Zinc ferrite brown spinel

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Zinc ferrite was tested for mutagenicity in a bacterial reverse mutation assay using Salmonella typhimurium, both in the absence and presence of S9 metabolism (Herbold, 1990). Zinc ferrite was assayed in tester strains TA98, TA100, T1535 and T 1537 and was reported to be negative.

The in vitro genetic toxicity of triiron tetraoxide has been evaluated in a series of three reliable studies: a bacterial reverse gene mutation assay (equivalent to OECD TG 471), in a mammalian cell gene mutation assay (acc. to OECD TG 476, GLP), and in a mammalian cell cytogenicity study (acc. to OECD TG 473, GLP). All studies returned an unequivocally negative result.

The in vitro genetic toxicity of the iron hydroxide oxide has been evaluated in a series of three reliable studies: a bacterial reverse gene mutation assay (acc. to OECD TG 471, GLP), in a mammalian cell gene mutation assay (acc. to OECD TG 476, GLP), and in a mammalian cell cytogenicity study (acc. to OECD TG 487, GLP). All studies returned an unequivocally negative result.

Details on the category justification are given in the read-across document attached in IUCLID section 13.2.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across based on grouping of substances (category approach)

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

Justification for type of information:

see attachment "Iron oxide category read-across concept-HH " in IUCLID section 13.2.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted: 21st July 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

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.

Target gene:

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

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

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.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

1% MC

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

1% MC

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

1% MC

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

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

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

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

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

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

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

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

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.

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

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.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

weight of evidence

Study period:

12 November 2018 - 22 March 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

see attachment "Iron oxide category read-across concept-HH " in IUCLID section 13.2.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted: 29 July 2016

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Test article stock solutions were prepared by formulating Iron Oxide Sicovit® Yellow 10 E172 under subdued lighting in RPMI 5%, with the aid of vortex mixing,
ultrasonication (approximately 10 minutes) and warming at 37°C (Mutation Experiment only), to give the maximum required concentration. Subsequent dilutions
were made using RPMI 5%. The test article solutions were protected from light and used within approximately 2.5 hours of initial formulation.

Target gene:

Hprt

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

The master stock of L5178Y tk+/- (3.7.2C) mouse lymphoma cells originated from Dr Donald Clive, Burroughs Wellcome Co. Cells supplied to Covance were stored as frozen stocks in liquid nitrogen. Full details of the supplier are documented in central records. Each batch of frozen cells was purged of mutants and confirmed to be mycoplasma free. For each experiment, at least one vial was thawed rapidly, the cells diluted in RPMI 10 and incubated at 37±1ºC. When the cells were growing well, subcultures were established in an appropriate number of flasks. Karyotype analysis on the master stock of cells confirmed a modal number of 40 chromosomes. The average doubling time of L5178Y tk+/- (3.7.2C) cells at Covance Laboratories is 10-12 hours.

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 nominal, 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 150 mM KCl or 10% S-9 mix respectively. The final S-9 volume in the test system was 1% (v/v).

Test concentrations with justification for top dose:

- Range finder: 63.28, 126.6, 253.1, 506.3, 1013, and 2025 µg/mL (maximum recommended test concentration)
- Mutation experiment: 0.1955, 0.3909, 0.7819, 1.564, 3.128, 6.255, 12.51, 25.02, 50.04, and 100.1 µg/mL (test item precipitation)

Vehicle / solvent:

- Vehicle used: RPMI 1640 culture medium supplemented with 5% heat inactivated horse serum (RPMI 5%)

- Justification for choice of vehicle: The test article was found to be insoluble in all commonly used vehicles and was therefore formulated as a suspension in the chosen vehicle.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

RPMI 5%

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

RPMI 5%

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Details on test system and experimental conditions:

CYTOTOXICITY RANGE-FINDER EXPERIMENT
Treatment of cell cultures for the cytotoxicity Range-Finder Experiment was as described below for the Mutation Experiment. However, single cultures only were used and positive controls were not included. The final treatment volume was 20 mL. Following 3 hour treatment, cells were centrifuged (200 g) for 5 minutes. The resulting supernatant was removed to appropriate sterile culture vessels and retained. Samples from this supernatant were taken for further turbidity assessment and for measurement of pH and osmolality. Osmolality was measured using a Fiske 2020 Osmometer and pH was measured using a Mettler Toledo Five Easy Plus pH meter. All cultures were washed with tissue culture medium and resuspended in 20 mL RPMI 10. Following resuspension of the cultures, a sample was taken from the highest non-precipitating concentration (observed by eye) and assessed using a haemocytometer to confirm the absence of precipitate. Cell concentrations were adjusted to 8 cells/mL and, for each concentration, 0.2 mL was plated into each well of a 96-well microtitre plate for determination of relative survival. The plates were incubated at 37±1ºC in a humidified incubator gassed with 5±1% v/v CO2 in air for 8 days. Wells containing viable clones were identified by eye using background illumination and counted.

MUTATION ASSAY
- Treatment of cell cultures: In the Mutation Experiment at least 10^7 cells were placed in a series of sterile disposable 50 mL centrifuge tubes. For all treatments 18 mL test article, vehicle or positive control solution (0.2 mL positive control plus 17.8 mL of RPMI 5%) was added. S-9 mix or 150 mM KCl was added as described. Each treatment, in the absence or presence of S-9, was in triplicate (single cultures only used for positive control treatments) and the final treatment volume was 20 mL. After 3 hours’ incubation at 37±1°C with gentle agitation in the Mutation Experiment, cultures were centrifuged (200 g) for 5 minutes. The resulting supernatant was removed to appropriate sterile culture vessels and retained. Samples from this supernatant were taken for further turbidity assessment. All cultures were washed with the appropriate tissue culture medium, centrifuged again (200 g) for 5 minutes and finally resuspended in 20 mL RPMI 10 medium. Following resuspension of the cultures, a sample was taken from the highest non-precipitating concentration (observed by eye) and assessed using a haemocytometer to confirm the absence of precipitate. Cultures were centrifuged again (200 g) for 5 minutes and resuspended in 20 mL RPMI 10 medium. Cell densities were determined using a Coulter counter and the concentrations adjusted to 2 x 10^5 cells/mL. Cells were transferred to flasks for growth throughout the expression period or were diluted to be plated for survival as described.

- Plating for survival: Following adjustment of the cultures to 2 x 10^5 cells/mL after treatment, samples from these were diluted to 8 cells/mL. Using a multichannel pipette, 0.2 mL of the final concentration of each culture was
placed into each well of 2 x 96-well microtitre plates (192 wells, averaging 1.6 cells/well). The plates were incubated at 37±1ºC in a humidified incubator gassed with 5±1% v/v CO2 in air until scoreable (7 days). Wells containing viable clones
were identified by eye using background illumination and counted.

- Expression period: Cultures were maintained in flasks for a period of 7 days during which the hprt- mutation would be expressed. Sub-culturing was performed as required with the aim of retaining an appropriate concentration of cells/flask. From observations on recovery and growth of the cultures during the expression period, the following cultures were selected to be plated for viability and 6TG resistance: S-9: 0, 0.1955, 0.3909, 0.7819, 1.564, 3.128, and 6.255 µg/mL and NQO (0.15 and 0.20 µg/mL; +S9: 0, 0.1955, 0.3909, 0.7819, 1.564, and 3.128 µg/mL and B[a]P (2 and 3 µg/mL).

