Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A full batery of genotoxcity studies was available, composed of an Ames test with iron sulfide, an Ames test for iron dichloride, and an Ames test and mammalian gene mutation for iron trichloride. Both iron dichloride and iron trichloride were used as source substances for iron sulfide. All tests were negative for test article related effects, therefore iron sulfide is considered to have no genotoxic potential.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012
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.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
hisitdine; (Salmonella typhimurium)
tryptophan; (Escherichia coli)
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Details on mammalian cell type (if applicable):
- Type and identity of media :Soft agar containing 0.5 mM histidine and biotin or 5 μg/mL tryptophan
- Properly maintained: yes
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
- Type and identity of media :Soft agar containing 0.5 mM histidine and biotin or 5 μg/mL tryptophan
- Properly maintained: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Preliminary toxicity test: Salmonella typhimurium tester strain TA100 at 50, 100, 200, 400, 800, 1600, 3200 and 5000 µg/plate test doses along with a sterile water control
Mutation assay: 50, 158, 500, 1580 and 5000 µg/plate (plate incorporation)
Confirmatory mutation assay: 100, 266, 707, 1880 and 5000 µg/plate (preincubation).
In a preliminary toxicity test, the mean number of revertant colonies was more or less comparable to the SW control plates up to the highest tested dose of 5000 μg/plate, both in the presence and absence of metabolic activation. No toxicity of the test item was seen as the intensity of the bacterial background lawn was comparable to that of the SW control plates up to 5000 μg/plate, both in the presence and absence of metabolic activation. The test item did not cause precipitation on the basal agar plates in any of the tested doses either in the presence or in the absence of metabolic activation. Based on these observations, 5000 µg/plate was tested as the maximum dose in the initial as well as the confirmatory mutation assay.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: sterile water
- Justification for choice of solvent/vehicle: Sterile water is one of the compatible vehicles with this test system.
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoantrhacene
Remarks:
Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 with S9 (4µg/plate) and E.Coli WP2uvrA (pKM101) with S9 (30µg/plate)
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
Salmonella typhimurium TA 98 without S9 (2µg/plate)
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Salmonella typhimurium TA 100, TA 1535 without S9 (1µg/plate)
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Salmonella typhimurium TA 1537 without S9 (50µg/plate)
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 4-Nitroquinoline-1-oxide
Remarks:
E.Coli WP2uvrA (pKM101) without S9 (4µg/plate)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) in the initial mutation assay;
Preincubation in the confirmatory assay

DURATION
- Preincubation period: no data
- Exposure duration: 67 hours

SELECTION AGENT (mutation assays): histidine/ tryptophan

NUMBER OF REPLICATIONS: 3 (initial and confirmatory mutation assay); 2 (preliminary toxicity test)

DETERMINATION OF CYTOTOXICITY
- Method: other: mean number of revertant colonies/plate compared to the respective vehicle controls; intensity of the bacterial background lawn compared to that of the controls; precipitation
Evaluation criteria:
To determine a positive result, there should be a dose related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of the test item either in the presence or absence of the metabolic activation system.
The test will be judged positive, if the increase in mean revertants at the peak of the dose response is equal to or greater than 2 times the mean vehicle control value for strains TA98, TA100 and WP2uvrA (pKM101) or equal to or greater than 3 times the mean vehicle control value for strains TA1535 and TA1537.
An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose responsive increase that does not achieve the respective threshold cited above or a non dose responsive increase that is equal to or greater than the respective threshold cited. A response will be evaluated as negative, if it is neither positive nor equivocal.
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Stability and homogeneity analysis results of dose formulations were provided.
The test item was found to be homogeneous and stable in sterile water after 24 hours at 500 and 100000 μg/mL at room temperature.
The concentration analysis results for the initial mutation assay indicate that the actual mean concentrations of the analyzed dose levels were between 91.1 and 104.8 % of their respective nominal target dose. The concentration analysis results of the dose formulation samples of the confirmatory mutation assay indicate that the actual mean concentrations of the analyzed dose levels were between 93.5 and 108.4 % of their respective nominal target concentrations.
Remarks on result:
other: all strains/cell types tested

Table 1. Summary Results of Initial Bacterial Reverse Mutation Assay in the Presence of Metabolic Activation

