Hematite (Fe2O3)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline study, unpublished report available, no restrictions, fully adequate for assessment

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine and tryptopahn

Species / strain / cell type:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

S-9 homogenate of the liver of rats treated with Aroclor 1254

Test concentrations with justification for top dose:

first trial: 50, 158, 500, 1581 and 5000 µg/plate
second trial: 100, 266, 707, 1880 and 5000 µg/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene; 9-aminoacridine; 2-nitrofluorene; 4-nitroquinoline-1-oxide; sodium azide

Details on test system and experimental conditions:

Preliminary Toxicity Test
One hundred microlitres each of the respective dilutions and the stock equivalent to a concentration of 16, 32, 64, 128, 256, 512, 1024, 2048 and 5000 µg was mixed with 2 ml of soft agar containing Histidine and Biotin, 500 µl of S-9 mix (for the test in the presence of metabolic activation) or 500 µl of PBS (for the test in the absence of metabolic activation), 0.1 ml of overnight TA 100 culture and overlaid onto pre-labeled VB agar plates in duplicate. A DMSO control, similarly treated, was maintained. After the agar had set, these plates were incubated at 37°C for 48 hours.
The number of revertant colonies on the VB agar plates was counted and the bacterial background lawn was observed. Toxicity was detectable as a decrease in the number of revertant colonies per plate and/or by a thinning or disappearance of the bacterial background lawn. If the test item is toxic, the highest concentration of the test item used in the subsequent mutagenicity assay will be that which gives a detectable reduction in the number of revertants on the selective agar plates and/or a thinning or disappearance of the bacterial background lawn.

Mutation Test
No. of Replicates: 3
The bacterial suspension of each tester strain was diluted up to 10-6 (six serial dilution) dilution in phosphate buffered saline. After dilution, 0.1 ml from the highest dilution of each tester strain was plated onto nutrient agar plates in triplicate. The plates were incubated at 37°C for 48 hours for both the trials mutation test. After incubation, the number of colonies in each plate were counted and colony forming units per ml of the suspension.

OBSERVATIONS
Effect on Bacterial Background Lawn: The condition of the bacterial background lawn was evaluated for evidence of the test item toxicity using the code system.
Number of Revertants: Revertant colonies for all given strains, for all groups were counted manually.
Viable Counts: Viable counts for the different bacterial strains on nutrient agar plates were counted manually.

Evaluation criteria:

Conditions necessary for determining a positive result are: there should be a concentration related increase over the range tested and/or a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain, either in the presence or absence of the metabolic activation system.

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeds the threshold level of twice [strains TA 98, TA100 and WP2 uvrA (pKM 101)], or thrice (strains TA 1535 and TA 1537) the colony count when compared to the corresponding vehicle control and this should be evident at a minimum of three dose levels.

Statistics:

Data were analyzed for differences among vehicle control, treatment and positive control groups using ANOVA. Differences between individual treatment and vehicle control was tested by Dunnet's 't' test at a 5% level (p < 0.05) of significance.

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

but tested up to the limit concentration

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

but tested up to the limit concentration

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

but tested up to the limit concentration

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

but tested up to the limit concentration

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

but tested up to the limit concentration

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

PRELIMINARY TOXICITY TEST
The number of revertant colonies was comparable to that of the respective vehicle control plates up to the highest tested concentration of 5000 µg/plate, both in the presence and absence of metabolic activation. Similarly, the intensity of the bacterial background lawn was comparable to that of the vehicle control plates up to 5000 µg/plate, both in the presence and absence of metabolic activation.

