Iron sulphide

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to GLP and valid methods, therefore it is considered relevant, reliable and adequate for classification. The data are from a secondary source of reliable instance and the level of detail was high.

Data source

Reference

Reference Type:

other: OECD SIDS

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:

Iron dichloride

EC Number:

231-843-4

EC Name:

Iron dichloride

Cas Number:

7758-94-3

IUPAC Name:

iron(2+) dichloride

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): : Iron dichloride
- Molecular formula (if other than submission substance): Cl2-Fe
- Molecular weight (if other than submission substance): 126.7516
- Smiles notation (if other than submission substance): [Fe](Cl)Cl
- InChl (if other than submission substance): 1S/2ClH.Fe/h2*1H;/q;;+2/p-2
- Structural formula attached as image file (if other than submission substance): See Fig.
- Substance type: Inorganic monoconstituent substance
- Physical state: Powder
- Analytical purity: 98 %
- Lot/batch No.: Sigma-Aldrich Corporation, LOT No. – 14330TA

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 8 weeks
- Weight during the study: 269.23 – 302.18 g for males and 191.34 –221.60 g for females
- Fasting period before study: No data

Administration / exposure

Route of administration:

oral: gavage

Duration of treatment / exposure:

42 days for male animals and 42 to 54 days for female animals

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

125 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Doses / concentrations 4

Dose / conc.:

500 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

70 animals for each sex in the main test: per dose group as follows G0:20M + 20 F, G1:15M + 15 F, G3:15M + 15F, G4:20M+ 20F
10 animals (5 males + 5 females) from G1 (0 mg/kg bw/day) and from G4 (500 mg/kg bw/day) groups were allocated as recovery groups.

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: In the preliminary tests all male rats in 1000 mg/kg bw/day treatment group were dead. For female rats, one rat was dead at the same dose level. Therefore, 500 mg/kg bw/day was chosen as the maximum dosage.
- Rationale for selecting satellite groups: 10 animals (5 males + 5 females) from G1 (0 mg/kg bw/day) and from G4 (500 mg/kg bw/day) groups were allocated as recovery groups.
- Post-exposure recovery period in satellite groups: 14 days

Examinations

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical symptoms were observed once a day and once a week in detail.
- The death rate was observed twice a day.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weights were measured once a week and right before the necropsy except mating period, but for pregnant females, it was measured on day 0, 7, 14, 20 of gestation period, date of delivery, and 4 days after the delivery.

FOOD CONSUMPTION AND COMPOUND INTAKE ):
- Consumption rate was measured once a week except during mating period.

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 18 hrs before necropsy.
- Anaesthetic used for blood collection: Yes ,ether
- Animals fasted: Yes
- How many animals: 5 males and 5 females from each test group (randomly selected)
- Parameters examined:
total erythrocyte count (RBC),
hemoglobin concentration (HEG),
hematocrit (HCT),
mean cell volume (MCV),
mean cell hemoglobin (MCH),
mean cell hemoglobin concentration (MCHC),
total leucocyte count (WBC),
platelet (PLT),
neutrophils (NEU),
eosinophils (EOS),
basophils (BASO),
lymphocytes (LYM),
monocytes (Mono).
After blood collection, sera were separated using a centrifuge to measure prothrombin time (PT) and activated partial thromboplastin time (APTT). Metheamoglobin (MH) was also analyzed.

CLINICAL CHEMISTRY: Yes
- Parameters examined:
alanine,
aminotransferase,
aspartate aminotransferase,
blood urea nitrogen,
creatinine,
total protein,
albumin,
sodium,
potassium,
triglycerides,
glucose,
phosphorus,
calcium,
cholinesterase.
Cholinesterase II activity was measured with S-butyrylthilcholine iodide as a substrate.

URINALYSIS: Yes
- Five males and five females were randomly selected from each test group.
- Following seven items were tested; color, specific gravity, pH, glucose, protein, leukocyte, erythrocyte.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Test for sensory organ: Five males and five females were randomly selected from each test group. Both auricle reflex test and corneal reflex test were performed before necropsy.
- Motor function test: Five males and five females were randomly selected from each test group for traction test. This test was performed before necropsy.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table)
- Organ weight: testes, epididymides(all males), liver, kidney, adrenal, thymus, spleen, brain and heart (5 male and 5 female animals from each test group).

HISTOPATHOLOGY: Yes
- Fixation: 21 tissues were preserved in 10 % buffered neutral formalin solution for histopathologic tests: brain, pituitary, spinal cord, heart, lung, trachea, stomach, ileum, liver, colon, spleen, thyroids, thymus, adrenals, kidneys, urinary bladder, sciatic nerve, bone marrow, uterus, ovaries and lymph node. Testes and epididymides were fixed in bouin’s fixative.