Treatment

[µg/plate]

No. of revertants/platea

TA98

TA100

TA1535

TA1537

WP2uvrA

(pKM101)

Mean

SD

Ratiob

Mean

SD

Ratiob

Mean

SD

Ratiob

Mean

SD

Ratiob

Mean

SD

Ratiob

 

Vehicle control -

SW(100 µL)

26

2

NA

115

8

NA

14

1

NA

10

1

NA

135

4

NA

 

50

27

1

1.01

112

3

0.98

15

2

1.02

11

1

1.06

135

8

1.00

 

158

27

2

1.04

111

9

0.97

12

2

0.81

11

1

1.03

138

3

1.02

 

500

24

2

0.92

116

5

1.01

13

1

0.93

10

1

1.00

137

9

1.01

 

1580

22

3

0.82

105

7

0.91

15

1

1.02

10

1

0.94

129

3

0.96

 

5000

26

2

1.00

114

12

0.99

14

2

1.00

11

1

1.06

132

3

0.98

 

Positive control

524c

18c

19.89c

868c

17c

7.55c

142c

17c

9.91c

123c

12c

11.90c

553d

4d

4.09d

aValues are means of three replicates and are rounded off to the nearest whole number.           

bRatio of treated/vehicle control (mean revertants per plate)         cTA98, TA100, TA1535, TA1537: 2-Aminoanthracene (4 µg/plate)

dWP2uvrA(pKM 101): 2-Aminoanthracene (30 µg/plate)            NA: Not applicable

Table 2. contd.Summary Results of Initial Bacterial Reverse Mutation Assay in the Absence of Metabolic Activation

Treatment

[µg/plate]

No. of revertants/platea

TA98

TA100

TA1535

TA1537

WP2uvrA

(pKM101)

Mean

SD

Ratiob

Mean

SD

Ratiob

Mean

SD

Ratiob

Mean

SD

Ratiob

Mean

SD

Ratiob

 

Vehicle control -

SW(100 µL)

25

3

NA

111

6

NA

12

2

NA

11

3

NA

139

2

NA

 

50

25

2

0.99

115

4

1.04

11

2

0.94

10

2

0.97

134

4

0.97

 

158

25

3

1.00

115

8

1.04

14

4

1.17

11

3

1.03

134

3

0.96

 

500

26

2

1.03

114

9

1.03

13

3

1.08

10

1

0.94

137

11

0.99

 

1580

27

2

1.08

107

12

0.97

12

3

1.03

11

1

1.00

137

3

0.99

 

5000

27

3

1.07

106

4

0.96

12

3

1.00

11

1

1.06

137

2

0.99

 

Positive control

237c

6c

9.34c

549d

16d

4.96d

144d

14d

11.97d

114e

9e

10.66e

557f

16f

4.01f

a Values are means of three replicates and are rounded off to the nearest whole number.           

b Ratio of treated/vehicle control (mean revertants per plate)         c TA98: 2-Nitrofluorene (2 µg/plate),                                             

d TA100, TA1535: Sodium azide (1 µg/plate)                                e TA1537: 9-Aminoacridine (50 µg/plate)                                       

f W P2uvrA(pKM 101): 4-Nitroquinoline-1-oxide (4 µg/plate)     NA: Not applicable                 

Table3.      Summary Results of Confirmatory Bacterial Reverse Mutation Assay in the Presence of Metabolic Activation

Treatment

[µg/plate]

No. of revertants/platea

TA98

TA100

TA1535

TA1537

WP2uvrA

(pKM101)

Mean

SD

Ratiob

Mean

SD

Ratiob

Mean

SD

Ratiob

Mean

SD

Ratiob

Mean

SD

Ratiob

 

Vehicle control -

SW(100 µL)

25

1

NA

105

9

NA

13

2

NA

10

2

NA

131

2

NA

 

100

24

3

0.96

104

7

0.99

15

3

1.13

11

2

1.06

129

2

0.98

 

266

26

5

1.05

109

10

1.03

13

2

1.00

10

1

0.97

134

2

1.02

 

707

23

3

0.95

102

3

0.97

14

4

1.10

10

1

0.97

135

4

1.03

 

