number of survived male neonates on Day 4post partum÷ number of survived female neonates on Day 4post partum) x 100

Historical control data:

Not reported.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males: Changes in body weight, at the dose of 500 mg/kg

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

effects observed, treatment-related

Description (incidence and severity):

Males: Changes in water consumption at the dose of 500 mg/kg

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Males: Changes in organ weight at the dose of 250 mg/kg
Females: Changes in organ weights were observed at the dose of 500 mg/kg

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Males/Females: Changes in histopathology at the dose of 500 mg/kg

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Unless stated, there were no effects in recovery group.

MORTALITY
There was no male mortality. Three females were found dead at 500 mg/kg; one in the main group took place on Day 38 and two in the recovery group on Day 46 and 51, respectively.

CLINICAL SIGNS
See Table 2-1 for the results. Blackish stool was found in all animals at all dose groups. Salivation was observed in 13 males and females, and all animals at the dose of 250 mg/kg and above. Soft stool was observed in male at all dose groups and in female at the dose of 500 mg/kg. Diarrhea was found in one female at the dose of 125 mg/kg, three females at 500 mg/kg and ten males at 500 mg/kg. Decrease in locomotion activity was observed in one female at the dose of 250 mg/kg. Paleness was observed in one male and three females, emaciation was observed in one male and one female, soiled perineal region in one female at the dose of 500 mg/kg.

BODY WEIGHT
See Tables 2-1 for the results. A significant decrease in body weight was observed in male at the dose of 250 mg/kg and above during the dosing period. This was observed in the recovery group at the dose of 500 mg/kg. Females showed a significant decrease in body weights at the dose groups of 125 and 500 mg/kg.

FOOD CONSUMPTION
There were no treatment-related effects on food consumption in male, however, there was a significant decrease in food consumption in female at the dose of 125 and 500 mg/kg on gestation day 0. Males in the recovery group at 500 mg/kg showed significant increase in food consumption on Day 3.

WATER CONSUMPTION
See Table 5-1 for the results.There was significant increase in water consumption at 500 mg/kg in both sexes.

HAEMATOLOGY
In males, there was a significant increase of MCV at the dose of 500 mg/kg. There was a significant increase of eosinophil values at the dose of 500 mg/kg recovery group.
In females, there was a significant increase in the number of platelet at the dose of 500 mg/kg in recovery group.

CLINICAL CHEMISTRY
In males, there was a significant decrease of cholinesterase at the dose of 250 mg/kg and above. In the male recovery group, a significant increase of triglyceride was observed at the dose of 500 mg/kg.

URINALYSIS
No treatment-related changes were detected in the urinary parameters examined.

NEUROBEHAVIOUR

ORGAN WEIGHTS
See Tables 12 and 13 for the results.In males, there was a significant increase in absolute and relative liver weight at the doses of 250 mg/kg and above. There was a significant increase in absolute and relative adrenal weight at 500 mg/kg, relative adrenal weight at 250 mg/kg and decrease in relative adrenal weight at 125 mg/kg.
In females, there was a significant increase in absolute and relative liver weight at the dose of 500 mg/kg and a significant decrease in absolute and relative thymus weight at the dose of 500 mg/kg, and absolute thumus weight at 125 mg/kg.
No significant effects of treatment were detected in the male sex organ weights.

GROSS PATHOLOGY
Diaphragmatic nodule in the liver was observed in both sexes in the dose groups and the control group (in males only). Diffuse black coloured liver was observed in all males, haemorrhage with diffuse black pigmentation in the stomach in 12 males, and unilateral atrophy in the testes in one male at the dose of 500 mg/kg. Mass of mesenteric lymphoid nodule was observed in one female at the dose of 500 mg/kg.
(Recovery group) Diaphragmatic nodule was observed in one male at the dose of 500 mg/kg, and in two females in the control group.
(Necropsy of three females found dead) A severe diffuse haemorrhagic grandular stomach was observed in the necropsy in one female found dead on Day 38. Severe distension of stomach were observed in two females dead in recovery group, but during the dosing period, therefore these findings were not considered as of recovery period.

HISTOPATHOLOGY: NON-NEOPLASTIC
See Tables 16-1 and 16-2 for the results.
(Males)
Adrenals: The following effects were observed in male adrenals at the dose of 500 mg/kg; unilateral hyperplasia of zone fasciculate in the adrenal cortex in 4 males, bilateral hyperplasia in hyperplasia in 4 males, and unilateral focal necrosis of adrenal cortex in one male.
Liver: Minimal diffuse haemosiderin deposit of liver parenchymal in 2 males, mild diffuse haemosiderin deposit in 8 males, moderate diffuse haemosiderin deposit in 4 males with one severe case were observed at the dose of 500 mg/kg.
Stomach: Moderate diffuse haemosiderin deposit in the granular in 9 males with 2 severe cases, moderate diffuse cellular infiltration (neutrophil) in submucosa in 3 males, mild hyperkeratosis of forestomach in 7 males, moderate hyperkeratosis of forestomach in one male with severe cases in 3 males.
(Females)
Minimal diffuse haemosiderin deposite in liver parenchymal was observed in one female at the dose of 250 mg/kg and above.
The following non treatment-related findings were observed; bilateral hyperplasia of zone fasciculate in the adrenal cortex in 4 females, bilateral hyperplasia of zone reticulosa in one female, minimal focal haemosiderin deposit of liver parenchymal in one female, mild diffuse haemosiderin deposit of liver parenchymal in one female, moderate multifocal haemosiderin deposit of liver parenchymal in mesenteric lymphnode in one female.
(Recovery group) Diffuse congestion of the adrenal were observed in 2 females. Hyperkeratosis of the forestomach, atrophy of the granularminimally focal haemosiderin deposit of liver parenchymal and focal oval cell proliferation were observed in one male, respectively.

CORPORA LUTEA AND IMPLANTATION
Pre-implantation rates were 14.4% in the control group, 9.4% at 125 mg/kg, 14.3% at 250 mg/kg and 9.8% at 500 mg/kg. Post-implantation loss rates were 6.0% for both the control group and the dose group of 125 mg/kg, 3.1% at 250 mg/kg and 7.0% at 500 mg/kg.

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity in males

Effect level:

125 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

organ weights and organ / body weight ratios

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity in females

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental toxicity in parents and offspring

Effect level:

>= 500 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks on result:

not determinable due to adverse toxic effects at highest dose / concentration tested

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity in males

Effect level:

55 mg/kg bw/day (nominal)

Based on:

element

Remarks:

Fe

Basis for effect level:

body weight and weight gain

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity in females

Effect level:

110 mg/kg bw/day (nominal)

Based on:

element

Remarks:

Fe

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental toxicity in parents and offspring

Effect level:

220 mg/kg bw/day (nominal)

Based on:

element

Remarks:

Fe

Remarks on result:

not determinable due to adverse toxic effects at highest dose / concentration tested

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No treatment-related effects were observed on mean live neonates, birth rates, survival rates and sex ratios on days 0 and 4 post partum. The only abnormalilty found in the external appearance examinations is an acaudate was observed in one neonate at 500 mg/kg. Crown Rump Length (CRL) of female neonates showed a significant decrease at 125 mg/kg on Day 4 post partum.

Dose descriptor:

NOAEL

Remarks:

Developmental toxicity

Effect level:

>= 500 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks on result:

not determinable due to adverse toxic effects at highest dose / concentration tested

Abnormalities:

not specified

Key result

Developmental effects observed:

no

Conclusions:

The oral administration of the test material to rats by gavage resulted in changes in body weight, water consumption, organ weight and histopathology in males at the dose of 500 mg/kg and changes in organ weight at the dose of 250 mg/kg. Therefore 125 mg/kg/day can be considered the No Observed Adverse Effect Level (NOAEL) in male rats. In females, changes in organ weights and histopathology were observed at the dose of 500 mg/kg. Therefore 250 mg/kg/day can be considered the NOAEL in female rats. The changes except body weight changes in males seemed to be reversible.
There was no treatment-related effects on reproductive functions in parental animals and development of neonates at any doses tested. the NOAEL for reproduction and developmental toxicity was considered to be ≥500 mg/kg/day.

