Iron sulphide

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test

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:

publication

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
- Weights during the study: 269.23 – 302.18 g for males and 191.34 –221.60 g for females

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

not specified

Details on mating procedure:

- M/F ratio per cage: 1/1
- Proof of pregnancy: The day after the copulating, mating was verified by sperm in a vaginal rinse.

Duration of treatment / exposure:

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

Premating exposure period : 2 weeks

Frequency of treatment:

Daily

Details on study schedule:

- Number of implantation and corpus luteum: while necropsied, the number of corpus luteum and implantation were counted; the former was measured in the ovary and the latter was measured in the uterus.
- Mating: Mating period was 14 days. The day after the copulating, mating was verified by sperm in a vaginal rinse. Mating rate, fertility rate for both sexes, and parturition rate were estimated.
- Pregnancy and delivery: The period of pregnancy was calculated from mating date (day 0). In addition, delivery period was estimated to identify the delivery (day 0).

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 (15 per dose level and additional 5 in low and high dose level)

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 fromG4 (500 mg/kg bw/day) groups were allocated as recovery groups.
- Post-exposure recovery period in satellite groups: 14 days

Examinations

Parental animals: Observations and examinations:

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:
- Consumption rate was measured once a week except mating period.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes , see Section 7.5.1

CLINICAL CHEMISTRY: Yes , see Section 7.5.1

URINALYSIS: Yes , see Section 7.5.1

NEUROBEHAVIOURAL EXAMINATION: Yes, , see Section 7.5.1

Litter observations:

STANDARDISATION OF LITTERS: No

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, postnatal mortality, presence of gross anomalies,

GROSS EXAMINATION OF DEAD PUPS:
yes, for external abnormalities

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals after 28 days of dosing
- Maternal animals: All surviving animals on day 4 of lactation

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

ORGAN WEIGHTS
- 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 (see table)
- 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.

Reproductive indices:

- Number of implantation and corpus luteum: while necropsied, the number of corpus lutea and implantations were counted; the former was measured in the ovary and the latter was measured in the uterus.
- Mating: Mating period was 14 days. The day after the copulating, mating was verified by sperm in a vaginal rinse. Mating rate, fertility rate
for both sexes, and parturition rate were estimated.
- Pregnancy and delivery: The period of pregnancy was calculated from mating date (day 0). In addition, delivery period was estimated to identify
the delivery (day 0).

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

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.

Mortality:

mortality observed, treatment-related

Description (incidence):

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, non-treatment-related

Description (incidence and severity):

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):

no effects observed

Description (incidence and severity):

There was no significant difference between the control and the treated groups, and no dose-related change was observed in both sexes.

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

effects observed, treatment-related

Description (incidence and severity):

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

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

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 biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

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 findings:

no effects observed

Description (incidence and severity):

There were no specific findings.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Sensory reflex test: Both auricle reflex test and corneal reflex test were performed evaluating sens
ory 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 resultin 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.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

See Behaviour

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

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.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

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)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
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 FOOD CONSUMPTION (PARENTAL ANIMALS)
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.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS): No effects (gavage dosing)

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS): No data

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS): No data

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- Number of implantation and corpus luteum: Pre-implantation loss rates (%) were 14.4, 9.4, 14.3, and 9.8 at the 0, 125, 250, 500 mg/kg bw/day treatment groups, respectively. Post-implantation loss rates (%) were 6.0, 6.0, 3.1, and 7.0.
- Estimation of mating data: Mating rates (%) for the control group, 125, 250, 500 mg/kg bw/day treatment groups were 93.3, 86.7, 100, and 100, respectively. Fertility rates (%) were 73.3, 80.0, 93.3, and 73.3 for male rats, and 78.6, 92.3 , 93.3, and 73.3 for female rats. Parturition rates (%) were identical with the fertility rates for female animals.
- Pregnancy: The period of pregnancy was 22.0, 22.1, 22.2, and 22.1 days for the control group, 125, 250, 500 mg/kg bw/day treatment groups, respectively.

ORGAN WEIGHTS (PARENTAL ANIMALS)
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 (PARENTAL ANIMALS)
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 (PARENTAL ANIMALS)
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 (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: mating index; fertility index; number of implantation sites; number of corpus luteum; duration of pregnancy.