- Plating for viability: At the end of the expression period, cell concentrations in the selected cultures were determined using a Coulter counter and adjusted to give 1 x 10^5 cells/mL in readiness for plating for 6TG resistance. Samples from these were diluted to 8 cells/mL. Using a multichannel pipette, 0.2 mL of the final concentration of each culture was placed into each well of 2 x 96-well microtitre plates (192 wells averaging 1.6 cells/well). The plates were incubated at 37±1ºC in a humidified incubator gassed with 5±1% v/v CO2 in air until scoreable (8 days). Wells containing viable clones were identified by eye using background illumination and counted.

- Plating for 6TG resistance: At the end of the expression period, the cell densities in the selected cultures were adjusted to 1 x 10^5 cells/mL. 6TG (1.5 mg/mL) was diluted 100-fold into these suspensions to give a final concentration of 15 μg/mL. Using a multichannel pipette, 0.2 mL of each suspension was placed into each well of 4 x 96-well microtitre plates (384 wells at 2 x 10^4 cells/well). Plates were incubated at 37±1ºC in a humidifiedincubator gassed with 5±1% v/v CO2 in air until scoreable (13 days) and wells containing clones were identified as above and counted.

ACCEPTANCE CRITERIA
The assay was considered valid if all of the following criteria were met:
1. The MF in the vehicle control cultures was considered acceptable for addition to the laboratory historical negative control database
2. The MF in the concurrent positive controls induced responses that were comparable with those generated in the historical positive control database and gave a clear, unequivocal increase in MF over the concurrent negative control
3. The test was performed with and without metabolic activation
4. Adequate numbers of cells and concentrations were analysable.

Evaluation criteria:

For valid data, the test article was considered to be mutagenic in this assay if:
1. The MF at one or more concentrations was significantly greater than that of the negative control (p≤0.05)
2. There was a significant concentration-relationship as indicated by the linear trend analysis (p≤0.05)
3. If both of the above criteria were fulfilled, the results should exceed the upper limit of the last 20 studies in the historical negative control database (mean MF
+ 2 standard deviations.

Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis.

Statistics:

Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines (Robinson et al., 1990)*. The control log mutant frequency (LMF)
was compared with the LMF from each treatment concentration and the data were checked for a linear trend in mutant frequency with test article treatment. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance.

*References:
- Robinson, W.D., Green, M.H.L., Cole, J., Garner, R.C., Healy, M.J.R. and Gatehouse, D. (1990). Statistical evaluation of bacterial/mammalian fluctuation tests. In Statistical Evaluation of Mutagenicity Test Data (Ed D J Kirkland) Cambridge University Press, pp 102-140

Key result

Species / strain:

mouse lymphoma L5178Y cells

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:

OSMOLALITY AND PH
No marked changes in osmolality or pH were observed in the Range-Finder at the highest concentration tested (2025 μg/mL) as compared to the concurrent vehicle controls.

CYTOTOXICITY RANGE-FINDER
Both upon addition of the test article to the cultures and following the treatment incubation period, precipitate was observed at all concentrations tested in the absence and presence of S-9 (63.28 to 2025 μg/mL). This precipitate was still present following the wash off procedure post treatment (confirmed by haemocytometer). Turbidity assessments pre and post treatment indicated an increased turbidity with increasing concentration of Iron Oxide Sicovit® Yellow 10 E172. Due to the nature of the compound all concentrations were plated for survival. The highest concentration tested (2025 μg/mL) gave 94% and 70% RS in the absence and presence of S-9, respectively (please refer to Table 1 in the field 'Any other information on results incl. tables').