Executive summary:

The test was intended to evaluate NOAEL (No Observed Adverse Effect Level) on reproduction process such as mating, conception, gestation, childbirth and development of neonates and the effect on the whole body including nerve and immune systems when female rats were orally dosed with Iron dichloride (CAS No.7758-94-3) once a day up topost partumday 4 from two weeks prior to the mating and male rats were orally dosed once a day with iron dichloride till two weeks before and after the mating.

 

Male and female SD rats were dosed with the test substance (0 (Control group), 125, 250 and 500 mg/kg/day) from two weeks before mating. Male SD rates were dosed once a day till two weeks after mating while female SD rats were dosed once a day up topost partumday 4. A total of 42 doses were provided for male rats while female rates had 42 to 54 dosages depending on mating and delivery of individuals. Clinical signs and mortality were observed and body weight and food and water consumption were measured. In the necropsy, gross examination of organs and tests on corpus luteum graviditatis and implantation rates were conducted. In addition, tests for sensory and motor functions, urinalysis and hematological and blood chemical tests were given and organ weights were measured for five individuals randomly selected from each group. External abnormalities, sex ratio, body weights, CRL (Crown Rump Length) and survival rate were observed onpost partumdays 0 and 4. 

 

During the observation period, the main group dosed with the substance showed signs such as melaena (black stool) and salivation but these signs were observed to disappear after dosing in the recovery group. There was no mortality in male SD rats, but three mortalities took place in female individuals at 500 mg/kg/day. The cause for mortalities was presumably the gastrointestinal damage by the substance. It was found that male individuals were more sensitive to body weight and food consumption than female counterparts. The change by the test substance was not recognized in mating data, sensory functions, motor functions, urine analysis and blood test. Gastric hemorrhage with blackened liver and black pigmentation of liver discovered in the necropsy findings was presumed to be caused by the test substance, but it was found to improve for the recovery period of two weeks. Weight changes in the liver and adrenal were observed in the absolute and relative organ weights of male individuals at 250 and 500 mg/kg and female individuals at 500 mg/kg. The histopathological test found parenchymal hemosiderosis and hyperplasia of adrenocortical zona fasciculate as well. It was found that the substance had no effect on birth rate, survival rate, body weight and CRL of neonates.

 

As a result of the test using iron dichloride (CAS No.7758-94-3), the NOAEL of repeated doses to male and female SD rats were 125 and 250 mg/kg/day, respectively. As there was no difference observed in reproductive functions of male and female SD rates and development of neonates between the control group and main group, NOAEL was thought to be 500 mg/kg/day. The effect of the test substance was none on reproductive functions, sensory functions, motor functions, urinalysis, and hematological and blood chemical findings which showed no differences between the control group and main group. Except the body weight change of male SD rats, clinical signs, water consumption, organ weights, necropsy findings and histopathological findings were reversible as they recovered after the dosing was over.   

Cross-reference 2

Reason / purpose for cross-reference:

read-across: supporting information

Reference

Iron can only be absorbed orally as the ferrous ion. Iron absorption in the rat is higher than humans. The presence of non-complexed iron in the diet rarely results in iron overload conditions. 
There are no reliable acute or repeated dose dermal studies that can be consulted for evidence of absorption via dermal route.
The water soluble inorganic iron salts do not undergo metabolism.
Iron can be inhaled as the ferric or ferrous ion. Ferric and ferrous ions precipitate in lysosomes of alveolar cells. However, it is not clear how fast the clearance of iron particles from pulmonary tissues is. 
Iron is uniformly distributed via blood. Greatest concentrations are in liver, bone marrow and spleen.
If there is an excess of the element within the body, there is no biochemical mechanism for its excretion and this may result in both severe and chronic symptoms if large amounts are ingested. About 1 mg of iron is lost each day through sloughing of cells from skin and mucosal surfaces, including the lining of the gastrointestinal tract. Only 0.01 to 0.02 % of the resorbed iron in humans are excreted daily. The daily losses of iron from the human body correspond to a biological half-time of iron of 10 to 20 years. 
The foetus is protected from the effects of excess iron in the mother.

Bioaccumulation potential:

low bioaccumulation potential

Justification for read-across

This endpoint is covered by the category approach for soluble iron salts (please see below for the category justification/report format).

In addition to the category members, the surrogate material ammonium iron(III) citrate was used for read-across (McCance & Widdowson 1938). This substance also dissociates at physiological pH to the same species produced by the ferric (iron III) salts. As discussed in the category justification section, the equilibrium to iron II species is quickly established. In result iron species burden is assumed to be comparable to equimolar exposure to the iron category member salts. The additional ammonium and citrate are considered not influencing the iron kinetics.

Introduction

Iron is an essential element, and plays an important role in biological processes, and iron homeostasis (biochemical mechanisms maintaining constant concentration in the cell) is under strict control (McCance & Widdowson 1938). Absorption, storage, mobilisation and excretion of iron are all regulated at the surface of cells by a homeostatic mechanism (Hostynek 1993). The counter ions of the soluble inorganic iron salts in question enter the body’s normal homeostatic processes, and are not discussed further.

 

Absorption

Oral

In humans the absorbance and uptake of iron salts from the digestive system is usually rather poor to the extent that treatment of simple anaemia by such means is of limited effectiveness. This is because iron can only be absorbed as the ferrous ion, but the ferrous ion can only exist in an acid medium. Therefore once in the small intestine the ferrous ion cannot exist. Iron absorption in the rat is higher than humans (Mahoney & Hendricks 1984); consequently, rat studies are considered unreliable models for iron toxicology in humans. Uptake is facilitated by the formation of iron chelates such as those with citrate and ascorbate that are present in the diet and in their absence iron absorption by the small intestine is very poor. Additionally, the presence of appreciable amounts of plant tannins may complex iron and further prevents its absorption. The result of this low solubility and low uptake by the human gut means that for healthy individuals, the presence of non-complexed iron in the diet rarely results in iron overload conditions.

There is some evidence that water-soluble iron salts are better absorbed than water-insoluble iron compounds. In both humans and animals, iron absorption from the digestive tract is adjusted to a fine homeostasis with low iron stores resulting in increased absorption and, alternately, sufficient body stores of iron decreasing absorption (Elinder 1986).

Significant differences in iron absorption from salts and food have been noted between rats and humans, with uptake significantly higher from identical meals in rats (Reddy and Cook, 1991), although rats poorly absorb haem (Bjorn-Rassmussen 1974). Dietary enhancers and inhibitors appear to affect non-haem iron absorption in humans to a greater extent than in rats (Reddy & Cook 1991). Growth requirements for iron in the rat are greater, and the dietary intake is about 100 times greater than that of humans, expressed on a body weight basis (WHO 1983).

EFSA (2012) concludes for FeSO4 on rapid absorption (10 % up to 60 % in case of iron deficiency) within 2 to 6 hours.

Dermal

The water solubility (estimated at 300 g/L) of ferric chloride sulphate suggests that it is unlikely to be absorbed across the lipid-rich stratum corneum. However, there are no reports of percutaneous absorption of iron in non-chelated form to support this prediction. Percutaneous absorption of iron has been reported only for chelated forms administered as ointments in mice (Hostynek 1993). There are no reliable acute or repeated dose dermal studies that can be consulted for evidence of absorption via the dermal route.