Statistics:

Homogeneity of variance was evaluated using Levene’s test in terms of body weight, food and water consumption, biochemical test of blood and organ weight. When the assumption of homogeneity of variance was met, ANOVA was used. If significant result was observed, Dunnett’s test was used. When the assumption of heterogeneity of variance was met, appropriate data transformation was carried out, then Levene’s test was performed on re-transformed data. If significant result was observed, Dunnett’s test was used.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

reversible decrease in locomotion activity at 500 mg/kg bw in females more than males

Mortality:

mortality observed, treatment-related

Description (incidence):

No death was observed for male animals. Three female rats in 500 mg/kg bw/day treatment group were found dead on the day 38, 46 and 51 of administration.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

decreased at 250 and 500 mg/kg bw/day male groups; no significant changes in females except on day 7 of premating period and the day 4 of lactation period

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):

increased in 500 mg/kg bw dosed males and females

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

Description (incidence and severity):

increased liver weights at 250 males and 500 mg/kg b.w. males and females; increased adrenal glands weights in 250 and 500 mg/kg b.w. dosed males

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

severe diffuse hemorrhagic grandular stomach/distension of stomach in dead animals, and diffuse black colored liver and hemorrhage with diffuse black pigmentation in 500 mg/kg males; mass of mesenteric lymph node in 500 mg/kg dosed females

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

hemosiderin deposit of hepatocyte and granular, hyperplasia of zona fasciculate in adrenal cortex, hyperkeratosis of forestomach, hemosiderin deposit of grandular stomach, neutrophil infiltration of submusoca in 500 mg/kg dosed males and less in females

Details on results:

CLINICAL SIGNS, BEHAVIOUR AND MORTALITY
No death was observed for male animals. Three female rats in 500 mg/kg bw/day treatment group were found dead on the day 38, 46 and 51 of administration.
Clinical signs such as blackish stool and salivation were observed in both the control and the treated groups. In the early stages of administration, cases of decrease in locomotion activity were found in 500 mg/kg bw/day groups of both sexes, but these were recovered to normal states. The female rats were more sensitively affected than the male rats in locomotion activity decrease, paleness, emaciation and soiled perineal region. However, these symptoms were reversible within the test period.

BODY WEIGHT AND WEIGHT GAIN
The rate of body weight gain was significantly decreased in 250 and in 500 mg/kg bw/day male groups. For females, there was no significant changes except on the day 7 of pre-mating period and the day 4 of lactation period. Further, no dose-dependent changes were shown.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
There was no significant difference between the control and the treated groups, and no dose-related change was observed in both sexes.

WATER CONSUMPTION (See Table 1 & 2)
In 500 mg/kg bw/day treatment group, the amount of water consumption was increased for both male and female animals.

HAEMATOLOGY
Statistically significant differences were found in mean cell volume (MCV), eosinophils (EOS) and platelet (PLT). But these were within the biologically normal range and no dose dependent changes were evident.

CLINICAL CHEMISTRY
Statistically significant differences were found in cholinesterase (CS), and triglycerides (TG). But these were within the biologically normal range and no dose dependent changes were evident.

URINALYSIS
There were no specific findings.

NEUROBEHAVIOUR
- Sensory reflex test: Both auricle reflex test and corneal reflex test were performed evaluating sensory reflex; no specific reaction was observed in comparison with the control group.
- Motor function test: Significant decrease was observed in female 125 and 500 mg/kg bw/day treatment groups. But these decreased values were higher than male control group since the mean value of female control group was higher than the male control group.
There was no significant result in female 250 mg/kg bw/day group and all male rats. Because there were no dose-dependent changes, motor function was not considered to be affected by iron dichloride.

ORGAN WEIGHTS (See Table 3 & 4)
Both absolute and relative weights of liver were increased in 250 and 500 mg/kg bw/day male groups and in 500 mg/kg bw/day female group. Also, for male rats, absolute adrenal glands weights were increased in 500 mg/kg bw/day group, and relative adrenal glands weights were increased in 250 and in 500 mg/kg bw/day group. Because of hemosiderin deposit in hepatocyte and hyperplasia of zona fasciculate in adrenal cortex, the increased weights of liver and adrenla glands were influnced by the test substance. In 125 mg/kg bw/day male group, liver weight did not differ from the control group, but adrenal glands weights were decreased as compared to the control group. For thymus, absolute weight was decreased in female 125 and 500mg/kg bw/day groups, and relative weight was decreased in 500 mg/kg bw/day group. However, these changes were considered to be individual variations and not due to the test substance.

