Mill scale (ferrous metal)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

23 July 2018 - 11 August 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted: 21st July 1997

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 hydroxide oxide yellow

EC Number:

257-098-5

EC Name:

Iron hydroxide oxide yellow

Cas Number:

51274-00-1

Molecular formula:

Fe(OH)O

IUPAC Name:

Iron hydroxide oxide

Test material form:

solid: nanoform

Details on test material:

Appearance: yellow powder

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Test article stock suspensions were prepared by formulating Iron Oxide Sicovit® Yellow 10 E172 under subdued lighting in 1% MC, with the aid of Silverson mixing, to give the maximum required treatment concentration. Subsequent dilutions were made using 1% methyl cellulose. All suspensions were homogenized by inversion prior to dilution or treatment. The test article suspensions were protected from light and used within approximately 5 hours of initial formulation.

Method

Target gene:

histidine operon genes (Salmonella strains); tryptophan operon genes (E. coli)

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 98

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 100

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 1535

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 1537

Species / strain 5

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

The mammalian liver post-mitochondrial fraction (S-9) used for metabolic activation was obtained from Molecular Toxicology Incorporated, USA where it was prepared from male Sprague Dawley rats induced with Aroclor 1254. The S-9 was supplied as lyophilized S-9 mix (MutazymeTM), stored frozen at <-20°C, and thawed and reconstituted with purified water to provide a 10% S-9 mix just prior to use. Each batch was checked by the manufacturer for sterility, protein content, ability to convert ethidium bromide and cyclophosphamide to bacterial mutagens, and cytochrome P-450-catalysed enzyme activities (alkoxyresorufin-O-dealkylase activities). Treatments were carried out both in the absence and presence of S-9 by addition of either buffer solution or 10% S-9 mix respectively

Test concentrations with justification for top dose:

- 5, 16, 50, 160, 500, 1600 and 5000 μg/plate (maximum recommended test concentration)

Vehicle / solvent:

- Vehicle used: 1% (w/v) methyl cellulose (MC)
- Justification for choice of vehicle: The Sponsor indicated that the test material was insoluble in water and organic vehicles compatible with the assay system; the test article was therefore prepared as a homogenous suspension in MC.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

1% MC

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

1% MC

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

1% MC

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

1% MC

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

1% MC

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Controls 6

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

1% MC

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF TREATMENT
For all assays, bacteria were cultured at 37±1°C for 10 hours in nutrient broth, containing ampicillin (TA98, TA100 and WP2 uvrA pKM101) as appropriate to provide bacterial cultures in the range of approximately 10^8 to 10^9 cells/mL, based on cell count data from testing of each strain batch.

Iron Oxide Sicovit® Yellow 10 E172 was tested for mutation (and toxicity) in four strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and one strain of Escherichia coli (WP2 uvrA pKM101) in two separate experiments at the concentrations detailed previously, using triplicate plates without and with S-9 for test article, vehicle and positive controls. These platings were achieved by the following sequence of additions to 2 mL supplemented molten top agar at 45±1°C: 0.1 mL bacterial culture, 0.1 mL test article suspension/vehicle control or 0.05 mL of positive control, and 0.5 mL 10% S-9 mix or buffer solution followed by rapid mixing and pouring on to Vogel-Bonner E agar plates. When set, the plates were inverted and incubated at 37±1°C protected from light for 3 days. Following incubation, these plates were examined for evidence of toxicity to the background lawn, and where possible revertant colonies were counted.

As the results of Experiment 1 were negative, treatments in the presence of S-9 in Experiment 2 included a pre-incubation step. Quantities of test article, vehicle control solution (reduced to 0.05 mL) or positive control, bacteria and S-9 mix detailed above, were mixed together and incubated for 20 minutes at 37±1°C, with shaking, before the addition of 2 mL molten agar at 45±1°C. Plating of these treatments then proceeded as for the normal plate-incorporation procedure. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected in the assay.

CYTOTOXICITY
The background lawns of the plates were examined for signs of toxicity. Moreover, the plates were analysed for marked reduction in revertant numbers.