Inhalation

In contrast to the wealth of data available on the human toxicology of ingested iron salts, there is only one available study (Johansson et al. 1992) on the potential for adverse health effects via inhalation. In this 2-month repeated dose inhalation study in rabbit, only local pulmonary effects were investigated. Here, macrophages were affected due to ferric chloride (FeCl3). Alveolar macrophages were increased in number in both exposed groups. There were prominent changes in the macrophages such as enlarged Lysosomes containing fibrous-looking structures or iron-rich inclusions leading to accumulation of iron. Since the lungs have a neutral pH of approximately 7.4, it is assumed that ferrous ion following a progressive oxidation in the presence of oxygen will transform to insoluble Fe(OH)3. Since a recovery group in Johansson (1992) study was not investigated, no information is available in regard to reversibility of occurring effects and the clearance of iron particles. Elinder (1986) indicated that yearly lung clearance of iron dust in humans is estimated to be 20 - 40 % of the deposited amount (data obtained from iron welders). This reference could be a hint that clearance of deposited iron in oxidized form from the lungs is relatively slow and possibly not complete.

Distribution

The average adult human stores about 1 to 3 grams of iron in the body. Iron is almost never found in the free ionic state in living cells in appreciable concentrations; it is chaperoned in the form of protein complexes immediately it is absorbed from the diet. In the blood plasma it is transported (as FeIII) by the protein transferrin, which passes it on to dividing cells, particularly the cells in the bone marrow that are the precursors of the red blood cells. This is mediated by the transferrin receptor. Transferrin, which binds iron with high affinity is only 20-35 % saturated, thus the concentration of unbound iron is very low (0.5–1.5 mg/L or 9–27 μmol/L), Tenenbein 2001). Iron is stored principally in the liver in the large proteins haemosiderin and ferretin, although these are also found in all cells and in the blood in lower concentrations. Ferritin exists as hollow spheres of 24 protein subunits and iron is taken up in the FeII state but stored as FeIII. As with transferrin, it is stored in a redox-inactive (and therefore non-toxic) form. Ferritin is also important in recycling iron within the body and is an important biological indicator of iron balance. One consequence of the parsimonious conservation of iron is that if there is an excess of the element within the body, there is no biochemical mechanism for its excretion and this may result in both severe and chronic symptoms if large amounts are ingested.

Foetal exposure

It has been found that extremely elevated maternal serum iron concentrations are not accompanied by corresponding increases in foetal serum iron levels (Curryet et al.1990). This finding suggests that the foetus is protected from the effects of excess iron in the mother.

Metabolism

These water soluble inorganic iron salts do not undergo metabolism per se. As already mentioned iron is bound to transferrin for transport to the bone marrow or contained within storage forms.

Excretion

About 1 mg of iron is lost each day through sloughing of cells from skin and mucosal surfaces, including the lining of the gastrointestinal tract (EVM 2003). Menstruation increases the average daily iron loss to about 2 mg per day in pre-menopausal female adults (Bothwell & Charlton, 1982). No physiological mechanism of iron excretion exists. Consequently, absorption alone regulates body iron stores (McCance and Widdowson 1938).

The daily losses of iron from the human body correspond to a biological half-time of iron of 10 to 20 years. The yearly lung clearance of iron dust is estimated to be 20-40 % of the deposited amount (data obtained from iron welders) (Elinder 1986).

Additional references of this section, not entered as studies

• Bjorn-Rassmussen et al. (1974). Food iron absorption in man. Applications of the two-pool extrinsic tag method to measure heme and nonheme iron absorption form the whole diet. J. Clin. Invest. 53:247-55.
• Bothwell and Charlton (1982). A general approach of the problems of iron deficiency and iron overload in the population at large. Seminars in Hematology 19, 54.
• Curryet et al (1990). An ovine model of maternal iron poisoning in pregnancy. Ann. Emerg. Med 19:632-38.
• EFSA European Food Safety Authority (2012). Conclusion on Pesticide Peer Review. Conclusion on the peer review of the pesticide risk assessment of the active substance iron sulphate. Self-published, Parma, Italy. EFSA Journal 10(1):2521. 48 p.
• Elinder (1986) Iron. IN: Friberg L, Nordberg GF, Vouk VB, eds., 1986. Handbook on the toxicology of metals. 2nd ed., the: Elsevier, 277-297 (Vol II).
• EVM Expert Group on Vitamins and Minerals (2003). Safe upper levels for vitamins and minerals. Report of the Expert Group on Vitamins and Minerals. ISBN 1-904026-11-7 Self-published in May by the Food Standards Agency, U.K. 360 p. http://cot.food.gov.uk/pdfs/vitmin2003.pdf
• Hostynek (1993). Metals and the Skin. Critical Reviews in Toxicology 23(2):171-235
• Johansson A, Curstedt T, Rasool O, Jarstrand C, Camner P (1992). Macrophage Reaction in Rabbit Lung following Inhalation of Iron Chloride. PMID 1597169 Environ Res 58(1):66-79.
• Mahoney AW, Hendricks DG (1984). Potential of the rat as a model for predicting iron bioavailability for humans. DOI 10.1016/S0271-5317(84)80067-6 Nutrition Res. 4(5):913-22.
• McCance RA,Widdowson EM (1938). The absorption and excretion of iron following oral and intravenous administration. DOI 10.1113/jphysiol.1938.sp003669 PMID 16995028 J. Phys. 94(1):148 -54. URL http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1393919/pdf/jphysiol01545-0174.pdf
• Reddy MB, Cook JD (1991). Assessment of dietary determinants of nonheme-iron absorption in humans and rats. PMID 1654740 Am. J. Clin. Nutr. 54(4):723-8. URL ajcn.nutrition.org/content/54/4/723.full.pdf
• Tenenbein M (2001). Hepatotoxicity in Acute Iron Poisoning. PMID 11778670 Clin. Toxicol. 39(7):721-6.
• WHO (1983) 571. Iron. Toxicological evaluation of certain food additives and contaminants. WHO Food Additives Series, No. 18, 1983, nos 554-573 on INCHEM URL http://www.inchem.org/documents/jecfa/jecmono/v18je18.htm

Chemical Category Reporting Format according to ECHA Guidance

The following definition complies with the ECHA (2008, chapter R.6) Guidance on QSARs and grouping of chemicals. It should be used in the discussions of the IUCLID 5 section 7 (Toxicology).

Table: Reporting Format for the Chemical Category According to ECHA Guidance R.6.2.6.2