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

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: No. of neonate

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Table 1. Mating, fertility, gestation, and postpartum data

DOSE (mg/kg)	0	125	250	500
Pairs started (N) Mating rate (%) Male fertility rate (%) Female fertility rate (%) Parturition rate (%) Pre-implantation loss rate (%) Post-implantation loss rate (%) No. of corpora lutea No. of implantations No. of neonate Mean gestation period (day)	14 93.3 73.3 78.6 78.6 14.4 6.0 17.6 15.1 14.2 23.6	13 86.7 80.0 92.3 92.3 9.4 6.0 16.8 15.3 14.3 22.1	15 100 93.3 93.3 93.3 14.3 3.1 17.2 14.8 14.3 22.3	15 100 73.3 73.3 71.4* 9.8 7.0 17.4 15.7 14.6 22.1

*: Animal death before parturition was excluded in calculation of ‘Parturition rate’ and ‘Mean gestation period’

 

Table 2. Historic control data of mating, fertility and gestation

- Mating method & method: Monogamy & 2 weeks

- Species/ strain: Rat/ Sprague Dawley

	No. of Male	No. of female	No. of coitus confirmed female	No. of pregnant female	Mating rate (%)	Male fertility rate (%)	Female fertility rate (%)
	15 25 25 25 16 16 21	15 25 25 25 16 16 21	14 24 25 24 16 16 21	11 24 25 20 15 16 18	93.3 96.0 100.0 96.0 100.0 100.0 100.0	73.3 96.0 100.0 80.0 98.8 100.0 85.7	78.6 100.0 100.0 83.3 93.8 100.0 85.7
Total	143	143	140	129 Mean±S.D.	97.9 97.9±2.8	90.2 89.8±10.4	92.1 91.6±9.0

 Mating rate (%) = (No. of female confirmed about coitus/ No. of male using mating) × 100

Male fertility rate (%) = (No. of pregnant female/ No. of male using mating) × 100

Female fertility rate (%) = (No. of pregnant female/ No. of female confirmed about coitus) × 100

Applicant's summary and conclusion

Conclusions:

There was no significant difference in mating data, pre and post implantation loss rates between the control group and the treatment groups. Therefore, NOAEL of iron dichloride for reproductive function was 500 mg/kg bw/day for both sexes.

Executive summary:

A combined repeated dose/reproductive & developmental toxicity study was conducted with iron dichloride (98% purity) in male and female Sprague-Dawley rats daily dosed by oral gavage at 0, 125, 250 and 500 mg/kg bw/ day for 42 days (male rats) and 42 to 54 days (female rats). Additional 5 animals per sex were allocated to control and high dose groups as (14 days) recovery groups. No death was observed for male animals, whereas 3 female rats in the high dose treatment group were found dead on the day 38, 46 and 51. 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. In 500 mg/kg bw/day treatment group, the amount of water consumption was increased for both male and female animals. There were no specific findings for haematology, serum analysis and urinalysis. No specific changes were observed for behaviour and motoric activity. 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 influenced 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.

Pre-implantation loss rates (%) were 14.4, 9.4, 14.3, and 9.8 at the 0, 125, 250, 500 mg/kg bw/day treatment groups, respectively. Post-implantation loss rates (%) were 6.0, 6.0, 3.1, and 7.0. Mating rates (%) for the control group, 125, 250, 500 mg/kg bw/day treatment groups were 93.3, 86.7, 100, and 100, respectively. Fertility rates (%) were 73.3, 80.0, 93.3, and 73.3 for male rats, and 78.6, 92.3 , 93.3, and 73.3 for female rats. Parturition rates (%) were identical with the fertility rates for female animals. The period of pregnancy was 22.0, 22.1, 22.2, and 22.1 days for the control group, 125, 250, 500 mg/kg bw/day treatment groups, respectively.

In conclusion, there was no significant difference in mating data, pre and post implantation loss rates between the control group and the treatment groups. Therefore, NOAEL of reproductive function was 500 mg/kg bw/day for both sexes.