COLONY COUNTING
Colonies were counted electronically using a Sorcerer Colony Counter (Perceptive Instruments) or manually where confounding factors such as bubbles or a split in the agar or precipitation affected the accuracy of the automated counter.

ACCEPTANCE CRITERIA
The assay was to be considered valid if the following criteria were met:
1. The vehicle control counts fell within the laboratory’s historical control ranges as reported
2. The positive control chemicals induced increases in revertant numbers of ≥2-fold (in strains TA98, TA100 and WP2 uvrA pKM101) or ≥3-fold (in strains TA1535 and TA1537) the concurrent vehicle control confirming discrimination between different strains, and an active S-9 preparation.

Evaluation criteria:

For valid data, the test article was considered to be mutagenic if:

1. A concentration related increase in revertant numbers was ≥2-fold (in strains TA98, TA100 and WP2 uvrA pKM101) or ≥3-fold (in strains TA1535 and TA1537) the concurrent vehicle control values

2. The positive trends/effects described above were reproducible.

The test article was considered positive in this assay if both of the above criteria were met.

The test article was considered negative in this assay if neither of the above criteria
were met.

Statistics:

not performed

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

STUDY RESULTS
- Although the test article was prepared as a suspension in 1% MC, precipitation (defined for this study as an aggregation of particulates visible to the unaided eye) was observed on the test plates at concentrations of 500 μg/plate and above.
- In both experiments, no evidence of toxicity was observed, as would normally manifest as a thinning of the background bacterial lawn or a marked reduction in revertant numbers.
- In both experiments, following Iron Oxide Sicovit® Yellow 10 E172 treatments of all the test strains in the absence and presence of S-9, the revertant colony number (please refer to Tabs. 1-4 in the field 'Any other information on results incl. tables') was not increased according to the evaluation criteria.

DATA ACCEPTABILITY AND VALIDITY
From the results of the mutation experiments, it can be seen that vehicle control counts (please refer to Tabs. 1-4 in the field 'Any other information on results incl. tables') fell within the laboratory’s historical ranges (please refer to attached background material). The positive control chemicals all induced increases in revertant numbers of ≥2-fold (in strains TA98, TA100 and WP2 uvrA pKM101) or ≥3-fold (in strains TA1535 and TA1537) the concurrent vehicle control confirming discrimination between different strains, and an active S-9 preparation. The study therefore demonstrated correct strain and assay functioning and was accepted as valid.

Any other information on results incl. tables

Table 1. Mutagenicity experiment 1; without metabolic activation

Strain	Compound	Concentration (µg/plate)	Mean	SD	Fold increase
TA 98	1% MC	-	26.0	3.6	-
	Iron Oxide Sicovit Yellow 10 E172	5	25.0	6.1	1.0
		16	21.0	7.8	0.8
		50	22.7	7.8	0.9
		160	20.0	3.6	0.8
		500	14.0	0.0	0.5
		1600	23.7	3.5	0.9
		5000	18.0	3.0	0.7
	2NF	5	783.7	94.2	30.1
TA 100	1% MC	-	130.0	15.7	-
	Iron Oxide Sicovit Yellow 10 E172	5	129.3	4.0	1.0
		16	129.7	3.2	1.0
		50	127.7	10.4	1.0
		160	114.3	23.9	0.9
		500	125.7	6.8	1.0
		1600	136.0	9.5	1.0
		5000	137.3	4.7	1.1
	NaN3	2	823.3	32.2	6.3
TA 1535	1% MC	-	24.7	3.2 -	-
	Iron Oxide Sicovit Yellow 10 E172	5	24.7	3.2	1.0
		16	23.0	4.0	0.9
		50	24.0	12.2	1.0
		160	22.0	6.9	0.9
		500	17.0	1.0	0.7
		1600	23.7	3.5	1.0
		5000	20.7	1.5	0.8
	NaN3	2	722.3	19.3	29.3
TA 1537	1% MC	-	11.3	3.1	-
	Iron Oxide Sicovit Yellow 10 E172	5	10.0	2.6	0.9
		16	11.7	4.2	1.0
		50	9.0	2.6	0.8
		160	9.3	2.5	0.8
		500	10.7	4.0	0.9
		1600	10.0	1.7	0.9
		5000	8.3	1.5	0.7
	AAC	50	554.3	75.1	48.9
WP2 uvrA	1% MC	-	192.7	19.3	-
	Iron Oxide Sicovit Yellow 10 E172	5	180.0	17.4	0.9
		16	167.7	5.5	0.9
		50	174.0	8.7	0.9
		160	165.0	12.2	0.9
		500	156.3	14.6	0.8
		1600	163.0	8.0	0.8
		5000	162.0	11.5	0.8
	NQO	2	1106.0	25.2	5.7