1.	Category definition and its members: Dissociating, inorganic and non-toxic iron compounds
1.1.	Category Definition
a.	Category Hypothesis In solid form iron element exists free, or in iron-containing compounds. In aqueous solution, it exists in one or two oxidation states, Fe2+, the ferrous form, and Fe3+, the ferric form. Many of the key biological functions of iron in living systems rely on the high redox potential, enabling rapid conversion between the Fe2+ and Fe3+ forms. The redox potential is relatively harmful in terms of the capacity for oxidative damage to cellular compound such as fatty acids, proteins and nucleic acids. However, iron within the body is normally bound to carrier proteins and/or molecules with antioxidant properties, which minimise the capacity of the free ion to cause oxidative stress (EVM 2003). In the first assumption, due to the neutral pulmonary pH (of about 7.4) the iron ions will precipitate in hydroxide or oxide of ferrous or ferric form and undergo rapid oxidation to ferric form. Therefore iron salts will act as identical material for the both iron oxidation forms. In the second assumption, independently of exposure route, the anions of iron salts are irrelevant for the toxicity: this category covers as well the anions of inorganic ferrous and ferric salts, i.e. chloride, sulphate and their crystalohydrate forms. All of these salts will dissociate immediately in contact with aqueous media to the respective anions and kations, and then be subject to further change of oxidation and speciated state according to the conditions. In the third assumption, the local effects could be slightly different depending on iron oxidation forms, but it should be not relevant in the regulatory context. In the fourth assumption, bioavailability of iron is regulated by homeostasis mechanisms. It is also known that the ferrous ion (Fe2+) has a higher oral bioavailability than the ferric ion (Fe3+) that could impair the category approach. However, this difference is not that significant to be relevant in the regulatory context. Regarding the long-term exposure, a tolerance development could be assumed. Therefore it is not necessary to make any differentiation between category members and read-across between the salts can be used freely for the toxicological property data sets.
b.	Applicability Domain of the category This category comprises five soluble iron salts (ferric and ferrous chloride and ferrous and ferric sulphate and ferric chloride sulphate, including their various hydrated forms).
c.	List of endpoints covered (numbers refer to the IUCLID 5 sections) 7. Toxicological information 7.1 Toxicokinetics, metabolism and distribution 7.2 Acute toxicity 7.3 Irritation / corrosion (limitation: differently as in this category, FeSO4 is irritant for eye) 7.4 Sensitization 7.5 Repeated dose toxicity 7.6 Genetic toxicity 7.7 Carcinogenicity 7.8 Toxicity to reproduction
1.2	Category Members apart from Hydronium jarosite (EC 940-441-4)
	Common name	EC number				CAS number
	Ferric chloride	231-729-4				7705-08-0
	Ferrous chloride	231-843-4				7758-94-3
	Ferric sulphate	233-072-9				10028-22-5
	Ferrous sulphate	231-753-5				7720-78-7
	Ferric chloride sulphate	235-649-0				12410-14-9
	The definition of these salts also covers a number of chemical identities relating to specific hydrates. Under REACH all hydrates are covered by the registration of the anhydrous salt. As the hydrates influence the molecular weight, correction should apply as the hydrate weight is considered in CLP according to ECHA Guidance on the Application of the CLP Criteria Version 2.0 (2012, p 535, Example D). The following hydrates are identified in the public domain though not all are necessarily commercially relevant:
	Chemical name (CAS name)		CAS number		Molecular formula
	Iron chloride (FeCl3), monohydrate (9CI)		60684-13-1		Cl3 Fe . H2O
	Iron chloride (FeCl3), hydrate (2:3) (9CI)		58694-76-1		Cl3 Fe . 3/2 H2O
	Iron chloride (FeCl3), dihydrate (9CI)		54862-84-9		Cl3 Fe . 2 H2O
	Iron chloride (FeCl3), trihydrate (9CI)		58694-75-0		Cl3 Fe . 3 H2O
	Iron chloride (FeCl3), hexahydrate (8CI, 9CI)		10025-77-1		Cl3 Fe . 6 H2O
	Iron chloride (FeCl3), nonahydrate (9CI)		58694-79-4		Cl3 Fe . 9 H2O
	Iron chloride (FeCl3), dodecahydrate (9CI)		58694-80-7		Cl3 Fe . 12 H2O
	Iron chloride (FeCl3), hydrate (8CI, 9CI)		24290-40-2		Cl3 Fe . x H2O
	Iron chloride (FeCl2), hydrate		23838-02-0		Cl2 Fe . x H2O
	Iron chloride (FeCl2), monohydrate		20049-66-5		Cl2 Fe . H2O
	Iron chloride (FeCl2), dihydrate		16399-77-2		Cl2 Fe . 2 H2O
	Iron chloride (FeCl2), tetrahydrate		13478-10-9		Cl2 Fe . 4 H2O
	Iron chloride (FeCl2), hexahydrate		18990-23-3		Cl2 Fe . 6 H2O
	Sulphuric acid, iron(3+) salt (3:2), nonahydrate		13520-56-4		Fe2(SO4)3.9H2O
	Sulphuric acid, iron(3+) salt (3:2), hydrate		15244-10-7		Fe2(SO4)3.xH2O
	Sulphuric acid, iron(3+) salt (3:2), hexahydrate		13761-89-2		Fe . 3/2 H2SO4 . 3 H2O
	Sulphuric acid, iron(3+) salt (3:2), heptahydrate		35139-28-7		Fe . 3/2 H2SO4 . 7/2 H2O
	Sulphuric acid, iron(3+) salt (3:2), tetrahydrate		230310-51-7		Fe . 3/2 H2SO4 . 2 H2O
	Sulphuric acid, iron(2+) salt (1:1), hexahydrate		59261-48-2		Fe . H2SO4 . 6 H2O
	Sulphuric acid, iron(2+) salt (1:1), trihydrate		58694-83-0		Fe . H2SO4 . 3 H2O
	Sulphuric acid, iron(2+) salt (1:1), tetrahydrate		20908-72-9		Fe . H2SO4 . 4 H2O
	Sulphuric acid, iron(2+) salt (1:1), monohydrate		17375-41-6		Fe . H2SO4 . H2O
	Sulphuric acid, iron(2+) salt (1:1), hydrate		13463-43-9		Fe . H2SO4 . x H2O
	Sulphuric acid, iron(2+) salt (1:1), dihydrate		10028-21-4		Fe . H2SO4 . 2 H2O
	Sulphuric acid, iron(2+) salt (1:1), heptahydrate		7782-63-0		Fe . H2SO4 . 7 H2O
1.3	Purity / Impurities Iron salts are not toxic to the population (cut off category 4 and upwards) and generally only substances with comparable purity or impurity profiles can be assessed together. Technical grade materials may contain relevant quantities of impurities, which exceed the low toxicological effects of the pure compounds comprised in this category. In cases where the sum of impurities triggers the toxicity, assessment has to be based on these impurities considering their concentration and the category assessment is restricted to the iron salt components. The purity of the category members is generally > 80 % w/w. Deviations below this purity value would normally not be acceptable since this would suggest that the substance should not be considered as a mono-constituent substance. Impurities comprise salts of other metals up to ≤ 1 % w/w and free acid up to < 10 % w/w.
2.	Category justification The iron salts category comprises a directly analogous group of iron Fe3+and Fe2+salts, with counter-ions (chloride and sulphate) which are ubiquitous and do not require additional consideration. The formation of a category is justified on the basis of a self-consistent model of the behaviour and properties of these substances. All of these salts will dissociate immediately in aqueous media to the respective anions and kations. The chloride and sulphate anions are of no further interest since they are already ubiquitous in vivo and do not represent health hazard. The ferric and ferrous ions will inter-convert rapidly according to the in vivo conditions that they are found in, and if one ion is introduced into a system, any effects observed may be due to that ion, the other oxidation state, or a mixture of the two. Although each form of iron could be assessed for a particular endpoint and the test substance well-characterised, as a consequence of the inter-conversion it is not always possible to determine the form of the iron responsible for a particular endpoint. In general, under oxygenated conditions, ferrous will be converted to ferric, and in the presence of water ferric hydroxides will precipitate initially. Even where the form of the iron present when the endpoint is reached can be determined there will be uncertainty regarding the species responsible, unless the toxicity can be followed as a function of this species, which means that a dose response-relationship can be established. The majority of toxicological endpoints are covered with experimental tests showing similar mode of action between iron oxidation forms and salts having different counter-ions. Therefore it is logical to consider these five salts together within a single chemical category. Where a data gap may exist for an individual salt, it is considered that relevant data for one or more of the other salts are an acceptable surrogate for the missing data, taking due account of the oxidation state present initially. The iron content of a given salt or solution may be used to convert or compare data.