1880

25

4

1.01

105

4

1.00

13

3

1.03

11

1

1.03

136

3

1.04

 

5000

24

4

0.97

109

9

1.03

15

1

1.13

11

3

1.10

133

2

1.01

 

Positive control

520c

10c

21.07c

882c

34c

8.40c

153c

7c

11.79c

113c

6c

10.97c

559d

16d

4.25d

a Values are means of three replicates and are rounded off to the nearest whole number.           

b Ratio of treated/vehicle control (mean revertants per plate)         c TA98, TA100, TA1535, TA1537: 2-Aminoanthracene (4 µg/plate)

d WP2uvrA(pKM 101): 2-Aminoanthracene (30 µg/plate)            NA: Not applicable

Table 4. contd.Summary Results of Confirmatory Bacterial Reverse Mutation Assay in the Absence of Metabolic Activation

Treatment

[µg/plate]

No. of revertants/platea

TA98

TA100

TA1535

TA1537

WP2uvrA

(pKM101)

Mean

SD

Ratiob

Mean

SD

Ratiob

Mean

SD

Ratiob

Mean

SD

Ratiob

Mean

SD

Ratiob

 

Vehicle control -

SW(100 µL)

25

2

NA

104

3

NA

14

4

NA

10

4

NA

134

7

NA

 

100

24

2

0.95

107

9

1.03

13

3

0.93

11

2

1.03

129

7

0.97

 

266

27

3

1.07

101

1

0.97

12

5

0.90

11

2

1.03

135

5

1.01

 

707

23

3

0.92

101

3

0.97

13

3

0.95

10

2

1.00

132

3

0.99

 

1880

25

3

0.99

105

5

1.01

14

3

1.02

11

3

1.10

133

3

1.00

 

5000

25

3

0.99

112

8

1.07

13

3

0.93

11

3

1.10

134

2

1.00

 

Positive control

239c

9c

9.55c

552d

20d

5.30d

145d

16d

10.63d

115e

9e

11.10e

565f

9f

4.21f

a Values are means of three replicates and are rounded off to the nearest whole number.           

b Ratio of treated/vehicle control (mean revertants per plate)         c TA98: 2-Nitrofluorene (2 µg/plate),                                             

d TA100, TA1535: Sodium azide (1 µg/plate)                                e TA1537: 9-Aminoacridine (50 µg/plate)                                       

f WP2uvrA(pKM 101): 4-Nitroquinoline-1-oxide (4 µg/plate)     NA: Not applicable                                                   

Conclusions:
Interpretation of results: negative with and without metabolic activation

All criteria for a valid study were met as described in the study plan. It is concluded that the test item, Tribotecc®-Ferrostar, was not mutagenic in this bacterial reverse mutation test at the tested doses and under the conditions of testing employed.
Executive summary:

The test item, iron sulfide (Tribotecc®-Ferrostar) was tested for its mutagenic potential in the bacterial reverse mutation assay. The study was conducted using TA98, TA100, TA1535 and TA1537 strains of Salmonella typhimurium and WP2uvrA (pKM101) strain of Escherichia coli. The study consisted of a preliminary toxicity test and two mutation assays (plate incorporation and preincubation) comprising four independent experiments. The bacterial tester strains were exposed to the test item in the presence and absence of metabolic activation system (S-9 fraction prepared from Aroclor 1254 induced rat liver).

In a preliminary toxicity test, the mean number of revertant colonies was more or less comparable to the SW control plates up to the highest tested dose of 5000 µg/plate, both in the presence and absence of metabolic activation. No toxicity of the test item was seen as the intensity of the bacterial background lawn was comparable to that of the SW control plates up to 5000 µg/plate, both in the presence and absence of metabolic activation. The test item did not cause precipitation on the basal agar plates in any of the tested doses either in the presence or in the absence of metabolic activation. Based on these observations, 5000µg/plate was tested as the maximum dose in the initial as well as the confirmatory mutation assay.