Table 2. Mutagenicity experiment 1; with metabolic activation

Strain	Compound	Concentration (µg/plate)	Mean	SD	Fold increase
TA 98	1% MC	-	32.0	4.4	-
	Iron Oxide Sicovit Yellow 10 E172	5	29.0	10.8	0.9
		16	35.0	10.5	1.1
		50	37.3	4.0	1.2
		160	36.7	3.1	1.1
		500	24.0	2.0	0.8
		1600	30.0	4.6	0.9
		5000	26.0	9.6	0.8
	B[a]P	10	390.3	25.2	12.2
TA 100	1% MC	-	143.7	9.2	-
	Iron Oxide Sicovit Yellow 10 E172	5	145.3	11.2	1.0
		16	130.7	5.7	0.9
		50	140.3	2.5	1.0
		160	148.3	5.5	1.0
		500	143.0	8.9	1.0
		1600	136.0	21.8	0.9
		5000	146.7	10.0	1.0
	AAN	5	2257.0	227.1	15.7
TA 1535	1% MC	-	18.3	3.2	-
	Iron Oxide Sicovit Yellow 10 E172	5	21.7	5.9	1.2
		16	32.0	3.6	1.7
		50	24.3	4.0	1.3
		160	24.0	8.2	1.3
		500	27.3	1.2	1.5
		1600	30.7	4.5	1.7
		5000	29.7	8.1	1.6
	AAN	5	228.7	6.4	12.5
TA 1537	1% MC	-	12.3	5.5	-
	Iron Oxide Sicovit Yellow 10 E172	5	18.0	4.4	1.5
		16	15.3	5.5	1.2
		50	10.7	5.1	0.9
		160	11.3	4.5	0.9
		500	12.7	4.0	1.0
		1600	12.3	2.1	1.0
		5000	9.0	1.7	0.7
	AAN	5	311.7	17.0	25.3
WP2 uvrA	1% MC	-	227.3	13.4	-
	Iron Oxide Sicovit Yellow 10 E172	5	228.3	14.2	1.0
		16	243.7	19.9	1.1
		50	239.3	12.4	1.1
		160	226.7	5.1	1.0
		500	236.3	36.2	1.0
		1600	251.3	41.0	1.1
		5000	235.3	33.6	1.0
	AAN	10	781.7	54.0	3.4