GROSS PATHOLOGY
Diaphragmatic nodules of liver were sporadically noted in the control and the treated groups. It is a congenital malformation, which is a morphological change and doesn’t have physiological effects.
The following necropsy opinions were caused by the test substance; severe diffuse hemorrhagic grandular stomach and severe distension of stomach in dead animals, and diffuse black colored liver and hemorrhage with diffuse black pigmentation in scheduled necropsy of 500 mg/kg bw/day male group. For females, a case of mass of mesenteric lymph node was observed in 500 mg/kg bw/day group.

HISTOPATHOLOGY: NON-NEOPLASTIC
For 500 mg/kg bw/day groups of both sexes, hemosiderin deposit of hepatocyte and grandular, hyperplasia of zona fasciculate in adrenal cortex, hyperkeratosis of fore stomach, hemosiderin deposit of grandular stomach, neutrophil infiltration of submucosa were observed. These conditions were induced by the test substance and were weaker in females. There was no specific findings in the recovery groups. In case of dead rats, due to severe villous atrophy of fore stomach, gastric function was abnormal. Therefore, it was concluded that female rats were dead by a physical irritancy of test substance.

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

125 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: overall effects

2

Key result

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: overall effects

Target system / organ toxicity

Critical effects observed:

no

Lowest effective dose / conc.:

250 mg/kg bw/day (actual dose received)

System:

hepatobiliary

Organ:

adrenal glands

liver

stomach

Any other information on results incl. tables

Table 1. Water consumption of male rats (mL/animal/day)

Group/Dose (mg/kg)	Day after treatment
		0	6	13	40
G1 0	Mean S.D. N	30.06   4.03 10	35.87   6.65 10	41.05 10.22 10	44,36 16.35 10
G2 125	Mean S.D. N	29.51  8.62 8	35.36  5.05 8	39.78  9.20 8	43.42  5.66 8
G3 250	Mean S.D. N	31.34   4.56 8	33.73   4.74 8	39.07   8.96 8	36.78   5.00 8
G4 500	Mean S.D. N	31.93    3.48 10	45.29 *   7.56 10	57.04 * 12.20 10	38.10 * 23.08 10

N: Number of cages

*: Statistical significance was observed.

 

Table 2. Water consumption of female rats (mL/animal/day)

Group/ Dose (mg/kg)		Premating period			Gestation period				Lactation period
		0	6	13	0	6	13	20	0	3
G1 0	Mean S.D. N	54.98 9.06 10	53.21 13.90 10	61.45 9.61 10	31.98 4.91 11	39.04 7.17 11	44.36 9.54 11	48.85 9.97 11	41.26 8.18 11	56.25 22.95 11
G2 125	Mean S.D. N	53.43 16.89 8	52.14 18.55 8	60.49 19.20 8	30.06 4.64 12	35.91 7.36 12	41.67 9.55 12	48.02 9.69 12	44.80 7.13 12	65.99 20.21 12
G3 250	Mean S.D. N	58.21 10.44 8	58.00 10.59 8	68.64 6.92 8	32.59 6.35 14	40.41 9.47 14	44.87 6.56 14	50.43 8.73 14	47.45 9.27 14	66.83 15.07 14
G4 500	Mean S.D. N	58.16 8.14 10	81.04* 10.68 10	82.60* 11.51 10	36.10 6.91 10	47.62 8.04 10	47.20 10.53 10	65.01* 13.43 10	51.81* 12.22 10	66.33 26.55 10

N: Number of cages (pre-mating period), Number of animals (Gestation period & Lactation period)

*: Statistical significance was observed.

Table 3. Absolute organ weights of males (group mean)

Dose (mg/kg)	0	125	250	500
Brain (g) Heart (g) Liver (g) Spleen (g) Thymus (g) Lt. Kidney (g) Rt. Kidney (g) Lt. Adrenal gland (g) Rt. Adrenal gland (g) Lt. Testes (g) Rt. Testes (g) Lt. Epididymis (g) Rt. Epididymis (g)	2.11 1.39 11.32 0.90 0.49 1.41 1.44 27.00 24.66 1.70 1.70 0.60 0.60	2.01 1.34 11.96 1.03 0.49 1.44 1.45 26.64 24.76 1.73 1.76 0.61 0.62	2.02 1.30 13.30* 1.03 0.47 1.42 1.46 28.90 28.90 1.70 1.92 0.62 0.60	1.95 1.25 14.04* 0.89 0.36 1.36 1.39 33.32* 34.26* 1.73 1.77 0.59 0.58

Number of animals: 5/group

*: Statistical significance was observed.