In the initial mutation assay, the test item was exposed in triplicate to 50, 158, 500, 1580 and 5000 µg/plate test doses in the presence and absence of metabolic activation using direct plate incorporation procedure. In the confirmatory assay, the test item was exposed in triplicate to concentrations of 100, 266, 707, 1880 and 5000 µg/plate in the presence and absence of metabolic activation using pre-incubation procedure. The vehicle control (SW) and the appropriate positive controls were tested simultaneously. The mean and standard deviation of numbers of revertant colonies were calculated for each test dose and the controls for all the tester strains.

The results from the initial as well as from the confirmatory assays, indicate the tested doses showed no positive mutagenic increase in the mean numbers of revertant colonies for all tester strains when compared to the respective vehicle control plates, either in the presence or absence of metabolic activation.The mean numbers of revertant colonies neither doubled for strains TA98, TA100 and WP2uvrA (pKM101) nor tripled for strains TA1535 and TA1537 when compared to the respective vehicle control plates, either in the presence or in the absence of the metabolic activation at any of the tested doses.The study indicated that Tribotecc®-Ferrostar was not mutagenic in this Bacterial Reverse Mutation Assay up to the highest tested dose of 5000 µg/plate.

Endpoint:
in vitro gene mutation study in mammalian cells
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
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
With the exception of electrolytic Fe, treatment of the mouse lymphoma cells with Ferric chloide and all other Fe compounds resulted in positive responses. With FeCl3 in the absence of S9, there was no increase in the mutant frequency. With the addition of S9, there was an increase in the number of induced mutants. This can be attributed to an increase in the production of Fe 2+, from the Fe 3+ in the presence of the NADP and the reductase in the liver S9 mix. The change in the equilibrium reaction, that is, increasing the concentration of Fe 2+ from the oxidation of Fe 3+, increases the absorption or uptake of Fe into the cell.

It was previously suggested that mutagenesis induced by metals may proceed by the generation of reactive intermediates of reduced oxygen. The activation of the oxygen species, H2O2 and superoxide (O2-) by Fe may result in the formation of the hydroxyl radical ( °OH), the most powerful DNA-damaging radical. Using spin-trapping ESR spectroscopy, Rashba-Step et al. [1993] reported that there was production of reactive oxygen intermediates when rat liver microsomes were incubated with NADPH as a cofactor and an Fe catalyst. Thus, in the presence of Fe, NADPH, and liver microsomes, O2- and H2O2 may serve as precursors for the production of the° OH through the Fenton reaction:
Fe 2+ + H202 --> Fe 3+ + °OH +1 OH-

Without metabolic activation, there was no increase in the number of induced mutants. However, in the presence of S9, there was a dose-related mutagenic response, with a marked increase in cytotoxicity.

Colony size was also determined for each compound. There was no increase in the number of small colonies, and the proportion of large and small colonies remained constant for all compounds.

Table 1. Results Ferric chloride (Fe+++) without S9 (Dunkel et al.)

 

Dose

(µg/mL)

Absolute cloning efficiency

Relative total growth (% of control)

Average number TFT colonies

Mutant frequency per 106survivors

Solvent control (Distilled water)

0

0.54/0.59/0.63/0.51

100.0

17/17/12/12

26

Ferric chloride

309.0

412.0

515.0

618.0

1030.0

0.63/0.66

0.69/0.70

0.83/0.57

0.68

0.48/0.62

49.5

50.5

32.5

35.0

12.5

14/22

15/15

25/15

28/22

25/28

28

24

28

32

49

Positive control (EMS)

 

0.36

49.0

162

450

 

Table 2. Results Ferric chloride (Fe+++) with S9 (Dunkel et al.)

 

Dose

(µg/mL)

Absolute cloning efficiency

Relative total growth (% of control)

Average number TFT colonies

Mutant frequency per 106survivors

Solvent control (Distilled water)

0

0.71/0.74/0.70

100.0

33/27/35

44

Ferric chloride

0.206

0.412

0.824

1.030

1.236

0.81/0.81

0.80/0.76

0.68/0.69

0.67/0.72

0.66/0.69

115.5

94.5

85.5

76.5

66.0

41/31

70/40

64/70

75/73

70/92

45

50

98

107

121

Positive control (MCA)

 

0.29

16.0

149

447

 

Conclusions:
Interpretation of results:
negative without metabolic activation
positive with metabolic activation

