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Description of key information

In an oral OECD 422 study on iron sulfate heptahydrate, the NOAEL for repeated dose toxicity was 100 mg/kg bw/day (equivalent to 20 mgFe/mg kg/day) based on the extramedullary hematopoiesis of the spleen in males and increased levels of inorganic phosphate in females. 
The subchronic study in which ferric chloride hexahydrate was administered to Fischer 344 rats (10/sex/dose) in their drinking water at concentrations of 0.12, 0.25, 0.5, 1.0 and 2.0% (equivalent to approximately 80, 154, 277, 550 and 1231 mg/kg bw/day in male rats, 88, 176, 314, 571 and 1034 mg/kg bw/day in female rats), there were no deaths or clinical signs of toxicity. There was a significant reduction in body weight gains at the two highest doses, increased serum iron content and higher red blood cell numbers for treated males . Microscopic pathological staining indicated iron overload effects at high doses and the NOAEL was determined as 0.5% or 57 mg Fe/kg bw/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to an appropriate OECD test guideline, with acceptable restrictions. No information on the GLP status of the study.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
No information on GLP, limited details on environmental conditions, limited details on examinations conducted, and no urinalysis.
Principles of method if other than guideline:
Range-finding study performed for a 2-year carcinogenicity study
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Japan Inc
- Age at study initiation: Six weeks
- Weight at study initiation: No data
- Fasting period before study: No data
- Housing: No data
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: One week


ENVIRONMENTAL CONDITIONS
- Temperature (°C): No data
- Humidity (%): No data
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): No data


IN-LIFE DATES: No data
Route of administration:
oral: drinking water
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Ferric chloride was dissolved in distilled water at concentrations of 0 (control), 0.12, 0.25, 0.5, 1.0 or 2.0 % (w/v).
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Continuous
Remarks:
Doses / Concentrations:
0.12, 0.25, 0.5, 1.0 and 2.0% w/v
Basis:
nominal in water
Remarks:
Doses / Concentrations:
80, 154, 277, 550 and 1231 mg/kg bw/day in male rats, 88, 176, 314, 571 and 1034 mg/kg bw/day in female rats
Basis:
other: approximate dose received
No. of animals per sex per dose:
Ten
Control animals:
other: Drinking water only
Details on study design:
- Dose selection rationale: This is a dose-range finding study to determine doses for a two-year carcinogenicity study.
- Rationale for animal assignment (if not random): No data
- Rationale for selecting satellite groups: No satellite group
- Post-exposure recovery period in satellite groups: None
- Section schedule rationale (if not random): No data
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the exposure period
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: All survivors
- Parameters examined: no data


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the exposure period
- Animals fasted: No data
- How many animals: All survivors
- Parameters examined: no data

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, for all survivors. No further details.
HISTOPATHOLOGY: Yes, all major organs and tissues. No further details.
Other examinations:
no further information
Statistics:
Body weight and the daily water intake data were analysed statistically using Student's t-test.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY: No deaths or clinical signs of toxicity were observed.


BODY WEIGHT AND WEIGHT GAIN: In the 1.0 and 2.0% groups there was a reduced body weight gain of at least 10% compared with the controls at the termination of treatment.


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): There was a significant suppression in the intake of drinking water observed in groups given concentrations of 0.5% or more (no further detail).


HAEMATOLOGY: Males of the treated groups had significantly increased red blood cell counts (no further details).


CLINICAL CHEMISTRY: Higher levels of serum iron were observed in males (control: 102±5 g/dl; 0.12%: 107±11 g/dl;
0.25%: 109±8  g/dl; 0.5%: 129±22  g/dl; 1.0%: 139±10  g/dl; 2.0%: 156 g/dl).


GROSS PATHOLOGY: No findings reported.


HISTOPATHOLOGY: NON-NEOPLASTIC: In examinations of sections stained with haematoxylin and eosin, brown pigment deposition was observed only in the keratin layers of the oesophageal mucosa in the groups given concentrations of 0.25% or higher, and in the laminae propriae of the large intestine in the 2.0% group. In sections stained with Berlin blue, increased numbers of positive pigments were also observed in the hepatocytes and Kupffer cells of the liver, the cartilage of the trachea and bronchus, the keratin mucosal layers of the tongue, the forestomach, the mucous layers of the small intestines, the white pulp of the spleen, the tubular epithelium of the kidney and the adipose tissues of the groups given 0.25% and higher. The intensity of the staining was marked in the intestine and liver.


HISTOPATHOLOGY: NEOPLASTIC: not investigated in this range-finding study.
Dose descriptor:
NOAEL
Effect level:
0.5 other: %
Based on:
test mat.
Sex:
male/female
Dose descriptor:
NOAEL
Effect level:
5 000 ppm
Based on:
test mat.
Sex:
male/female
Dose descriptor:
NOAEL
Effect level:
>= 277 - <= 314 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Equivalent to 277 and 314 mg/kg bw/day in males and females, respectively.
Dose descriptor:
NOAEL
Effect level:
>= 57 mg/kg bw/day (actual dose received)
Based on:
element
Sex:
male/female
Basis for effect level:
other: NOAEL for Fe (III), based on iron content in the test material
Critical effects observed:
not specified

Doses: The dose of ferric chloride in mg/kg bw/day was calculated from the drinking water intake and the final weight of male and female rats

