Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

A key 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). Although there was some paternal and maternal toxicity, 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, which is even considered to be higher for iron sulfide based on its lower water solubility and absorption rate.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
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.
Justification for type of information:
See attached read-across justification
Reason / purpose:
read-across source
Clinical signs:
no effects observed
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
Clinical biochemistry 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.
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):
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 result in female 250 mg/kg bw/day group and all male rats. Because there were no dose-dependent cha
nges, motor function was not considered to be affected by iron dichloride.
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, non-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 wer e 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: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed
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.




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.
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
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
Reproductive effects observed:
not specified

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

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 read-across substance 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 read-across substance 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 read-across substance iron dichloride of reproductive function was 500 mg/kg bw/day for both sexes.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
High quality study
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

A key combined repeated dose/reproductive & developmental toxicity study was available for 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) (OECD SIDS, 2004). 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 adrenal 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.

 

From a systemic viewpoint, iron dichloride can be considered to be a (worst case) read across source chemical for iron sulfide. Taking into account that water solubility and absorption of iron chloride is much higher than iron sulfide, systemic toxicity of iron chloride is considered to be higher than iron sulfide. From the read across for the acute studies, a benefit factor of at least 6 for the NOAEL of iron sulfide could be argumented. Therefore, if the NOAEL is assumed to be at least 6 times higher, NOAEL for iron sulfide would exceed the limit dose. In any terms, there is no reason for classification.

Effects on developmental toxicity

Description of key information

A key 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). Although there was some paternal and maternal toxicity, there was no significant difference in mating data, pre and post implantation loss rates between the control group and the treatment groups. Therefore, NOAEL for developmental toxicity was 500 mg/kg bw/day for both sexes, which is even considered to be higher for iron sulfide based on its lower water solubility and absorption rate. In addition, there is the fact that iron is supplemented in pregnant women, therefore further testing is considered unnecessary from a scientific viewpoint.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
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.
Justification for type of information:
See attached read-across justification
Reason / purpose:
read-across source
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 8 weeks
- Weights during the repeated dose toxicity study: 269.23 – 302.18 g for males and 191.34 – 221.60 g for females
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
Frequency of treatment:
Daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
125 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
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 vehicle
Details on study design:
- Dose selection rationale: In the preliminary tests all male rats in 1000 mg/kg bw/day treatment g
roup 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
Maternal 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 th
e necropsy except mating period, but for pregnant females, it was measured on day 0, 7, 14, 20 of g
estation period, date of delivery, and 4 days after the delivery.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Consumption rate of fodder was measured once a week except mating period.
POST-MORTEM EXAMINATIONS: Yes
- Organs examined: : 21 tissues were preserved in 10 % buffered neutral formalin solution for histop
athologic 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.
- Organ weight: liver, kidney, adrenal, thymus, spleen, brain and heart ( 5 female animals from each
test group).
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No data
- Number of late resorptions: No data
Fetal examinations:
- Birth rate and survival rate
- Body weight and crown rump length(CRL): measured on the day 0 and 4 at postpartum
- Sex ratio
- External findings in neonates
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. W
hen the assumption of heterogeneity of variance was met, appropriate data transformation was carrie
d out, then Levene’s test was performed on re-transformed data. If significant result was observed,
Dunnett’s test was used.
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 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.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
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. Because of hemosiderin deposit in hepatocyte , the inc
reased weights of liver were influnced by the test substance. 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 pathological findings:
effects observed, treatment-related
Description (incidence and severity):
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 schedualed necropsy of 500 mg/kg b.w./day male group. For females, a case of mass of mesenteric lumph node was observed in 500 mg/kg b.w./day group.
Neuropathological findings:
no effects observed
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
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
See Section 7.8.1
Total litter losses by resorption:
no effects observed
Early or late resorptions:
not specified
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
Litter size, birth rate, survival rate, and sex rate: All data were within the normal range.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: body weight changes, target organ changes in stomach and liver at 250 and 500 mg/kg bw

Details on maternal toxic effects:
CLINICAL SIGNS AND MORTALITY
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
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
There was no significant difference between the control and the treated groups, and no dose-related change was observed in both sexes.

WATER CONSUMPTION
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
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. Because of hemosiderin deposit in hepatocyte , the increased weights of liver were influnced by the test substance. 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. 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.
Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Basis for effect level:
other: developmental toxicity
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Body weight and crown rump length (CRL) of neonates: CRL of neonates were significantly decreased as compared with that of the control group for postpartum day 4 in 125 mg/kg bw/day treatmen
t group (p < 0.01). But the decrease did not correlate with body weights that is a main growth and developmental index. Since there was no dose-dependence of CRL decrease, it was concluded that
the test substance did not influence the growth of neonates.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
not specified
Changes in litter size and weights:
not specified
Changes in postnatal survival:
no effects observed
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
A case of acaudate was observed at 500 mg/kg bw/day treatment group. However, because of the low frequency of occurrence, it was not a teratogenic effect.
Skeletal malformations:
not examined
Visceral malformations:
not examined
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
- Litter size, birth rate, survival rate, and sex rate: All data were within the normal range.
- Body weight and crown rump length (CRL) of neonates: CRL of neonates were significantly decreased as compared with that of the control group for postpartum day 4 in 125 mg/kg bw/day treatment group (p < 0.01). But the decrease did not correlate with body weights that is a main growth and developmental index. Since there was no dose-dependence of CRL decrease, it was concluded that the test substance did not influence the growth of neonates.
- External findings in neonates: A case of acaudate was observed at 500 mg/kg bw/day treatment group. However, because of the low frequency of occurrence, it was not a teratogenic effect.
Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: developmental toxicity
Abnormalities:
not specified
Developmental effects observed:
not specified

