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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
migrated 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.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004

Materials and methods

Test guideline
Qualifier:
according to
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

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
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 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
Vehicle:
water
Details on oral 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.
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.
Doses / concentrations
Remarks:
Doses / Concentrations:
125, 250 and 500 mg/kg bw/day
Basis:
nominal 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.

Others:
Mating method and copulation confirmation: The mating period lasted 2 weeks. One male and one female rats were put in one cage for spontaneous copulation. 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.

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.

Positive control:
No.

Examinations

Observations and examinations performed and frequency:
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.

OTHER:
Sacrifice and pathology:
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.
Other examinations:
Mating:
The mating data was obtained by observing rats during mating, gestation and delivery period.

Corpora lutea and implantation:
The corpus luteum graviditatis was counted in the ovary removed in the necropsy and the number of implantation was counted in the uterus.

Observation of neonates:
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.

Reproductive indices are in 'any other information on materials and methods incl. tables' section.
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.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
(mortality) females only, local effects
Mortality:
no mortality observed
Description (incidence):
(mortality) females only, local effects
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
males only
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
males only
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
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.

OTHER FINDINGS
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.

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 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.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
125 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks:
FeCl2
Sex:
male
Basis for effect level:
organ weights and organ / body weight ratios
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks:
FeCl2
Sex:
female
Basis for effect level:
organ weights and organ / body weight ratios
Dose descriptor:
NOAEL
Remarks:
reproductive and development toxicity
Effect level:
>= 500 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks:
FeCl2
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
There were no treatment-related effects on reproductive functions in parental animals and development of neonates at any doses tested.
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:
other: recalculated value from the FeCl2 level
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:
other: recalculated value from the FeCl2 level
Dose descriptor:
NOAEL
Remarks:
reproductive and developmental toxicity
Effect level:
220 mg/kg bw/day (nominal)
Based on:
element
Remarks:
Fe
Sex:
male/female
Basis for effect level:
other: recalculated value from the FeCl2 level

Target system / organ toxicity

Critical effects observed:
not specified

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 to post partum day 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 on post partum days 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. 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.