 Dose of ferric chloride hexahydrate     Final weight (kg)     Final daily water intake (ml/d)     Dose (mg/kg bw/day)   
 %  mg/l Males   Females  Males  Females  Males Females 
 0  0  0.35  0.195  23.5  15  0
 0.12  1.2 0.345   0.19  23  14  80  88
 0.25  2.5  0.34  0.185  21 13   154  176
 0.5  5.0  0.325  0.175  18  11  277  314
 1.0  10.0  0.3  0.175  16.5  10  550  571
 2.0  20.0  0.195  0.145  12 7.5   1231  1034
Conclusions:
ferric chloride hexahydrate was administered to rats in their drinking water for 13 weeks, the NOAEL was 0.5% (equivalent to 277 and 314 mg/kg bw/day in males and females, respectively).
The NOAEL was 0.5% (equivalent to 277 and 314 mg/kg bw/day in males and females, respectively) based on the reduced body weight gain.
The equivalent NOAEL doses expressed as iron trinitrate would be 246 mg/kg bw/day for males and 281 mg/kg bw/day for females.
Executive summary:

Ferric chloride hexahydrate was administered to Fischer 344 rats (10/sex/dose) in their drinking water for 13 weeks, at concentrations of 0.12, 0.25, 0.5, 1.0 and 2.0% (equivalent to approximately 80, 154, 277, 550 and 1231 mg/kg bw/day in male rats, 88, 176, 314, 571 and 1034 mg/kg bw/day in female rats). All deaths and clinical signs of toxicity were recorded. Body weights were measured weekly. At the end of the dosing period, all survivors were killed for haematological, clinical chemistry and pathological exminations. All major organs and tissues were examined microscopically.

There were no deaths or clinical signs of toxicity. There was a significant reduction in body weight gains at the two highest doses at the end of the treatment period. Treated males had increased levels of serum iron and higher red blood cell counts compared with controls. In microscopic examinations of sections stained with haematoxylin and eosin, brown pigment deposition was observed only in the keratin layers of the oesophageal mucosa in the groups given concentrations of 0.25% or higher, and in the laminae propriae of the large intestine in the 2.0% group. In sections stained with Berlin blue, increased numbers of positive pigments (an indication of iron overload) were also observed in the hepatocytes and Kupffer cells of the liver, the cartilage of the trachea and bronchus, the keratin mucosal layers of the tongue, the forestomach, the mucous layers of the small intestines, the white pulp of the spleen, the tubular epithelium of the kidney and the adipose tissues of the groups given 0.25% and higher. The intensity of the staining was marked in the intestine and liver. The NOAEL was 0.5% (equivalent to 277 and 314 mg/kg bw/day in males and females, respectively) based on the reduced body weight gain.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
57 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Two good quality studies (90-day toxicity, screening study) performed with read-across substances are available for this endpoint.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Information for oral toxicity of iron trinitrate was obtained by read across to data generated for ferrous sulphate. Information was available from a subacute administration to rats in a screening reproductive toxicity study, a preliminary 13 week study and a chronic drinking water exposure study in rats to primarily detrmine carcinogenic effects.

Subacute toxicity information was available from a screening test for reproductive and developmental effects in rats. While there were no reproductive effects apparent at the limit dose of 1000 mg/kg bw/day, the parental animals did show signs of systemic toxicity and the NOAEL for systemic effects in this study was considered to be 100 mg/kg bw/day (equivalent to circa 91 mg/kg bw/day for iron trinitrate).

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 key repeated dose toxicity study is the oral 13-week dose-range finding study on ferric chloride. While intended to be a range-finding study for the carcinogenicity study, in the full two-year study there were no haematology and clinical chemistry investigations, so the NOAEL from this 13 week study (that included more thorough investigations in accordance with the OECD guideline for repeated dose toxicity) has been used as the starting point for the DNEL derivation.

In this 13-week study ferric chloride hexahydrate was administered to Fischer 344 rats (10/sex/dose) in their drinking water at concentrations of 0.12, 0.25, 0.5, 1.0 and 2.0% (equivalent to approximately 80, 154, 277, 550 and 1231 mg/kg bw/day in male rats, 88, 176, 314, 571 and 1034 mg/kg bw/day in female rats).

There were no deaths or clinical signs of toxicity. There was a significant reduction in body weight gains at the two highest doses at the end of the treatment period. Treated males had increased levels of serum iron and higher red blood cell counts compared with controls. In microscopic examinations of sections stained with haematoxylin and eosin, brown pigment deposition was observed only in the keratin layers of the oesophageal mucosa in the groups given concentrations of 0.25% or higher, and in the laminae propriae of the large intestine in the 2.0% group. In sections stained with Berlin blue, increased numbers of positive pigments (an indication of iron overload) were also observed in the hepatocytes and Kupffer cells of the liver, the cartilage of the trachea and bronchus, the keratin mucosal layers of the tongue, the forestomach, the mucous layers of the small intestines, the white pulp of the spleen, the tubular epithelium of the kidney and the adipose tissues of the groups given 0.25% and higher. The intensity of the staining was marked in the intestine and liver.

The NOAEL was 0.5% (equivalent to 277 and 314 mg/kg bw/day in males and females, respectively, and 57 and 65 mg Fe/kg bw/day, respectively) based on the reduced body weight gain. The equivalent NOAEL doses expressed as iron trinitrate would be 246 mg/kg bw/day for males and 281 mg/kg bw/day for females.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Study provides the longest duration of exposure and the lowest NOAEL

Repeated dose toxicity: via oral route - systemic effects (target organ) cardiovascular / hematological: spleen

Justification for classification or non-classification

Available data for a read-across substance do not indicate classification for iron trinitrate.