MUTATION TEST
Trial 1 was conducted using 5000 µg/plate as the maximum concentration, both in the presence and in the absence of metabolic activation. Since, the results of the Trial 1 were negative, a confirmatory trial (Trial 2) was conducted by modifying the method of exposure to treatment and also the concentration spacing of the test item up to a maximum of 5000 µg/plate, both in the presence and in the absence of metabolic activation.
Viable Counts:
The viable counts determined for all the tester strains were within the required range of 1 - 2 x 10E9 CFU/ml, in the first as well as the confirmatory trial.
Number of Revertants:
TRIAL 1: The mean number of revertant colonies/plate in the DMSO control was within the range of in-house spontaneous revertant counts for all the tester strains. For all the tester strains, the mean number of revertant colonies was statistically comparable to or lesser than that of the vehicle control at all the tested concentrations, both in the presence and absence of metabolic activation. The intensity of the bacterial background lawn was comparable to that of the vehicle control plates up to 5000 µg/plate, both in the presence and absence of metabolic activation. The specific positive control chemicals tested simultaneously produced a significantly high increase in the mean revertant colonies compared to the vehicle control plates.
TRIAL 2: The mean number of revertant colonies/plate in the DMSO control was within the range of in-house spontaneous revertant counts for all the tester strains. For all the tester strains, the mean number of revertant colonies was statistically comparable to or lesser than that of the vehicle control at all the tested concentrations, both in the presence and absence of metabolic activation. The intensity of the bacterial background lawn was comparable to that of the vehicle control plates up to 5000 µg/plate both in the presence and absence of metabolic activation. The specific positive control chemicals tested simultaneously produced a significantly high increase in the mean revertant colonies compared to the vehicle control plates.

Mean number of Revertant Colonies: The means and standard deviations of the two trials indicated no doubling of mean numbers of revertant colonies in strains TA98, TA100 and WP2 uvrA (pKM 101) or tripling of mean numbers of revertant colonies in strains TA1535 and TA1537 in any of the five concentrations tested as compared to the respective vehicle controls, presence or absence of metabolic activation.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Summary of Bacterial Reverse Mutation Test

 

Group	Test Item concentration (µg/plate)	No. of revertants/plate*
		Presence of Metabolic activation
		TA98		TA100		TA1535		TA1537		WP2 uvrA (pKM 101)
		Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
G1	Vehicle control – DMSO	19	1	113	5	16	1	16	0	102	6
G2	50	18	2	111	4	16	1	16	1	100	3
G3	158	18	1	105	5	15	0	15	1	98	5
G4	500	18	2	136	2	14-	1	14	1	95	2
G5	1581	17	2	93-	3	15	0	13-	2	87-	7
G6	5000	15-	1	90-	3	12-	1	11-	1	83-	7
G7	Positive control	615+	24	805+	37	118+	6	127+	13	563+	34
*: Values are means of three replicates                                           +/-: Significantly higher/lower than the vehicle control

 

Group	Test Item concentration (µg/plate)	No. of revertants/plate*
		Absence of Metabolic activation
		TA98		TA100		TA1535		TA1537		WP2 uvrA (pKM 101)
		Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
G1	Vehicle control – DMSO	18	1	109	3	15	1	15	1	98	7
G2	50	18	1	108	3	15	1	15	1	96	3
G3	158	17	1	103	3	14	2	14	0	97	5
G4	500	15	2	102	2	15	1	14	1	87	4
G5	1581	14-	2	98-	5	13	1	11-	2	85-	5
G6	5000	11-	2	83-	7	12-	1	8-	1	82-	4
G7	Positive control	172+	16	578+	38	139+	11	132+	9	589+	37
*: Values are means of three replicates                                           +/-: Significantly higher/lower than the vehicle control

 

 

Group	Test Item concentration (µg/plate)	No. of revertants/plate*
		Presence of Metabolic activation
		TA98		TA100		TA1535		TA1537		WP2 uvrA (pKM 101)
		Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
G1	Vehicle control – DMSO	17	1	109	3	15	2	13	2	98	6
G2	100	17	2	107	4	15	1	14	1	96	4
G3	266	17	1	104	9	15	1	14	2	94	2
G4	707	16	2	97	5	14	2	13	2	94	5
G5	1880	16	1	95	7	13	2	12	1	88	3
G6	5000	13-	2	85-	4	11	1	11	1	80-	7
G7	Positive control	+597	47	+776	24	+114	9	+138	18	+571	25
*: Values are means of three replicates                                           +/-: Significantly higher/lower than the vehicle control