MUTATION EXPERIMENT
- Cytotoxicity and precipitation:
In the Mutation Experiment, ten concentrations were tested in the absence and presence of S-9 ranging from 0.1955 to 100.1 μg/mL. Precipitation was observed at the time of treatment at the highest five concentrations tested in the absence and presence of S-9 (6.225 to 100.1 μg/mL). Post treatment precipitate (observed by eye) was observed at the highest five concentrations tested in the absence of S-9 (6.225 to 100.1 μg/mL) and at the highest six concentrations tested in the presence of S-9 (3.128 to 100.1 μg/mL). Assessment by haemocytometer following the wash off procedure indicated that the highest non-precipitating concentrations were 6.255 μg/mL in the absence of S-9 and 1.564 μg/mL in the presence of S-9. The lowest concentration at which precipitate was observed (by eye) at the end of the treatment incubation period in the absence and presence of S-9 was retained and higher concentrations were discarded. Turbidity assessment before treatment confirmed a concentration related increase in turbidity compared to the vehicle control. Turbidity assessment post treatment indicated no increase in turbidity across the concentrations analysed. Seven days after treatment all concentrations retained in the absence and presence of S-9 were selected to determine viability and 6TG resistance. The highest concentrations analysed were 6.255 μg/mL in the absence of S-9 and 3.128 μg/mL in the presence of S-9 which gave 83% and 109% RS, respectively (please refer to Table 2 in the field 'Any other information on results incl. tables').
- Mutant frequency:
Following 3 hour treatment up to precipitating concentrations, no statistically significant increases in MF were observed following treatment with Iron Oxide Sicovit® Yellow 10 E172 at any concentration analysed in the absence and presence of S-9 and there were no statistically significant linear trends (please refer to Table 2 in the field 'Any other information on results incl. tables').

ASSAY VALIDITY
Vehicle and positive control treatments were included in the Mutation Experiment in the absence and presence of S-9. Mutant frequencies (MF) in vehicle control cultures fell within acceptable ranges (please refer to attached background material) and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline 1-oxide (NQO) (without S-9) and benzo(a)pyrene (B[a]P) (with S-9). Therefore the study was accepted as valid. All acceptance criteria were met and the study was accepted as valid.

Table 1. Range-Finder Experiment - 3 Hour Treatment in the Absence and Presence of S-9

Concentration (µg/mL)	%RS -S-9	%RS +S-9
0	100	100
63.28 P, PP	83	78
126.6 P, PP	104	82
253.1 P, PP	82	99
506.3 P, PP	68	91
1013 P, PP	77	101
2025 P, PP	94	70

%RS: Percent Relative Survival

P: Precipitation noted at time of treatment

PP: Precipitation noted at end of treatment incubation period

Table 2. Mutation Experiment - 3 Hour Treatment in the Absence and Presence of S-9

Concentration (µg/mL)	%RS	MF § (Day 7)	Concentration (µg/mL)	%RS	MF § (Day 7)
3-hour treatment (-S-9)			3-hour treatment (+S-9)
0	100	4.16	0	100	4.53
0.1955	87	5.36 NS	0.1955	102	2.12 NS
0.3909	89	2.69 NS	0.3909	91	2.60 NS
0.7879	89	2.47 NS	0.7819	105	1.61 NS
1.564	82	3.89 NS	1.564	114	3.32 NS
3.128	113	4.22 NS	3.128 P, PP	109	2.86 NS
6.255 P, PP	83	3.53 NS
NQO 0.15	36	27.87	B[a]P 2	55	34.26
NQO 0.2	27	35.63	B[a]P 3	58	12.35

Linear trends: Not significant (-/+S-9)

§: 6 -TG resistant mutants/10^6viable cells 7 days after treatment

%: RS Percent relative survival adjusted by post treatment cell counts

NS: Not significant

P: Precipitation (by eye) noted at time of treatment

PP: Precipitation (by eye) noted at end of treatment incubation period

Conclusions:

In the main test, the lowest concentration at which precipitate was observed (by eye) at the end of the treatment incubation period in the absence and presence of S-9 was retained and higher concentrations were discarded. The highest concentrations analysed were 6.255 μg/mL in the absence of S-9 and 3.128 μg/mL in the presence of S-9 which gave 83% and 109% RS, respectively. Following 3-hour treatment up to precipitating concentrations, no statistically significant increases in MF were observed following treatment with Iron Oxide Sicovit® Yellow 10 E172 at any concentration analysed in the absence and presence of
S-9 and there were no statistically significant linear trends. The solvent control values were within the acceptable limits in each assay. The positive controls showed distinct and significant increases in mutant frequency and demonstrated sensitivity of the test system. All validity criteria were met.
Based on the study results, it is concluded that Iron Oxide Sicovit® Yellow 10 E172 did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to a precipitating concentration for 3 hours in the absence and presence of a rat liver metabolic activation system (S-9) under the experimental conditions described.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

weight of evidence

Study period:

15 November 2018 - 22 March 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

see attachment "Iron oxide category read-across concept-HH " in IUCLID section 13.2.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

Adopted: 29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

31 May 2016

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Test article stock formulations of Iron Oxide Sicovit® Yellow 10 E172 were prepared under subdued lighting in McCoy's 5A to give the maximum required concentration. Subsequent dilutions were made using McCoy's 5A. The test article formulations were protected from light and used within approximately 1.5 hours of initial formulation. Turbidity of each treatment formulation in the Range-Finder and Micronucleus Experiment was assessed using a SpectraMax M2e plate reader. 100 μL of vehicle control and each test article formulation were added to a 96-well plate. Optical density readings were determined for each sample analysed (measured data not reported).

Target gene:

not applicable

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CHO cells, supplied by Dr. S Galloway, West Point, PA, USA, are maintained at Covance Laboratories Ltd. in tissue culture flasks containing McCoy's 5A medium
including 10% (v/v) heat inactivated foetal calf serum (FCS) and 0.52% penicillin / streptomycin. They are sub-cultured regularly at low density and before overgrowth occurs, to maintain low aberration frequencies. The measured cell cycle time of the cells used at Covance Laboratories Ltd. is approximately 13 hours. Stocks of cells preserved in liquid nitrogen are reconstituted for each experiment so as to maintain karyotypic stability. The cells are routinely screened for mycoplasma contamination.

Cell sheets were removed from stock cultures using trypsin/EDTA solution and subcultured at a low density (approximately 6 x 10^5 cells per flask) into 25 cm² tissue culture flasks (9 mL or 8.9 mL cell suspension per flask for the Range-Finder and Micronucleus Experiment, respectively). After one day of incubation in an atmosphere of 5% (v/v) CO2 in air, at 37±1°C, cultures at a suitable level of confluence were selected for treatment.

Cytokinesis block (if used):

Cytochalasin-B

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).

Test concentrations with justification for top dose:

- Range-Finder: 7.256, 12.09, 20.16, 33.59, 55.99, 93.31, 155.5, 259.2, 432.0, 720.0, 1200, and 2000 µg/mL (maximum recommended test concentration)
- Micronucleus experiments: 0.1465, 0.2930, 0.5859, 1.172, 2.344, 4.688, 9.375, 18.75, 37.50, 75.0, 150, and 300 µg/mL (test item precipitation)

Vehicle / solvent:

- Vehicles used: McCoy's 5A culture medium

- Justification for choice of solvent/vehicle: The test article was found to be insoluble in all commonly used vehicles and was
therefore formulated as a suspension in culture medium. A medium replacement methodology was performed to ensure that treatments were performed up to the maximum recommended concentration according to current regulatory test guidelines (2000 μg/mL) in the cytotoxicity Range-Finder Experiment.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: noscapine

Details on test system and experimental conditions:

CYTOTOXICITY RANGE-FINDER
Immediately prior to treatment, all cultures were pelleted (approximately 300 g, 10 minutes) and 9 mL of culture medium removed. S-9 mix or KCl (1 mL per culture) was added appropriately. Cultures were treated with the test article or vehicle (8 mL per culture). A further 0.9 mL or 1 mL of culture medium was added to the 24+0 hour and the 3+21 hour cultures, respectively. Positive control treatments were not included. Cyto-B, formulated in DMSO, was added directly (0.1 mL per culture) to all continuous (24+0 hour) cultures at the time of treatment to give a final concentration of 3 μg/mL per culture. The final culture volume was 10 mL. Cultures were incubated at 37±1°C for the designated exposure time.