Table 3. Mutagenicity experiment 2; without metabolic activation

Strain	Compound	Concentration (µg/plate)	Mean	SD	Fold increase
TA 98	1% MC	-	36.3	3.5	-
	Iron Oxide Sicovit Yellow 10 E172	31.25	31.7	1.5	0.9
		62.5	26.3	2.5	0.7
		125	20.3	2.3	0.6
		250	29.7	4.0	0.8
		500	27.3	2.9	0.8
		5000	21.7	4.9	0.6
	2NF	5	1473.0	116.6	40.5
TA 100	1% MC	-	131.0	17.1	-
	Iron Oxide Sicovit Yellow 10 E172	31.25	131.0	13.2	1.0
		62.5	138.0	16.8	1.1
		125	131.0	11.3	1.0
		250	143.0	15.7	1.1
		500	120.7	15.6	0.9
		5000	131.0	14.1	1.0
	NaN3	2	920.0	16.0	7.0
TA 1535	1% MC	-	25.3	0.6	-
	Iron Oxide Sicovit Yellow 10 E172	31.25	29.7	4.6	1.2
		62.5	30.3	3.8	1.2
		125	24.3	8.1	1.0
		250	30.3	6.7	1.2
		500	36.3	11.0	1.4
		5000	22.7	3.2	0.9
	NaN3	2	741.3	17.5	29.3
TA 1537	1% MC	-	13.0	1.7	-
	Iron Oxide Sicovit Yellow 10 E172	31.25	9.3	4.2	0.7
		62.5	15.7	3.1	1.2
		125	15.7	3.5	1.2
		250	13.3	2.3	1.0
		500	14.7	1.2	1.1
		5000	14.7	1.5	1.1
	AAC	50	698.7	152.0	53.1
WP2 uvrA	1% MC	-	170.7	7.4	-
	Iron Oxide Sicovit Yellow 10 E172	31.25	186.3	17.4	1.1
		62.5	154.7	19.9	0.9
		125	167.0	4.6	1.0
		250	162.7	9.0	1.0
		500	165.3	4.0	1.0
		5000	148.3	15.5	0.9
	NQO	2	1326.7	61.9	7.8

Table 4. Mutagenicity experiment 2; with metabolic activation

Strain	Compound	Concentration (µg/plate)	Mean	SD	Fold increase
TA 98	1% MC	-	46.0	7.8	-
	Iron Oxide Sicovit Yellow 10 E172	31.25	44.3	11.0	1.0
		62.5	50.0	8.7	1.1
		125	46.3	5.5	1.0
		250	42.3	11.9	0.9
		500	37.0	7.2	0.8
		5000	41.7	8.5	0.9
	B[a]P	10	429.0	33.6	9.3
TA 100	1% MC	-	137.3	10.6	-
	Iron Oxide Sicovit Yellow 10 E172	31.25	144.7	9.9	1.1
		62.5	152.0	20.1	1.1
		125	135.0	17.7	1.0
		250	138.7	5.0	1.0
		500	131.3	15.2	1.0
		5000	112.7	10.7	0.8
	AAN	5	2444.7	151.8	17.2
TA 1535	1% MC	-	15.0	2.6	-
	Iron Oxide Sicovit Yellow 10 E172	31.25	11.0	3.6	0.7
		62.5	14.3	4.0	1.0
		125	14.7	5.5	1.0
		250	11.7	4.9	0.8
		500	17.0	6.0	1.1
		5000	12.7	2.1	0.8
	AAN	5	215.0	75.8	14.3
TA 1537	1% MC	-	24.3	4.6	-
	Iron Oxide Sicovit Yellow 10 E172	31.25	17.7	3.1	0.7
		62.5	11.0	2.6	0.5
		125	19.7	2.9	0.8
		250	15.0	8.7	0.6
		500	10.7	4.0	0.4
		5000	10.0	2.0	0.4
	AAN	5	316.0	27.6	13.0
WP2 uvrA	1% MC	-	251.7	29.6	-
	Iron Oxide Sicovit Yellow 10 E172	31.25	274.3	21.2	1.1
		62.5	282.3	14.0	1.1
		125	272.0	26.9	1.1
		250	244.3	20.6	1.0
		500	194.7	33.3	0.8
		5000	219.7	14.0	0.9
	AAN	10	889.3	56.8	3.5

Applicant's summary and conclusion

Conclusions:

No toxicity (thinning of the background lawn or a reduction in the number of revertants) was found in both experiments. Precipitation (defined for this study as an aggregation of particulates visible to the unaided eye) was observed on the test plates at concentrations of 500 μg/plate and above. Iron Oxide Sicovit® Yellow 10 E172 did not show any evidence of mutagenic activity when tested using the preincubation and plate incorporation method with or without metabolic activation. The sensitivity of the test system was demonstrated. All validity criteria were met. The study was fully compliant with OECD 471 (1997).

Based on the study results, it is concluded that Iron Oxide Sicovit® Yellow 10 E172 was not mutagenic in this bacterial reverse mutation assay under the conditions of the test.