With read-acorss substance ferric chloride (FeCl3), there was an increase in mutant frequency only with S9. With the exception of electrolytic Fe, treatment of the mouse lymphoma cells with Ferric chloride and all other Fe compounds resulted in positive responses.
Executive summary:

The mutagenic activity of elemental and salt forms of iron (Fe), including compounds currently being used in dietary supplements and for food fortification, were evaluated for mutagenicity in L5178Y mouse lymphoma cells. Responses with the elemental forms of Fe were divergent. Electrolytic Fe with a relatively larger particle size and irregular shape was negative. The smaller-sized carbonyl Fe, which after 4 hr attached to and was taken up by the cells, induced mutagenic responses both with and without S9. With read-across substance ferric chloride (FeCl3) and other ferric salts, there was an increase in mutant frequency only with S9. This can be attributed to an increase in the production of Fe 2+, from the Fe 3+ in the presence of the NADP and the reductase in the liver S9 mix. The change in the equilibrium reaction, that is, increasing the concentration of Fe 2+ from the oxidation of Fe 3+, may increase the absorption or uptake of Fe into the cell. Further mechanism on the generation of reactive intermediates of reduced oxygen were discussed.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

A key in vivo Micronucleus assay with iron dichloride was used as source substance for iron sulfide. The test was negative, therefore iron sulfide is also considered to have no genotoxic potential.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Justification for type of information:
See attached read-across justification
Reason / purpose:
read-across source
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Positive controls validity:
valid

Main tests:

After first dosing, one animal of 50 mg/kg bw/day dose level was dead and piloerection was observed in all animals of 25 and 50 mg/kg bw/day groups. After second dosing, piloerection and hypoactivity were observed in three animals of 50 mg/kg bw/day group. Any clinical sign was not observed in 12.5 mg/kg bw/day group.

Table1. Summary of PCE/(PCE+NCE) ratio and MNPCE frequency

Treatment group

PCE/(PCE+NCE) (mean)

Group mean frequency of MNPCE per 2000 PCE (mean±S.D.)

 

Vehicle (10 mL/kg)

Iron dichloride (12.5 mg/kg)

Iron dichloride (25 mg/kg)

Iron dichloride (50 mg/kg)

Mitomycin C (2 mg/kg)

0.58

0.61

0.56

0.51

0.54

3.8 ± 2.3

3.8 ± 1.6

3.7 ± 1.5

2.0 ± 1.0

185.7 ± 16.3SS

SS: Statistical significance was observed (p ≤ 0.05)

Conclusions:
Interpretation of results: negative
Read-across substance iron dichloride did not induce micronuclei in the mice bone marrow cells under the test conditions.
Executive summary:

6 Male ICR mice per dose level were administered read-across substance iron dichloride by intraperitoneal injection at dose levels of 0, 12.5, 25 and 50 mg/kg bw once daily for two days. The negative controls were administered 10 mL/kg corn oil (vehicle) in the same way. The positive control animals received Mitomycin C 2 mg/kg only once. After first dosing, one animal of 50 mg/kg bw/day dose level was dead and piloerection was observed in all animals of 25 and 50 mg/kg bw/day groups. After second dosing, piloerection and hypoactivity were observed in three animals of 50 mg/kg bw/day group. Any clinical sign was not observed in 12.5 mg/kg bw/day group. Read-across substance iron dichloride did not induce micronuclei in the mice bone marrow cells under the test conditions.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