 

Group	Test Item concentration (µg/plate)	No. of revertants/plate*
		Absence of Metabolic activation
		TA98		TA100		TA1535		TA1537		WP2 uvrA (pKM 101)
		Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
G1	Vehicle control – DMSO	17	1	105	6	14	1	14	2	99	5
G2	100	17	1	103	1	15	1	15	1	93	5
G3	266	14	2	102	4	15	1	13	2	90	4
G4	707	15	2	94-	3	13	1	12	2	84-	3
G5	1880	13-	2	89-	3	12-	1	11	1	81-	5
G6	5000	10-	1	80-	6	10-	1	8-	1	77-	2
G7	Positive control	160 +	14	565 +	48	135 +	8	129 +	10	590+	38
*: Values are means of three replicates                                           +/-: Significantly higher/lower than the vehicle control

Conclusions:

Interpretation of results (migrated information):
negative without metabolic activation all strains tested
negative with metabolic activation all strains tested

Sulfur dust was not mutagenic in the Ames test up to the highest tested concentration of 5000 µg/plate.

Executive summary:

The genotoxic effect of sulfur dust was studied using the Ames test. The study, according to OECD guideline 471 and under GLP, was conducted using TA98, TA100, TA1535 and TA1537 strains of Salmonella typhimurium and WP2uvrA (pKM 101) strain of Escherichia coll. Two trials were carried out (with two experiments in each trial, in the presence and in the absence of metabolic activation). The test item was tested in triplicate at the concentrations of 50, 158, 500, 1581 and 5000 µg/plate in the first trial and 100, 266, 707, 1880 and 5000 µg/plate in the second trial using DMSO as vehicle. The vehicle control and the appropriate positive controls were tested simultaneously. The mean numbers of revertant colonies for the different concentrations of the test item in the different tester strains was statistically comparable with or lower than those of the respective vehicle control plates, for both the first and the confirmatory trials, either in the presence or in the absence of the metabolic activation, while there was a statistically significant increase in the mean number of revertant colonies in the positive controls under identical conditions.

The study indicated that the test item sulfur dust is not mutagenic in this Ames test up to the highest tested concentration of 5000 µg/plate.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Research publication. Well documented meets generally accepted scientific principles, acceptable for assessment.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

not specified

Principles of method if other than guideline:

Method: other: Clive et al/1975 and 1979

GLP compliance:

not specified

Type of assay:

mammalian cell gene mutation assay

Target gene:

thymidine kinase (TK) gene

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: Fischer's medium for leukemic cells of mice supplemented with 10% horse serum and 0.02% pluronic F-68
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver S9

Test concentrations with justification for top dose:

without S9: 309-1030 µg Fe/mL (i.e. mg Fe/L)
with S9: 0.206-1.236 µg Fe/mL (i.e. mg Fe/L)

Vehicle / solvent:

Vehicle used: Distilled water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

destilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

destilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: no data
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 10-12 days
- Fixation time (start of exposure up to fixation or harvest of cells): 12-14 days

SELECTION AGENT (mutation assays): trifluorothymidine (final concentration 3ug/ml) added to cloning mediumfor mutant selection

NUMBER OF REPLICATIONS: duplicate cultures

NUMBER OF CELLS EVALUATED: 1E+06 for mutant selection, 200/plate for mutant count, colony size range 0.2-1.1 mm, counted with an Artek automated colony counter

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Doses of test compound selected for mutagenicity assay were within the range yielding approximately 0-90% cytotoxicity

Evaluation criteria:

Doubling of mutant frequency over  concurrent solvent treated control value together with dose relationship.

