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Description of key information

A key combined repeated dose/reproductive & developmental toxicity study for subacute oral toxicity in rats was available for read across substance iron dichloride, showing NOAEL values of 125 and 250 mg/kg bw for males and females, respectively. Identified target organs were the liver, stomach and adrenal gland. Read across with iron dichloride is justified, although a factor 6 was considered in benefit of iron sulfide, which would bring the NOAEL to 750 mg/kg bw. The relevance of the stomach as target organ for iron sulfide is questioned, as it has no corrosive proporties. A 90-day study was waived based on the availability of a carcinogenicity study.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
See attached read-across justification
Reason / purpose:
read-across source
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.


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
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
Critical effects observed:
not specified

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.

Conclusions:
By the particular test results such as the rate of body weight gain, water consumption, organ weights, necropsy, and histopathology, NOAEL values of read-across substance Iron dichloride were determined to 125 mg/kg bw/day for males and 250 mg/kg bw/day for females.
Executive summary:

Repeated dose toxicity of read-across substance 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 of read-across substance Iron dichloride were determined to 125 mg/kg bw/day for males and 250 mg/kg bw/day for females.

For iron sulfide, read across with iron dichloride is justified, although a factor 6 was considered in benefit of iron sulfide, which would bring the NOAEL to 750 mg/kg.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
750 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
High quality study
System:
hepatobiliary
Organ:
adrenal glands
liver
stomach

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

A key combined repeated dose/reproductive & developmental toxicity 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). 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.

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. Target organs identified were the liver, the stomach and the adrenal gland.

 

The 90 -day subchronic oral toxicity study was waived, as a 24 -month oral carcinogenicity study with iron dichloride is available (see section 7.7). Subacute and subchronic inhalation and dermal toxicity testing were waived as the oral route was demonstrated to be the most appropriate route for testing based on toxicokinetic reasons.

 

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, a NOAEL of 750 mg/kg bw is assumed for iron sulfide. The relevance of the stomach as target organ for iron sulfide is questioned, as it has no corrosive properties. In any terms, there is no reason for classification.

Justification for classification or non-classification

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