A key study for Ames bacterial mutagenicity was done with iron sulfide (Tribotecc®-Ferrostar) using TA98, TA100, TA1535 and TA1537 strains of Salmonella typhimurium and WP2uvrA (pKM101) strain of Escherichia coli (Suresh,2012). In a preliminary toxicity test, no toxicity of the test item was seen as the intensity of the bacterial background lawn was comparable to that of the SW control plates up to 5000 µg/plate, both with and without metabolic activation. The test item did not cause precipitation on the basal agar plates in any of the tested doses. In the initial mutation assay, the test item was exposed in triplicate to 50, 158, 500, 1580 and 5000 µg/plate test doses in the presence and absence of metabolic activation using direct plate incorporation procedure. In the confirmatory assay, the test item was exposed in triplicate to concentrations of 100, 266, 707, 1880 and 5000 µg/plate in the presence and absence of metabolic activation using pre-incubation procedure. The vehicle control (SW) and the appropriate positive controls were tested simultaneously. The results from the initial as well as from the confirmatory assays, indicate the tested doses showed no positive mutagenic increase in the mean numbers of revertant colonies for all tester strains when compared to the respective vehicle control plates, either in the presence or absence of metabolic activation. The study indicated that Tribotecc®-Ferrostar was not mutagenic in this Bacterial Reverse Mutation Assay up to the highest tested dose of 5000 µg/plate. A support Ames test was available for iron dichloride (OECD SIDS, 2004). Preliminary tests were carried out using dose levels with 5-fold intervals of 1.6, 8, 40, 200, 1000 and 5000 μg/plate to determine the dose range. In the main test Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 and Escherichia coli WP2uvrA were tested in triplicate after preincubation at 33.3, 100, 300, 1000, 3000 and 5000 μg iron dichloride/plate in absence and presence of metabolic activation (S9 -mix). Precipitation was observed at and above 1000 μg/plate. Iron dichloride did not cause the reverse mutations in the Salmonella typhimurium (strains TA 98, TA 100, TA 1535 and TA 1537) and Escherichia coli (strain WP2 uvrA) in presence and in absence of metabolic activation (S9). The positive controls were valid. Based on this anchorpoint as well as previous anchorpoints, further genotoxicity studies were used from iron dichloride. A support Ames test was also available for iron trichlorid (Dunkel et al, 1999). The mutagenic activity of elemental and salt forms of iron (Fe), including compounds currently being used in dietary supplements and for food fortification, were evaluated for mutagenicity in Salmonella typhimurium. The mutagenic response of Ferric chloride in Salmonella typhimurium strains TA98, TA100, TA102, TA1535, TA1537 and TA1538 was negative under the test conditions of this test.

 

The mutagenic activity of elemental and salt forms of iron (Fe), including compounds currently being used in dietary supplements and for food fortification, were evaluated for mutagenicity in L5178Y mouse lymphoma cells (Dunkel et al, 1999). Responses with the elemental forms of Fe were divergent. Electrolytic Fe with a relatively larger particle size and irregular shape was negative. The smaller-sized carbonyl Fe, which after 4 hr attached to and was taken up by the cells, induced mutagenic responses both with and without S9. With ferric chloride (FeCl3) and other ferric salts, there was an increase in mutant frequency only with S9. This can be attributed to an increase in the production of Fe 2+, from the Fe 3+ in the presence of the NADP and the reductase in the liver S9 mix. The change in the equilibrium reaction, that is, increasing the concentration of Fe 2+ from the oxidation of Fe 3+, may increase the absorption or uptake of Fe into the cell. Further mechanism on the generation of reactive intermediates of reduced oxygen were discussed. However, as there is a 24 -month carcinogenicity study in rats with iron trichloride, showing negative results for test substance related tumors (see Section 7.7), the in vitro results is considered to be overruled by in vivo results and therefore not relevant.

 

A key in vivo Micronucleus test was available for iron dichloride tested in male ICR mice dosed by intraperitoneal injection at 0, 12.5, 25 and 50 mg/kg bw once daily for two days. The negative controls were administered 10 mL/kg corn oil (vehicle) in the same way. The positive control animals received Mitomycin C 2 mg/kg only once.After first dosing, one animal of 50 mg/kg bw/day dose level was dead and piloerection was observed in all animals of 25 and 50 mg/kg bw/day groups. After second dosing, piloerection and hypoactivity were observed in three animals of 50 mg/kg bw/day group. Any clinical sign was not observed in 12.5 mg/kg bw/day group. Iron dichloride did not induce micronuclei in the mice bone marrow cells under the test conditions.

 

The read across between the acute toxicity of iron dichloride (source chemical) and iron sulfide (target chemical), was further elaborated with the results of iron dichloride and iron trichloride in the Ames test, which both showed comparable (negative) results to those of iron sulfide. Also in this case, iron dichloride and iron trichloride are considered as worst case comparators for the mammalian gene mutation and chromosomal aberration endpoints.

Justification for classification or non-classification

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