Statistics:

Colonies larger than 0.1 mm diameter were counted with an Artek automated colony counter. Colony size was also determined.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: L5178Y TK+/- 3.7.C mouse lymphoma cells

Table 1: Number of revertants per plate (mean of 2 plates) 

Concentration µg Fe/ml	Mutant frequency*	Growth**	Concentration µg Fe/ml	Mutant frequency*	Growth**
	-S9	-S9		+S9	+S9
0*	25	100	0	27	100
20.1	25	84.5	0.804	53	61.5
50.25	29	72.5	1.005	67	54.5
100.5	32	51.5	1.206	70	41.5
150.75	46	27.5	1.508	88	5.0
201.0	80	10.5
Positive control	430	46	Positive control	171	52

* per 1E+06 survivors

** as % of control

PRECIPITATION CONCENTRATION: None reported

Conclusions:

Interpretation of results (migrated information):
negative

In the mouse lymphoma TK+/- assay ferrous sulphate FeSO4 x 7H2O showed a weak positive response in the absence of metabolic activation at cytotoxic concentrations and a dose-related increase in mutant frequency in the presence of metabolic activation, with marked increase of cytotoxicity.

Executive summary:

A L5178Y TK+/- mouse lymphoma cell assay was conducted with FeSO4 x 7H2O. The cytotoxicity of the test substance was determined with and without metabolic activation (rat liver S9 mix) prior to the mutagenicity test in order to determine the appropriate testing concentrations. The mutagenicity assay was performed in duplicates. As indication of a positive effect doubling of the mutant frequency was used. The mouse lymphoma cells showed with and without metablic activation a weak increase in the number of induced mutants at cytotoxic concentrations.

FeSO4 x 7H2O is negative for mutagenicity in absence and presence of metabolic activation under the conditions of this test system.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline study, unpublished report available, no restrictions, fully adequate for assessment

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Chinese Hamster (Cricetulus griseus) ovary cell line CHO-K1

Metabolic activation:

with and without

Metabolic activation system:

S-9 homogenate of the liver of rats treated with Aroclor 1254

Test concentrations with justification for top dose:

main test, first trial:
- S9: 4, 8, 16 µg/ml
+ S9: 4, 8, 16 µg/ml

main test, second trial:
- S9: 2, 4, 8 µg/ml
+ S9: 4, 8, 16 µg/ml

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ethylmethane sulphonate and cyclophosphamide

Details on test system and experimental conditions:

PRELIMINARY CYTOTOXICITY TEST
The first preliminary cytotoxicity test was conducted with the test concentrations of 75, 150, 300, 600, 1200, 2400 and 5000 µg/ml and DMSO as the vehicle control. Since, there was toxicity to the cells even at the lowest tested concentration of 75 µg/ml, the test was repeated with still lower concentrations.
Exposure of Target Cells to Treatment: The target cells were exposed to 5 concentrations of the test item and the vehicle control.
Group No. Test item conc. (µg/ml)
G1 DMSO (100 µl)
G2 4
G3 8
G4 16
G5 32
G6 64
Three sets of each of the above groups with two replicates in each, were prepared and exposed to the test item as follows:
Set No. Activation Treatment duration
1 +S-9 3 hrs
2 -S-9 3 hrs
3 -S-9 20 hrs and 15 min
All test solutions were prepared immediately before use in sterile test tubes.
Medium from each target cells flask was removed by aspiration and replaced with 4.5 ml and 5 ml of F12 FBS5 for tests in the presence and absence of metabolic activation respectively. For tests in the presence of metabolic activation, 0.5 ml of S-9 mix was added to each flask to achieve a concentration of 10% (v/v) of S-9 mix in each flask. One hundred microlitre of each of the above concentrations of the test item and the DMSO control was added to respective test flasks and gently mixed.
Treatment in the 1st and 2nd set of flasks was terminated by removal of the test solutions after incubation for 3 hours. The cell monolayer was washed twice with PBS and the flasks were refilled with 5 ml of F12 FBS10 and incubated for a further period of 16 hours and 15 minutes. Twenty hours and fifteen minutes after the start of the treatment, the medium from all the three sets of flasks (1, 2 and 3) was aspirated, the cell monolayer was trypsinized and the cells were suspended with 5 ml of F12 FBS5. The cell suspension from the replicates were pooled and the cell counts were determined separately using a hemocytometer. The effect of the test item on cell multiplication was estimated by expressing the number of cells in each treated culture as a percentage of the number in the solvent control.