3.	Data matrix The category approach was evaluated basing on the following experimental data for the acute oral toxicity endpoint. Studies are generally considered reliable or represent the sole data point available.
	Test substance	Identifier of the study		Animal species	LD50 [mg/kg bw] based on test substance	LD50 [mg Fe/kg bw] based on Fe
	FeCl2	Choi 2004a		rat	500 (300 – 2000, toxic classes tested)	220 (132-881, toxic classes tested)
	FeSO4.7H2O	MHLW 2002		rat	> 2000	> 401
	FeSO4	Anon 2000		rat	3200	643
	FeSO4	Parent 2000		rat	3200	1176
	FeSO4	Weaver 1961		rat	2625 (2323-2966, 95% CV)	964 (854-1090, 95% CV)
	FeSO4	Weaver 1961		mouse	1025 (802-1311, 95% CV)	377 (295-482, 95% CV)
	FeSO4	Boccio 1998		mouse	670 (females) 680 (males)	246 (females) 250 (males)
	FeCl3	Hosking 1970		mouse, female	1278 (871-1830, 95% CV)	440 (300-630, 95% CV)
	Fe2(SO4)3	ICI 1991		rat	500-2000 (females) >2000 (males)	140-559 (females) >559 males
	The category approach was evaluated basing on the following experimental data for the skin irritation / corrosion endpoint. Studies are generally considered reliable or represent the sole data point available.
	Test substance	Identifier of the study		Test method & animal species		Result
	FeSO4.7H2O	Clouzeau 1994		Skin irritation / corrosion in vivo, rabbit		Irritating, Category 2
	FeCl2	Park 2004		Skin irritation / corrosion in vivo, rabbit		Not irritating
	FeCl3	BASF 1977		Skin irritation / corrosion in vivo, rabbit		Irritating, Category 2
	The category approach was evaluated based on the following experimental data for eye irritation / corrosion. Studies are generally considered as reliable or represent the sole data point available. In the study of Bayer AG (1992) FeSO4. 7H2O is classified as non-irritating, however, according to Draft Assessment Report for Iron Sulphate (September 2008), FeSO4 is classified as irritating to eyes (R36). Therefore, FeSO4 should be adopted as irritant to eyes.
	Test substance	Identifier of the study		Test method & animal species	Result
	FeCl2	Jeong 2004		Eye irritation / corrosion in vivo, rabbit	Corrosive to rabbit eye, Category 1
	FeSO4.7H2O	Bayer AG 1992		Eye irritation in vivo, rabbit	(Not irritating) Irritating
	FeCl3water free solid	BASF 1977		Eye irritation / corrosion in vivo, rabbit	Corrosive to rabbit eye, Category 1
	The category approach was evaluated basing on the following experimental data for the skin sensitisation.
	Test substance	Identifier of the study		Test method & animal species	Result
	FeSO4	Ikarashi 1992		Skin sensitisation in vivo	negative
	FeSO4	Stitzinger 2010		Skin sensitisation – LLNA, mouse	negative
	FeCl3	Storck 1962		Skin sensitisation in vivo, guinea pigs	Ambiguous, 1 of 2 guinea pigs were positive
	The category approach was evaluated based on the following experimental data for repeated dose toxicity. Studies are generally considered reliable or represent the sole data point available.
	Test substance	Identifier of the study		Test method & animal species	Result based on iron salt	Result based on iron
	FeCl2	Beom 2004		Repeated dose toxicity: oral – OECD 422, rat	NOAEL: 125 mg/kg bw in males, 250 mg/kg bw in females LOAEL: 250 mg/kg bw in males 500 mg/kg bw in females	NOAEL: 55.1 mg/kg bw in males, 110.1 mg/kg bw in females LOAEL: 110.1 mg/kg bw in males 220.5 mg/kg bw in females
	FeSO4.7H2O	Furuhashi 2002		Repeated dose toxicity: oral – OECD 422, rat	NOAEL: 100 mg/kg bw, LOAEL: 300 mg/kg bw for the test item; NOAEL: 54.6 mg/kg bw, LOAEL: 163.9 mg/kg bw for anhydrous FeSO4	NOAEL: 20.1 mg/kg bw LOAEL: 60.3 mg/kg bw
	FeCl3	Sato 1992		Repeated dose toxicity: oral, 90 d rat	NOAEL: 277 mg/kg bw in males, 341 mg/kg bw in females	NOAEL: 95.4 mg/kg bw in males, 117.4 mg/kg bw in females
	The category approach was evaluated based on the following experimental data for the genetic toxicity. Studies are generally considered reliable or represent the sole data point available.
	Test substance	Identifier of the study		Test method & animal species	Result
	FeCl2	Kim 2004		Bacterial mutagenicity – Ames test	negative with and without metabolic activation
	FeCl2	Ji Yoon 2004		In vivo cytogenicity – micronucleus, mouse	negative; 2, 5, 10, 20, 50, 100 and 200 mg/ml in the dose range-finder; 1.25, 2.5 and 5 mg/mL in the micronucleus experiment
	FeSO4	Bianchini 1988		In vivo micronuclei induction in GI tract, oral, mouse	negative
	FeCl3.6H2O	Dunkel 1999		Bacterial mutagenicity – Ames test	negative; not a bacterial mutagen, tested up to 10’000 µg/plate (equivalent to 6001 µg/plate anhydrous FeCl3)
	FeCl3	Dunkel 1999		Mammalian gene mutation – mouse lymphoma assay	negative up to 1030 µg Fe/mL -S9, up to 1.236 µg Fe/mL +S9; cytotoxicity at the highest tested concentrations
	FeCl3	Schulz 2009		In vitro chromosome aberration – micronucleus assay	negative with and without metabolic activation
	FeCl3	Bianchini 1988		In vivo micronuclei induction in GI-tract, oral, mouse	negative; dose related toxic effects were seen in colons of feeding animals and colon and stomach of fasting animals
	The category approach was evaluated based on the following experimental data for the carcinogenicity. Studies are generally considered reliable or represent the sole data point available.
	Test substance	Identifier of the study		Test method & species	Result
	FeCl3	Sato 1992		Similar to OECD 451, rat	no carcinogenic potential
	Iron supplementation	Ullen 1997		Epidemiological, human	Protective effect of iron
	The category approach was evaluated based on the following experimental data for the reproduction toxicity. Studies are generally considered reliable or represent the sole data point available.
	Test substance	Identifier of the study		Test method & animal species	Result based on iron salt	Result based on iron
	FeCl2	Beom 2004		Screening, oral – OECD 422, rat	NOAEL: ≥ 500 mg/kg bw/day	NOAEL: ≥ 220.5 mg/kg bw/day
	FeSO4.7H2O	Furuhashi 2002		Screening, oral – OECD 422, rat	NOAEL: ≥ 1000 mg/kg bw/day	NOAEL: ≥ 200.9 mg/kg bw/day
4.	Conclusions per endpoint for C&L, PBT/vPvB and dose descriptor Considering the acute oral toxicity, the LD50 values show marked variability (220 – 964 mg Fe/kg bw) without clear difference between Fe2+and Fe3+salts. Nevertheless all these iron salt forms clearly have LD50 values > 300 mg/kg bw, that according to CLP support a classification from category 4 to not categorized range and it could be one of the reason to handle the corresponding iron salts as one chemical category. Considering skin irritation, all here mentioned irons salts according their mode of action and following CLP, could be classified as skin irritating – category 2. Considering eye irritation / corrosion, iron salts could be classified as causing irreversible effects on the eye – category 1. An exception may be made for FeSO4, which is already listed in Annex I of the European Plant Protection Products Directive and classified into category 2 (eye irritant). Considering sensitization, in general iron salts are deemed to have a no potential to cause sensitisation that is relevant for classification and could be regarded as one chemical category. Considering repeated dose toxicity, in all reliable oral studies the LOAEL of iron salts is above 100 mg/kg bw (not categorized range according to CLP) and no clear difference between Fe2+and Fe3+is observed. The apparent effect of slightly lower or equal toxicity of FeCl3 in 90-day study is in line with the expectation of bioregulation, i.e. tolerance development. Therefore iron salts could be regarded as one chemical category. In the case of repeated inhalation toxicity, the comparability of the Fe2+and Fe3+oxidation states depends particularly on assumption 1.1.a. of the Category Hypothesis. The scientific evidence of this assumption should be relying on the brother basis of data then they become available. Considering genetic toxicity, iron salts show no genotoxic potential and could be regarded as one chemical category. Considering carcinogenicity, human and animal data together are in agreement that iron is not carcinogenic and accordingly no classification is necessary for the iron salts of this category. Considering toxicity to reproduction, only for FeCl2 and FeSO4 reliable studies (conducted according to OECD TG 422) are available. As the bioavailability of ferrous iron is assumed to be initially higher than that of ferric iron, a read across from these two substances to the ferric salts of this category is deemed valid and conservative. For both substances no adverse effects were seen at the highest tested dose levels where moderate to strong parental toxicity was already present. Based on the available data it can therefore be assumed that the iron salts of this category are not reproductive toxicants. Iron is a bioessential element and uptake of iron is highly regulated by organism, therefore no concerns exist regarding bioaccumulation.

