Iron sulphide

Genetic toxicity: in vitro

Administrative data

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Iron sulphide

EC Number:

215-268-6

EC Name:

Iron sulphide

Cas Number:

1317-37-9

Molecular formula:

FeS

IUPAC Name:

sulfanylideneiron

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Method

Target gene:

hisitdine; (Salmonella typhimurium)
tryptophan; (Escherichia coli)

Species / strain

Species / strain 1

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 2

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.

Controls

Controls 1

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)

Controls 2

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Salmonella typhimurium TA 98 without S9 (2µg/plate)

Controls 3

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Salmonella typhimurium TA 100, TA 1535 without S9 (1µg/plate)

Controls 4

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Salmonella typhimurium TA 1537 without S9 (50µg/plate)

Controls 5

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.

Results and discussion

Test results

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

Any other information on results incl. tables

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

Treatment [µg/plate]	No. of revertants/plate^a
	TA98			TA100			TA1535			TA1537			WP2uvrA (pKM101)
	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b
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	524^c	18^c	19.89^c	868^c	17^c	7.55^c	142^c	17^c	9.91^c	123^c	12^c	11.90^c	553^d	4^d	4.09^d
^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/plate^a
	TA98			TA100			TA1535			TA1537			WP2uvrA (pKM101)
	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b
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	237^c	6^c	9.34^c	549^d	16^d	4.96^d	144^d	14^d	11.97^d	114^e	9^e	10.66^e	557^f	16^f	4.01^f
^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: 2-Nitrofluorene (2 µg/plate),                                              ^dTA100, TA1535: Sodium azide (1 µg/plate)                                ^eTA1537: 9-Aminoacridine (50 µg/plate)                                        ^fW 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/plate^a
	TA98			TA100			TA1535			TA1537			WP2uvrA (pKM101)
	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b
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	520^c	10^c	21.07^c	882^c	34^c	8.40^c	153^c	7^c	11.79^c	113^c	6^c	10.97^c	559^d	16^d	4.25^d
^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 4. contd.Summary Results of Confirmatory Bacterial Reverse Mutation Assay in the Absence of Metabolic Activation

Treatment [µg/plate]	No. of revertants/plate^a
	TA98			TA100			TA1535			TA1537			WP2uvrA (pKM101)
	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b
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	239^c	9^c	9.55^c	552^d	20^d	5.30^d	145^d	16^d	10.63^d	115^e	9^e	11.10^e	565^f	9^f	4.21^f
^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: 2-Nitrofluorene (2 µg/plate),                                              ^dTA100, TA1535: Sodium azide (1 µg/plate)                                ^eTA1537: 9-Aminoacridine (50 µg/plate)                                        ^fWP2uvrA(pKM 101): 4-Nitroquinoline-1-oxide (4 µg/plate)     NA: Not applicable

Applicant's summary and conclusion

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.