CHROMOSOME ABERRATION TEST
Exposure of Target Cells to Treatment: All test solutions and the positive control were prepared immediately before use in sterile test tubes and poured into cultures after removal of the existing medium.
The treatment medium consisted of 13.875 ml and 15 ml of F12 FBS5, in Trial 1 for experiments in the presence and absence of metabolic activation, respectively. Similarly, in Trial 2, it was 13.5 ml and 15 ml of F12 FBS5 for experiments in the presence and absence of metabolic activation, respectively.
For experiments in the presence of metabolic activation, 1.125 ml and 1.5 ml of S-9 mix was added to the respective test tubes to achieve a final concentration of 7.5 and 10% (vlv) in the test medium, in the first and second trial, respectively. To respective tubes 300 µl of the solvent control, test item and the positive control diluted to the appropriate concentrations in DMSO was added, mixed and poured into respective test flasks after removal of the existing medium.
The target cells in quintuplicate were exposed to the solvent control, positive control and the 3 concentrations of the test item as follows:
TRIAL 1: Cells both in the presence and absence of metabolic activation were exposed for 3 hours.
TRIAL 2: Cells in the presence of metabolic activation were exposed for 3 hours. Cells in the absence of metabolic activation were exposed continuously for 19 hours and 35 minutes.
After the treatment period, all the cultures from Trial 1 and the cultures treated in the presence of metabolic activation from Trial 2 were drained, washed with phosphate buffered saline and given fresh medium.

MITOTIC ARREST
At 19 hours and 35 minutes after the start of the treatment, 300 µI of Colchicine (10 µg/mI) was transferred into tubes containing 15 ml of F12 FBS10, mixed well and poured into respective flasks after removal of the existing medium.

CHROMOSOME PREPARATION
Each culture from the solvent control, positive control and treatment groups was harvested at 21 hours and 15 minutes after the beginning of the treatment and processed separately for the preparation of chromosomes.
At the end of the incubation period, mitotic cells were suspended in F12 FBS5 after trypsinization. Two hundred microlitres of mitotic cells of each group from individual replicates were pooled into respective test tubes for the determination of cell counts. The remaining cell suspension was centrifuged at 1000 rpm for 10 minutes and suspended in 0.56% warm potassium chloride and incubated for 10 minutes at room temperature.
After incubation, the cell suspensions were centrifuged at 2000 rpm for 10 minutes. The supernatant was removed and to each tube, 2 ml of freshly prepared cold methanol:acetic acid fixative (3:1) was added dropwise while shaking the tube gently to resuspend the cells. The tubes were incubated for 10 minutes at room temperature and then centrifuged at approximately at 2400 rpm for 10 minutes and the supernatant was discarded.
Once again, 2 ml of fixative was added dropwise and the tubes were allowed to stand in the refrigerator for a minimum period of 1 hour.
After refrigeration, the cell suspensions was centrifuged at 2400 rpm for 10 minutes, the supernatant was discarded and the cell button was resuspended in 2 ml of fixative and the tubes were incubated at room temperature for 10 minutes.
The above procedure was repeated and the cell button was resuspended in required quantity of fixative and the cell suspension was incubated at room temperature for 10 minutes prior to preparing the slides.

SLIDE PREPARATION
The cell suspension was dropped onto a clean chilled slide, flame dried and dried on a slide warmer maintained at approximately 40°C. The slides were marked with the study number, treatment group, activation, trial number and replicate number with a diamond point pencil.