Table 1. Litter size, birth rate, survival rate, and sex ratio

DOSE: (mg/kg)

0

125

250

500

Mean litter size

Birth rate (%)

Postpartum 0 day’s survival rate (%)

Postpartum 4 day’s survival rate (%)

Postpartum 0 day’s sex ratio (%)

Postpartum 4 day’s sex ratio (%)

14.2

96.8

96.8

97.4

  1.0

 1.0

   14.3

100

100

   98.8

   1.3

   1.3

14.4

98.0

98.0

98.5

 1.1

 1.1

14.6

97.3

97.3

98.6

 0.9

 0.8

Table 2. Body weight(B.W.) and Crown rump length(CRL) of neonates

Sex of neonates

 

Male

Female

Group/

Dose(mg/kg)

 

G1

0

G2

125

G3

250

G4

500

G1

0

G2

125

G3

250

G4

500

B.W. (g)

(postpartum day 0)

Mean

7.13

7.04

7.21

6.99

6.70

6.63

6.61

6.69

B.W. (g)

(postpartum day

4)

 

Mean

11.50

10.87

11.60

10.62

10.84

10.39

10.65

10.05

CRL (cm)

(postpartum day

0)

 

Mean

4.17

4.27

4.34

4.27

4.11

4.31

4.20

4.16

CRL (cm)

(postpartum day

4)

 

Mean

5.29

5.09

5.28

5.22

5.10

4.86*

5.13

5.01

*: Statistical significance was observed.

 

Table 3. External findings in neonates

Organs

Findings

Group/

Dose (mg/kg)

postpartum day 0

postpartum day 4

 

G1/

0

G2/

125

G3/

250

G4/

500

G1/

0

G2/

125

G3/

250

G4/

500

Tails

Acaudate

 

No. of

neonates

examined

No. of

neonates

founded

 

151

 

 

0

172

 

 

0

197

 

 

0

142

 

 

1

147

 

 

0

170

 

 

0

194

 

 

0

140

 

 

1

 0: No abnormalities detected

1: One case of Acaudate was detected

Conclusions:
NOAEL for developmental toxicity of read-across substance iron dichloride after oral gavage under this test conditions was 500 mg/kg bw/day.
Executive summary:

A combined repeated dose/reproductive & developmental toxicity study was conducted with read-across substance 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 adrenal 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.

Litter size, birth rate, survival rate, and sex ratewere within the normal range. The crown rump lengths (CRL) of neonates were significantly decreased as compared with that of the control group for postpartum day 4 in 125 mg/kg bw/day treatment group (p < 0.01). But the decrease did not correlate with body weights that is a main growth and developmental index. Since there was no dose-dependence of CRL decrease, it was concluded that the test substance did not influence the growth of neonates. A case of acaudate was observed at 500 mg/kg bw/day treatment group. However, because of the low frequency of occurrence, it was not a teratogenic effect.

NOAEL for developmental toxicity of read-across substance iron dichloride after oral gavage under this test conditions was 500 mg/kg bw/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
High quality study
Additional information

A key 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) (OECD SIDS, 2004). 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 adrenal 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. Litter size, birth rate, survival rate, and sex rate were within the normal range. The crown rump lengths (CRL) of neonates were significantly decreased as compared with that of the control group for postpartum day 4 in 125 mg/kg bw/day treatment group (p < 0.01). But the decrease did not correlate with body weights that is a main growth and developmental index. Since there was no dose-dependence of CRL decrease, it was concluded that the test substance did not influence the growth of neonates. A case of acaudate was observed at 500 mg/kg bw/day treatment group. However, because of the low frequency of occurrence, it was not a teratogenic effect. NOAEL for developmental toxicity of iron dichloride after oral gavage under this test conditions was 500 mg/kg bw/day.

 

From a systemic viewpoint, iron dichloride can be considered to be a (worst case) read across source chemical for iron sulfide. Taking into account that water solubility and absorption of iron chloride is much higher than iron sulfide, systemic toxicity of iron chloride is considered to be higher than iron sulfide. From the read across for the acute studies, a benefit factor of at least 6 for the NOAEL of iron sulfide could be argumented. Therefore, if the NOAEL is assumed to be at least 6 times higher, NOAEL for iron sulfide would exceed the limit dose. In any terms, there is no reason for classification. Based on the low absorption rate of iron sulfide (see Section 7.1) and based on the need of iron supplementation during pregnancy (see separate document on iron supplementation), further developmental toxicity testing of iron sulfide is considered unnecessary from a scientific viewpoint.

Justification for classification or non-classification

Iron sulfide does not have to be classified for reproductive toxicity according the CLP Regulation No. 1272/2008 of 16 December 2008.