• Anon. 2000. Acute toxicity data submission 96-95. Int J Toxicol 19: No 5. Available from the secondary source OECD (2007, table 145).
• EVM Expert Group on Vitamins and Minerals (2003). Safe upper levels for vitamins and minerals. Report of the Expert Group on Vitamins and Minerals. ISBN 1-904026-11-7 Self-published in May by the Food Standards Agency, U.K. 360 p. http://cot.food.gov.uk/pdfs/vitmin2003.pdf
• ICI (1991). Acute range oral toxicity study to the rat (CT20-126). Unpublished report. Testing laboratory: ICI Central Toxicology Laboratories. Report no.: Internal data Report No CTL/L/4392. Study number: AR5351. Report date: 1991-12-12. Available from the secondary source OECD (2007, table 145).
• OECD Organisation for Economic Co-operation and Development (2007). Chemical Category: Iron Salts. SIDS Initial Assessment Report for SIAM 24, held in Paris, France, 17-20 April. Self-published, Paris, France. 138 p.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

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

Test material information

Constituent 1

Reference substance name:

Ferrous chloride

IUPAC Name:

Ferrous chloride

Constituent 2

Reference substance name:

Iron dichloride

EC Number:

231-843-4

EC Name:

Iron dichloride

Cas Number:

7758-94-3

Molecular formula:

FeCl2

IUPAC Name:

iron(2+) dichloride

Details on test material:

- Name of test material : Iron dichloride.
- Substance type: Tan powder.
- Physical state: Solid.
- Analytical purity: 98%.
- Lot/batch No.: 14330TA.
- Stability under test conditions: Not applicable, analysis was not conducted.
- Storage condition of test material: 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: Sam: Strain of TacN(SD), Samtako Bio Korea, 77-1 Seorang-dong, Osan-si, Gyeonggido.

- Age at study initiation: 8 weeks old.

- Weight at study initiation: Male: 269.23 - 302.18 g., Female: 191.34 - 221.60 g.

- Fasting period before study: The day before the necropsy.

- Housing:
Facility used:Stainless steel wired cage (260W X 350L X 210H (mm), Daejong Lab), polycarbonate cage (260W X 420L X 180H (mm), MJLTD).
Pre-mating period: 1 - 2 individuals (separate female and male rats), in stainless steel wire cage.
Mating period: One female and one male in one cage, in stainless steel wire cage.
Gestation and nursing period (females): Females individually housed in polycarbonate cage.
Post-mating period (males): 1 - 2 individuals, in stainless steel wire cage.
recovery group: 1 - 2 individuals, in stainless steel wire cage.

- Diet : The lab rat feed 5057 (Agri Purina Korea, 627 Jangdang-dong, Pyeongtaek, Gyeonggido), ad libitum.Analysis of the diet met the criteria of the protocol.

- Water (e.g. ad libitum): Mains water, filtred and sterilised, ad libitum. Analysis of the water met the criteria of the drinking water quality (Ministry of Environment).

- Acclimation period: 7 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.7 - 23.0ºC.
- Humidity (%): 40.8 ± 69.1%.
- Air changes (per hr): 10 - 15 changes per hour, front ventilation.
- Photoperiod (hrs dark / hrs light): 12 hrs light (7am - 7pm) / 12 hrs dark. 150 - 300 lux was used.

IN-LIFE DATES:
- Males: From Day 0 To: Day 43.
- Female: From: Day 0 Up To: Day 5 post partum, (female mated but did not show gestation signs) Up To: gestation Day 27.
(Recovery group)
- Males: From: Day 0 To: Day 57.
- Females. From: Day 0 To: Day 69.

Administration / exposure

Route of administration:

oral: gavage

Type of inhalation exposure (if applicable):

other: not applicable

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The substance was suspended in the water for injection by dose and formulated just before the dosing.

VEHICLE (Water for injection)
- Amount of vehicle : 10 ml/kg/bw.
- Lot/batch no. : AAW4AB, Choongwae Pharma Corporation.

Details on mating procedure:

- M/F ratio per cage: 1:1.

- Length of cohabitation: 2 weeks.

- Proof of pregnancy: Not defined. The copulation was confirmed in the morning and afternoon by the fall of vaginal plug. The date when copulation was confirmed was set to gestation Day 0.

- After successful mating each pregnant female was caged : Individually housed in polycarbonate cage.

- Others: Individuals which did not deliver up to gestation Day 26 and showed the signs of gestation such as inflated mammary glands and abdominal enlargement were regarded as not pregnant. The final gestation was decided by the implantation signs in the uterus in the necropsy. Delivery signs were observed every day. When the delivery was confirmed up to the end of observation (5pm), the day was set on post partum Day 0. When delivery was made after the observation was over, the day after delivery day was set on post partum Day 0.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

Not applicable.

Duration of treatment / exposure:

(Males) From Day 0 To: Day 42.
(Female) From: Day 0 Up To: Day 4 post-partum, (female mated but did not show gestation signs) Up To: gestation Day 26.

Frequency of treatment:

Once daily.

Details on study schedule:

- Age at mating of the mated animals in the study: At least 10 weeks.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

125 mg/kg bw/day (nominal)

Remarks:

in water

Doses / concentrations 2

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

in water

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

in water

No. of animals per sex per dose:

15 animals per sex per dose and the control group.
(Recovery group; 5 animals per sex at 500 mg/kg/day and control group.)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on the results in the preliminary test (mortality took place at the dose level of 1000 mg/kg).

- Rationale for animal assignment (if not random): Based on the body weights measured at the end of the quarantine and purification period, rats were deployed as G1 - G2 - G3 - G4 - G4 - G3 - G2 - G1 - G1 - G1 - G3 - G4, where G1 of control, G2 125 mg/kg, G3 of 250 mg/kg and G4 of 500 mg/kg.

- Post-exposure recovery period in satellite groups: For two weeks.

Positive control:

No.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice a day (30 minutes after dosing, and just before the job was over on the day) during the test period.
- Cage side observations checked: Presence of dead animals or animals in critical condition.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once a day.

BODY WEIGHT: Yes
- Time schedule for examinations: Once daily except mating period.

FOOD CONSUMPTION : Yes
- Time schedule:
(male) The day before the dosing and once a week after the dosing started. After mating, food was supplied on the day of confirming copulation and the day before necropsy. The remains were measured the day after the food supply. Not measured during mating period.
(female) The day before the dosing and once a week after the dosing started. Food was supplied on gestation days 0, 6, 13 and 20 and on post partum days 0 and 3. The remains were measured the day after the food supply. Not measured during mating period.

FOOD EFFICIENCY: No.