STAINING
The slides were stained with freshly prepared 5% Giemsa stain in distilled water for 20 minutes, rinsed with tap water, air dried, immersed in xylene and mounted with DPX. The slides were then coded before evaluation.

MICROSCOPIC ANALYSIS
Chromosome aberrations for the three concentrations of the test item and the positive and solvent controls cultures were scored. The slides were scanned and each metaphase spread was examined under the 100x objective. The number of chromosomes in each spread was counted and those containing 18 to 22 centromeres were evaluated for aberrations. A total of 200 such metaphases from quintuplicate cultures were evaluated for each group. The microscope coordinates were recorded for each aberrant metaphase. The total number of metaphases showing one or more aberrations both including and excluding gaps was calculated from a set of 200 metaphases for each group.

MEASURES OF CYTOTOXICITY
Concurrent cytotoxicity for all treated and control cultures was recorded soon after trypsinization, based on cell counts as explained under preliminary cytotoxicity study. At the time of chromosome preparation, about 0.2 ml of mitotic cells of each group from individual replicates were pooled into respective test tubes, mixed well and the cell counts were determined separately using a hemocytometer.

Evaluation criteria:

Biological relevance of the results should be considered first. Statistical methods may be used as an aid in evaluating the test results, but statistical significance should not be the only determining factor for a positive response. However, there are certain reservations based on the differences in ranking the types of aberrations. Gaps are ranked lowest and exchange configurations are ranked highest.
A positive result if any, is strengthened by the demonstration of a dose-related increase of the effect. This is particularly crucial if only the frequency of gaps is increased.
Exchanges are such rare events (<1 in 1000 cells) that they are seldom observed in control samples. Thus, the observation of exchanges in experimental groups, even without a dose-related increase, is a strong indication for a positive response

Statistics:

The data analysed were the proportions of aberrant metaphases in each sample, both including and excluding gaps as aberrations. The pooled data from each test concentration and the positive control data were compared with the solvent control using one-tailed Fisher exact test.
All analysis and comparisons was evaluated at 5% (P<0.05) level.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Taken together, the results of the two trials suggest that the test item does not have the potential to cause chromosome damage either including gaps or excluding gaps either in the presence or absence of metabolic activation.
In both the trials, at the highest concentration tested, the reduction in the cell growth was in the range of 55.78 to 58.15% over the DMSO control, both in the presence and absence of metabolic activation.
In each of these trials, the positive control items produced a statistically significant increase in aberrant metaphases, demonstrating that the system was able to detect the effect of known mutagens.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Concurrent Cytotoxicity test results with Sulfur Dust on the growth of CHO cells in the presence of metabolic activation

 

Test Item conc. (µg/ml)	Trial I		Trial II
	Cell count (x10^6/flask)	% Over Control	Cell count (x10^6/flask)	% Over Control
DMSO (0.3 ml)	6.07	100.00	6.40	100.00
4	5.37	88.47	4.22	65.94
8	4.23	69.69	3.79	59.22
16	2.54	41.85	2.83	44.22
CPA 55	4.82	79.41	4.27	66.72

 

Concurrent Cytotoxicity test results with Sulfur Dust on the growth of CHO cells in the absence of metabolic activation

 

Trial I			Trial II
Test Item conc. (µg/ml)	Cell count (x10^6/flask)	% Over Control	Test Item conc. (µg/ml)	Cell count (x10^6/flask)	% Over Control
DMSO (0.3 ml)	5.73	100.00	DMSO (0.3 ml)	5.99	100.00
4	5.47	95.46	2	3.85	64.27
8	3.95	68.94	4	3.44	57.43
16	2.45	42.76	8	2.54	42.40
EMS600	4.92	85.86	EMS600	3.62	60.43

 

Summary of results ofIn vitroMammalian Chromosome Aberration Test with Sulfur Dust in the presence of metabolic activation (TRIAL I)

 