 

Table 4. Absolute organ weight of females (group mean)

Dose (mg/kg)	0	125	250	500
Brain (g) Heart (g) Liver (g) Spleen (g) Thymus (g) Lt. Kidney (g) Rt. Kidney (g) Lt. Adrenal gland (g) Rt. Adrenal gland (g)	1.93 1.03 9.77 0.86 0.34 0.89 0.88 3.50 3.47	1.97 0.97 9.81 0.73 0.23* 0.88 0.89 3.53 3.42	1.91 0.96 10.22 0.72 0.27 0.86 0.86 3.85 3.46	1.93 0.98 11.36* 0.74 0.25* 0.94 0.96 3.95 3.78

Number of animals : 5/group

*: Statistical significance was observed.

Applicant's summary and conclusion

Conclusions:

By the particular test results such as the rate of body weight gain, water consumption, organ weights, necropsy, and histopathology, NOAEL values were determined to 125 mg/kg bw/day for males and 250 mg/kg bw/day for females.

Executive summary:

Repeated dose toxicity of Iron dichloride (98% purity) was tested in male and female Sprague-Dawley rats by daily oral gavage at dose levels of 0, 125, 250 and 500 mg/kg bw/ day for 42 days (male rats) and 42 to 54 days (female rats). 15 animals per sex per dose level were used and in the high and low dose group additional 5 animals per sex were allocated as (14 days) recovery groups. No death was observed for male animals. Three female rats in the high dose treatment group were found dead on the day 38, 46 and 51 of administration. Clinical signs such as blackish stool and salivation were observed in both the control and the treated groups. In the early stages of administration, cases of decrease in locomotion activity were found in 500 mg/kg bw/day groups of both sexes, but these were recovered to normal states. The female rats were more sensitively affected than the male rats in locomotion activity decrease, paleness, emaciation and soiled perineal region. However, these symptoms were reversible within the test period.

The rate of body weight gain was significantly decreased in 250 and in 500 mg/kg bw/day male groups. For females, there was no significant changes except on the day 7 of pre-mating period and the day 4 of lactation period. Further, no dose-dependent changes were shown. There was no significant difference in food consumption between the control and the treated groups, and no dose-related change was observed in both sexes.

In 500 mg/kg bw/day treatment group, the amount of water consumption was increased for both male and female animals.

Statistically significant differences were found in mean cell volume (MCV), eosinophils (EOS), platelet (PLT), cholinesterase (CS) and triglycerides (TG). But these were within the biologically normal range and no dose dependent changes were evident. There were no specific findings in the urinalysis.

Both auricle reflex test and corneal reflex test were performed evaluating sensory reflex; no specific reaction was observed in comparison with the control group. Significant decrease was observed in female 125 and 500 mg/kg bw/day treatment groups. But these decreased values were higher than male control group since the mean value of female control group was higher than the male control group.

There was no significant result in motor function test in female 250 mg/kg bw/day group and all male rats. Because there were no dose-dependent changes, motor function was not considered to be affected by iron dichloride.

Both absolute and relative weights of liver were increased in 250 and 500 mg/kg bw/day male groups and in 500 mg/kg bw/day female group. Also, for male rats, absolute adrenal glands weights were increased in 500 mg/kg bw/day group, and relative adrenal glands weights were increased in 250 and in 500 mg/kg bw/day group. Because of hemosiderin deposit in hepatocyte and hyperplasia of zona fasciculate in adrenal cortex, the increased weights of liver and adrenal glands were influenced by the test substance. In 125 mg/kg bw/day male group, liver weight did not differ from the control group, but adrenal glands weights were decreased as compared to the control group. For thymus, absolute weight was decreased in female 125 and 500mg/kg bw/day groups, and relative weight was decreased in 500 mg/kg bw/day group. However, these changes were considered to be individual variations and not due to the test substance.

Diaphragmatic nodules of liver were sporadically noted in the control and the treated groups. It is a congenital malformation, which is a morphological change and doesn’t have physiological effects.

The following necropsy opinions were caused by the test substance; severe diffuse hemorrhagic grandular stomach and severe distension of stomach in dead animals, and diffuse black colored liver and hemorrhage with diffuse black pigmentation in scheduled necropsy of 500 mg/kg bw/day male group. For females, a case of mass of mesenteric lymph node was observed in 500 mg/kg bw/day group.

For 500 mg/kg bw/day groups of both sexes, hemosiderin deposit of hepatocyte and grandular, hyperplasia of zona fasciculate in adrenal cortex, hyperkeratosis of fore stomach, hemosiderin deposit of grandular stomach, neutrophil infiltration of submucosa were observed. These conditions were induced by the test substance and were weaker in females. There was no specific findings in

the recovery groups. In case of dead rats, due to severe villous atrophy of fore stomach, gastric function was abnormal. Therefore, it was concluded that female rats were dead by a physical irritancy of test substance.

By the particular test results such as the rate of body weight gain, water consumption, organ weights, necropsy, and histopathology, NOAEL values were determined to 125 mg/kg bw/day for males and 250 mg/kg bw/day for females.