WATER CONSUMPTION : Yes
- Time schedule for examinations:
(male) The day before the dosing and once a week after the dosing started. After mating, food was supplied on the day of confirming copulation and the day before necropsy. The remains were measured the day after the food supply. Not measured during mating period.
(female) The day before the dosing and once a week after the dosing started. Food was supplied on gestation days 0, 6, 13 and 20 and on post partum days 0 and 3. The remains were measured the day after the food supply. Not measured during mating period.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY / CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: For 18 hours the day before the necropsy.
- Anaesthetic used for blood collection: Yes (ether).
- Animals fasted: Yes
- How many animals: 5 males and 5 females of each group.
- Parameters checked:
- Haematology, paramerers examined: RBC, HGB, HCT, MH, MCV, MCH, MCHC, WBC, PLT, NEU, LYM, MONO, EOS, BASO, PT and APTT
- Clinical chemistry, paramerers examined: ALT, AST, Cs, Glu, BUN, Crea, T-Bil, TG, TP, Alb, A/G, P, Ca, Na, K and Cl.

URINALYSIS: Yes
- Time schedule for collection of urine: For 3 to 4 hours the day before the necropsy
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters checked: colour, pH, protein, erythrocyte, specific gravity, glucose and leukocyte.
A certain amount of fresh urine was collected from five males and five females of each group and the recovery group. Comvur10 Test M (Roche, Germany) was used for analysis.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: The day before necropsy. (motor functions and autonomic nervous system were observed once a day).
- Battery of functions tested: sensory activity and motor fuctions. The auricle reflex and corneal reflex tests were conducted on five males and five females randomly selected from each group and the recovery group to evaluate sensory functions. The traction test was conducted to evaluate motor function.

Oestrous cyclicity (parental animals):

No reported.

Sperm parameters (parental animals):

As a part of histopathological test of the gonad of male rats, sperm generation was examined.

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in offspring: birth rate, surviving rate, mortality on Day 0 and 4 post partum, body weight Day 0 and 4 post partum, external appearance and sex ratio were examined.

GROSS EXAMINATION OF DEAD PUPS: Not reported.

Postmortem examinations (parental animals):

GROSS PATHOLOGY: Yes
The day after the dosing was over (two weeks after the dosing was over for the recovery group), five rats were placed under ether anesthesia for external appearance examination. After blood collection from the dorsal aorta, the subjects were placed for bloodletting and killed. The organ tissues were observed with macroscopically; external surface and all orifices / cranial cavity and external surface of the brain / nasal cavity and paranasal simus / thoracic, abdominal and pelvic cavities and their viscera / cervical tissues and organs.
Necropsy was immediately performed on dead animals or animals in critical condition.

- Organ weights: Organs in tables 12 and 13 were removed from 5 males and 5 females of each group at the necropsy and weighed. Testes and epididymides were examined from all male rats.

HISTOPATHOLOGY: Yes
After a uniformly 3 mm thick tissues were removed from fixed tissues of 5 males and 5 females of the control and the dose group of 500 mg/kg, dead bodies of 500 mg/kg group, organs observed macroscopically for abnormality that showed abnormality in organ weight measurement, 1 4 μm histopathological section was stained with haematoxylin and eosin and observed wuith the optic microscope (Olympus BX50, Olympus Optical Co., Japan) for histopathological test. The gonads of male rats were examined for sperm generation and interstitial cells. See Tables 16-1 and 16-2 for the organs examined.

Postmortem examinations (offspring):

GROSS NECROPSY
- Gross necropsy consisted of external appearance including measurement of crown rump length.

HISTOPATHOLOGY / ORGAN WEIGTHS
Not performed.

Statistics:

Levene's test. If significant in the one-way ANOVA due to homogeneous distribution, Dunnett's t-test was conducted. If the distribution was not homogeneous, the proper data transformation was conducted and the the Levene's test was repeated. If the distribution was homogeneous, the one-way ANOVA was conducted. In case of significance, Dunnett's t-test was repeated.

Reproductive indices:

Copulation rate (%) = (number of female and male rats confirmed for copulation ÷ number of male rats used for mating)x100

Male impregnation rate (%) = (number of pregnant female rats ÷ number of female rats used for mating)x100

Female impregnation rate (%) = (number of pregnant female rats ÷ number of female rats confirmed for copulation)x100

Delivery rate (%) = (number of female rats that delivered live neonatas ÷ number of female rats confirmed for copulation)x100

- Measurement of corpora lutea and implantation

% pre – implantation loss = [1 – (number of implantation ÷ number of corpora lutea)] x 100

% post – implantation loss = [1 - (number of neonates – number of corpora lutea)] x 100

Offspring viability indices:

Birth rate (%) = (Number of live neonates during delivery ÷ total number of neonates) x 100

Survival rate on Day 0post partum(
%) = (Number of live neonates on delivery date ÷Number of live neonates during delivery) x 100

Survival rate on Day 4post partum(%) = (Number of live neonates on Day 4post partum÷Number of live neonates on Day 0post partum) x 100

Sex ratio on Day 0post partum=(Number of survived male neonates on Day 0post partum÷ number of survived female neonates on Day 0post partum) x 100

Sex ratio on Day 4post partum=(Number of survived male neonates on Day 4post partum÷ number of survived female neonates on Day 4post partum) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Mortality:

mortality observed, treatment-related

Description (incidence):

Three mortalities took place in female individuals at 500 mg/kg/day

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

males at 500 mg/kg/day

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

effects observed, treatment-related

Description (incidence and severity):

males at 500 mg/kg/day

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Males/females at 500 mg/kg/day

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Details on results (P0)

Unless stated, there were no effects in recovery group.

MORTALITY
There was no male mortality. Three females were found dead at 500 mg/kg; one in the main group took place on Day 38 and two in the recovery group on Day 46 and 51, respectively.

CLINICAL SIGNS
See Table 2-1 for the results. Blackish stool was found in all animals at all dose groups. Salivation was observed in 13 males and females, and all animals at the dose of 250 mg/kg and above. Soft stool was observed in male at all dose groups and in female at the dose of 500 mg/kg. Diarrhea was found in one female at the dose of 125 mg/kg, three females at 500 mg/kg and ten males at 500 mg/kg. Decrease in locomotion activity was observed in one female at the dose of 250 mg/kg. Paleness was observed in one male and three females, emaciation was observed in one male and one female, soiled perineal region in one female at the dose of 500 mg/kg.

BODY WEIGHT
See Tables 2-1 for the results. A significant decrease in body weight was observed in male at the dose of 250 mg/kg and above during the dosing period. This was observed in the recovery group at the dose of 500 mg/kg. Females showed a significant decrease in body weights at the dose groups of 125 and 500 mg/kg.

FOOD CONSUMPTION
There were no treatment-related effects on food consumption in male, however, there was a significant decrease in food consumption in female at the dose of 125 and 500 mg/kg on gestation day 0. Males in the recovery group at 500 mg/kg showed significant increase in food consumption on Day 3.

WATER CONSUMPTION
See Table 5-1 for the results.There was significant increase in water consumption at 500 mg/kg in both sexes.

HAEMATOLOGY
In males, there was a significant increase of MCV at the dose of 500 mg/kg. There was a significant increase of eosinophil values at the dose of 500 mg/kg recovery group.
In females, there was a significant increase in the number of platelet at the dose of 500 mg/kg in recovery group.

CLINICAL CHEMISTRY
In males, there was a significant decrease of cholinesterase at the dose of 250 mg/kg and above. In the male recovery group, a significant increase of triglyceride was observed at the dose of 500 mg/kg.

URINALYSIS
No treatment-related changes were detected in the urinary parameters examined.

NEUROBEHAVIOUR

ORGAN WEIGHTS
See Tables 12 and 13 for the results.In males, there was a significant increase in absolute and relative liver weight at the doses of 250 mg/kg and above. There was a significant increase in absolute and relative adrenal weight at 500 mg/kg, relative adrenal weight at 250 mg/kg and decrease in relative adrenal weight at 125 mg/kg.
In females, there was a significant increase in absolute and relative liver weight at the dose of 500 mg/kg and a significant decrease in absolute and relative thymus weight at the dose of 500 mg/kg, and absolute thumus weight at 125 mg/kg.
No significant effects of treatment were detected in the male sex organ weights.