Test item conc. (µg/ml)	No. of metaphases scored	No. (%) of metaphases with aberrations							Total No. (%) of metaphases* with aberrations
		Gaps		Breaks		Exchanges		RC	With Gaps	Without Gaps
		Cs	Ct	Cs	Ct	Cs	Ct
DMSO (0.3 ml)	200	0	1 (0.5)	0	0	0	0	0	1 (0.5)	0
4	200	0	0	0	0	0	0	0	0	0
8	200	0	1 (0.5)	0	0	0	0	0	1 (0.5)	0
16	200	0	0	0	0	0	0	0	0	0
CPA 55	200	38 (19)	97 (48.5)	2 (1.0)	74 (37.0)	30 (15.0)	93 (46.5)	4 (2.0)	+163 (81.5)	+135 (67.5)

*: Metaphase plate with one or more than one aberrations considered as one metaphase plate with aberrations

Cs: Chromosome type

Ct: Chromatid type

RC: Ring chromosome

+: Significantly higher than control (p<= 0.05) by Fisher exact test

Summary of results ofIn vitroMammalian Chromosome Aberration Test with Sulfur Dust in the absence of metabolic activation (TRIAL I)

 

Test item conc. (µg/ml)	No. of metaphases scored	No. (%) of metaphases with aberrations							Total No. (%) of metaphases* with aberrations
		Gaps		Breaks		Exchanges		RC	With Gaps	Without Gaps
		Cs	Ct	Cs	Ct	Cs	Ct
DMSO (0.3 ml)	200	1 (0.5)	1 (0.5)	0	1 (0.5)	0	0	0	3 (1.5)	1 (0.5)
4	200	0	1 (0.5)	0	1 (0.5)	0	0	0	2 (1.0)	1 (0.5)
8	200	1 (0.5)	2 (1.0)	0	0	0	0	0	3 (1.5)	0
16	200	1 (0.5)	3 (1.5)	0	1 (0.5)	0	0	0	5 (2.5)	1 (0.5)
CPA 55	200	37 (18.5)	59 (29.5)	3 (1.5)	31 (15.5)	45 (22.5)	73 (36.5)	2 (1.0)	+157 (78.5)	+120 (60.0)

*: Metaphase plate with one or more than one aberrations considered as one metaphase plate with aberrations

Cs: Chromosome type

Ct: Chromatid type

RC: Ring chromosome

+: Significantly higher than control (p<= 0.05) by Fisher exact test

Summary of results ofIn vitroMammalian Chromosome Aberration Test with Sulfur Dust in the presence of metabolic activation (TRIAL II)

 

Test item conc. (µg/ml)	No. of metaphases scored	No. (%) of metaphases with aberrations							Total No. (%) of metaphases* with aberrations
		Gaps		Breaks		Exchanges		RC	With Gaps	Without Gaps
		Cs	Ct	Cs	Ct	Cs	Ct
DMSO (0.3 ml)	200	0	2 (1.0)	0	0	0	0	0	2 (1.0)	0
4	200	0	2 (1.0)	0	0	0	0	0	2 (1.0)	0
8	200	0	0	0	0	0	0	0	0	0
16	200	0	2 (1.0)	0	0	0	0	0	2 (1.0)	0
CPA 55	200	43 (21.5)	74 (37.0)	0	47 (23.5)	52 (26.0)	83 (41.5)	1 (0.5)	+162 (81.0)	+136 (68.0)

*: Metaphase plate with one or more than one aberrations considered as one metaphase plate with aberrations

Cs: Chromosome type

Ct: Chromatid type

RC: Ring chromosome

+: Significantly higher than control (p<= 0.05) by Fisher exact test

Summary of results ofIn vitroMammalian Chromosome Aberration Test with Sulfur Dust in the absence of metabolic activation (TRIAL II)

 

Test item conc. (µg/ml)	No. of metaphases scored	No. (%) of metaphases with aberrations							Total No. (%) of metaphases* with aberrations
		Gaps		Breaks		Exchanges		RC	With Gaps	Without Gaps
		Cs	Ct	Cs	Ct	Cs	Ct
DMSO (0.3 ml)	200	1 (0.5)	3 (1.5)	0	0	0	0	0	4 (2.0)	0
4	200	0	1 (0.5)	0	0	0	0	0	1 (0.5)	0
8	200	2 (1.0)	1 (0.5)	0	0	0	0	0	3 (1.5)	0
16	200	2 (1.0)	4 (2.0)	0	0	0	0	0	5 (2.5)	0
600	200	29 (14.5)	56 (28.0)	3 (1.5)	37 (18.5)	43 (21.5)	61 (30.5)	1 (0.5)	+153 (76.5)	+123 (61.5)

*: Metaphase plate with one or more than one aberrations considered as one metaphase plate with aberrations

Cs: Chromosome type

Ct: Chromatid type

RC: Ring chromosome

+: Significantly higher than control (p<= 0.05) by Fisher exact test

Conclusions:

Interpretation of results (migrated information):
negative without metabolic activation
negative with metabolic activation

Sulfur dust was not considered to have the potential to cause chromosome aberrations at the concentrations tested.

Executive summary:

The potential of the test item sulfur dust to induce chromosome aberrations in mammalian cells was evaluated using cultured Chinese Hamster Ovary (CHO) cells (OECD guideline 473, GLP compliant). In both trial 1 and 2, for experiments in the presence of metabolic activation, CHO cells were exposed to the test item in quintuplicate for 3 hours at concentrations of 4, 8 and 16 µg/ml of the medium in the presence of metabolic activation. These concentrations were based on results from preliminary range finding tests and the top dose of 16 µg/ml reflects the maximum non-cytotoxic concentration. In trial 1 for the experiment in the absence of metabolic activation, CHO cells were exposed to the test item at concentrations of 4, 8 and 16 µg/ml of the medium for 3 hours. In trial 2 for the experiment in the absence of metabolic activation, CHO cells were exposed to the test Item at concentrations of 2, 4, and 8 µg/ml continuously for 19 hours and 35 minutes. In a similar way, concurrent vehicle control (DMSO) and appropriate positive controls viz., cyclophosphamide in the presence of metabolic activation and ethylmethanesulfonate in the absence of metabolic activation were also tested in quintuplicate.There was no evidence of induction of chromosome aberrations by sulfur dust with experiments either in the presence or absence of metabolic activation. In each of these experiments, the respective positive control items produced a large and statistically significant increase in aberrant metaphases, under identical conditions. In both the trials, at the highest concentration tested, the reduction in the cell growth was in the range of 55.78 to 58.15% over the DMSO control, both in the presence and absence of metabolic activation.

The study indicated that the test item does not have the potential to cause chromosome aberrations at the concentrations tested.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to a test protocol that is comparable to the appropriate OECD test guideline. It was not compliant with GLP. Only a single sample was taken (at 24 hours).

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

Only a single sample was taken (at 24 hours)

Principles of method if other than guideline:

Method: other

GLP compliance:

not specified

Type of assay:

micronucleus assay

Species:

mouse

Strain:

other: Strain : ddy

Sex:

male

Route of administration:

intraperitoneal

Frequency of treatment:

Single injection

Post exposure period:

Sampling time 24 hours

Remarks:

Doses / Concentrations:
25, 50, 100, 180 mg/kg
Basis:

No. of animals per sex per dose:

6 males (test) 2 males (control)

Control animals:

yes

Positive control(s):

Mitomycin C

Sex:

male

Genotoxicity:

negative

Toxicity:

not specified

Remarks:

% PCEs appeared to be unaffected by substance administration

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Negative

Conclusions:

Interpretation of results (migrated information): negative
Iron sulphate has been tested in a micronucleus study. No increase in micronucleated PCEs was observed in any dose 24 hours after sampling. It is concluded that the test substance is negative for the induction of micronuclei under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

In relation to the information available the UVCB substance does not show Genetic toxicity