GROSS PATHOLOGY
Diaphragmatic nodule in the liver was observed in both sexes in the dose groups and the control group (in males only). Diffuse black coloured liver was observed in all males, haemorrhage with diffuse black pigmentation in the stomach in 12 males, and unilateral atrophy in the testes in one male at the dose of 500 mg/kg. Mass of mesenteric lymphoid nodule was observed in one female at the dose of 500 mg/kg.
(Recovery group) Diaphragmatic nodule was observed in one male at the dose of 500 mg/kg, and in two females in the control group.
(Necropsy of three females found dead) A severe diffuse haemorrhagic grandular stomach was observed in the necropsy in one female found dead on Day 38. Severe distension of stomach were observed in two females dead in recovery group, but during the dosing period, therefore these findings were not considered as of recovery period.

HISTOPATHOLOGY: NON-NEOPLASTIC
See Tables 16-1 and 16-2 for the results.
(Males)
Adrenals: The following effects were observed in male adrenals at the dose of 500 mg/kg; unilateral hyperplasia of zone fasciculate in the adrenal cortex in 4 males, bilateral hyperplasia in hyperplasia in 4 males, and unilateral focal necrosis of adrenal cortex in one male.
Liver: Minimal diffuse haemosiderin deposit of liver parenchymal in 2 males, mild diffuse haemosiderin deposit in 8 males, moderate diffuse haemosiderin deposit in 4 males with one severe case were observed at the dose of 500 mg/kg.
Stomach: Moderate diffuse haemosiderin deposit in the granular in 9 males with 2 severe cases, moderate diffuse cellular infiltration (neutrophil) in submucosa in 3 males, mild hyperkeratosis of forestomach in 7 males, moderate hyperkeratosis of forestomach in one male with severe cases in 3 males.
(Females)
Minimal diffuse haemosiderin deposite in liver parenchymal was observed in one female at the dose of 250 mg/kg and above.
The following non treatment-related findings were observed; bilateral hyperplasia of zone fasciculate in the adrenal cortex in 4 females, bilateral hyperplasia of zone reticulosa in one female, minimal focal haemosiderin deposit of liver parenchymal in one female, mild diffuse haemosiderin deposit of liver parenchymal in one female, moderate multifocal haemosiderin deposit of liver parenchymal in mesenteric lymphnode in one female.
(Recovery group) Diffuse congestion of the adrenal were observed in 2 females. Hyperkeratosis of the forestomach, atrophy of the granularminimally focal haemosiderin deposit of liver parenchymal and focal oval cell proliferation were observed in one male, respectively.

CORPORA LUTEA AND IMPLANTATION
Pre-implantation rates were 14.4% in the control group, 9.4% at 125 mg/kg, 14.3% at 250 mg/kg and 9.8% at 500 mg/kg. Post-implantation loss rates were 6.0% for both the control group and the dose group of 125 mg/kg, 3.1% at 250 mg/kg and 7.0% at 500 mg/kg.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

125 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

organ weights and organ / body weight ratios

2

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

organ weights and organ / body weight ratios

histopathology: non-neoplastic

3

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive and developmental toxicity

Effect level:

>= 500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to adverse toxic effects at highest dose / concentration tested

Remarks:

Generation: parents and offspring

4

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

55 mg/kg bw/day (nominal)

Based on:

element

Remarks:

Fe

Sex:

male

Basis for effect level:

organ weights and organ / body weight ratios

5

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

110 mg/kg bw/day (nominal)

Based on:

element

Remarks:

Fe

Sex:

female

Basis for effect level:

organ weights and organ / body weight ratios

histopathology: non-neoplastic

6

Dose descriptor:

NOAEL

Remarks:

reproductive and developmental toxicity

Effect level:

>= 220 mg/kg bw/day (nominal)

Based on:

element

Remarks:

Fe

Sex:

male/female

Remarks on result:

not determinable due to adverse toxic effects at highest dose / concentration tested

Remarks:

Generation: parents and offspring

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

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)

OBSERVATION OF NEONATES
No treatment-related effects were observed on mean live neonates, birth rates, survival rates and sex ratios on days 0 and 4 post partum. The only abnormalilty found in the external appearance examinations is an acaudate that was observed in one neonate at 500 mg/kg. Crown Rump Length (CRL) of female neonates showed a significant decrease only at 125 mg/kg on Day 4 post partum.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

The oral administration of the test material to rats by gavage resulted in changes in body weight, water consumption, organ weight and histopathology in males at the dose of 500 mg/kg and changes in organ weight at the dose of 250 mg/kg. Therefore 125 mg/kg/day can be considered the No Observed Adverse Effect Level (NOAEL) in male rats. In females, changes in organ weights and histopathology were observed at the dose of 500 mg/kg. Therefore 250 mg/kg/day can be considered the NOAEL in female rats. The changes except body weight changes in males seemed to be reversible.
There was no treatment-related effects on reproductive functions in parental animals and development of neonates at any doses tested. the NOAEL for reproduction and developmental toxicity was considered to be ≥500 mg/kg/day.

Executive summary:

The test was intended to evaluate NOAEL (No Observed Adverse Effect Level) on reproduction process such as mating, conception, gestation, childbirth and development of neonates and the effect on the whole body including nerve and immune systems when female rats were orally dosed with Iron dichloride (CAS No.7758-94-3) once a day up to post partum day 4 from two weeks prior to the mating and male rats were orally dosed once a day with iron dichloride till two weeks before and after the mating.  

Male and female SD rats were dosed with the test substance (0 (Control group), 125, 250 and 500 mg/kg/day) from two weeks before mating. Male SD rates were dosed once a day till two weeks after mating while female SD rats were dosed once a day up topost partumday 4. A total of 42 doses were provided for male rats while female rates had 42 to 54 dosages depending on mating and delivery of individuals. Clinical signs and mortality were observed and body weight and food and water consumption were measured. In the necropsy, gross examination of organs and tests on corpus luteum graviditatis and implantation rates were conducted. In addition, tests for sensory and motor functions, urinalysis and hematological and blood chemical tests were given and organ weights were measured for five individuals randomly selected from each group. External abnormalities, sex ratio, body weights, CRL (Crown Rump Length) and survival rate were observed onpost partumdays 0 and 4.   

During the observation period, the main group dosed with the substance showed signs such as melaena (black stool) and salivation but these signs were observed to disappear after dosing in the recovery group. There was no mortality in male SD rats, but three mortalities took place in female individuals at 500 mg/kg/day. The cause for mortalities was presumably the gastrointestinal damage by the substance. It was found that male individuals were more sensitive to body weight and food consumption than female counterparts. The change by the test substance was not recognized in mating data, sensory functions, motor functions, urine analysis and blood test. Gastric hemorrhage with blackened liver and black pigmentation of liver discovered in the necropsy findings was presumed to be caused by the test substance, but it was found to improve for the recovery period of two weeks. Weight changes in the liver and adrenal were observed in the absolute and relative organ weights of male individuals at 250 and 500 mg/kg and female individuals at 500 mg/kg. The histopathological test found parenchymal hemosiderosis and hyperplasia of adrenocortical zona fasciculate as well. It was found that the substance had no effect on birth rate, survival rate, body weight and CRL of neonates.  

As a result of the test using iron dichloride (CAS No.7758-94-3), the NOAEL of repeated doses to male and female SD rats were 125 and 250 mg/kg/day, respectively. As there was no difference observed in reproductive functions of male and female SD rates and development of neonates between the control group and main group, NOAEL was thought to be ≥ 500 mg/kg/day. The effect of the test substance was none on reproductive functions, sensory functions, motor functions, urinalysis, and hematological and blood chemical findings which showed no differences between the control group and main group. Except the body weight change of male SD rats, clinical signs, water consumption, organ weights, necropsy findings and histopathological findings were reversible as they recovered after the dosing was over.