MICRONUCLEUS EXPERIMENT
- Cell treatment:
Immediately prior to treatment, all vehicle and test article treated cultures were pelleted (approximately 300 g, 10 minutes) and 8.9 mL of culture medium removed. S-9 mix or KCl (1 mL per culture) was added appropriately. Cultures were treated with the test article (8 mL per culture) or vehicle (9 mL or 8.9 mL per culture for the 3+21 hour or 24+0 hour vehicle control cultures, respectively). A further 1.0 mL or 0.9 mL of culture medium was added to the 3+21 hour or 24+0 hour test article cultures, respectively. Positive control cultures were treated at 1% v/v (0.1 mL per culture), S-9 mix or KCl (1 mL per culture) and vehicle (8.9 mL per culture). Cyto-B, formulated in DMSO, was added directly (0.1 mL per culture) to all continuous (24+0 hour) cultures at the time of treatment to give a final concentration of 3 μg/mL per culture. The final culture volume was 10 mL. Cultures were incubated at 37±1°C for the designated exposure time (Table 1; please refer to the field 'Any other information on materials and methods incl. tables'). The number of cultures used in the different experiments is tabulated in Table 2 (please refer to the field 'Any other information on materials and methods incl. tables')

For 24+0 hour treatments, flasks were gassed with 5% (v/v) CO2 in air for approximately 10-20 seconds prior to returning to an incubator (set to 37±1°C). Treatment media remained on cultures receiving the continuous treatment until sampling, 24 hours after the beginning of treatment.

For 3 hour treatments with a recovery period, treatment medium was removed after 3 hours and cell monolayers washed twice with saline (pre-warmed in an incubator set to 37±1°C). Cultures were re-fed with fresh pre-warmed McCoy’s 5A medium containing foetal calf serum and penicillin / streptomycin and Cyto B (at a final concentration of 3 μg/mL). All culture flasks were gassed with 5% (v/v) CO2 in air for approximately 10-20 seconds prior to returning to an incubator at 37±1°C until cell harvest.

- Post-Treatment for Micronucleus Experiment Cultures:
For removal of the test article following 3 hour treatments, treatment medium was removed, cell monolayers washed twice with sterile saline (pre-warmed in an incubator set to 37±1°C), and re-fed with fresh pre-warmed McCoy's 5A medium containing inactivated foetal calf serum (10%) penicillin / streptomycin and Cytochalasin B (at a final concentration of 3 μg/mL). Cultures were incubated in an atmosphere of 5% (v/v) CO2 in air at 37±1°C until harvest. In all experiments, the solubility of the test article in culture medium was assessed, by eye and microscope, at the beginning and end of treatment (microscopic assessment at the end of treatment only).

- Harvesting:
Immediately prior to harvest in the Micronucleus Experiment, the test article from all 24+0 hour cultures was washed off immediately prior to harvest in order in an attempt to reduce the amount of precipitate at slide making. At the defined sampling time, the medium from each flask was transferred to a plastic centrifuge tube together with a saline rinse. The monolayers were removed using trypsin/EDTA and the suspension from each flask was transferred to the plastic centrifuge tube containing the culture medium and saline rinse. Cultures were centrifuged at approximately 200 g for 5 minutes, the supernatant removed and discarded and cells re-suspended in 4 mL (hypotonic) 0.075 M KCl at 37±1°C for 4 minutes to allow cell swelling to occur. Cells were fixed by dropping the KCl / cell suspension into fresh, cold methanol/glacial acetic acid (7:1 v/v). The fixative was changed by centrifugation (approximately 200 g, 5 minutes) and re-suspension. This procedure was repeated as necessary (centrifuging at approximately 1250 g, 2-3 minutes) until the cell pellets were clean. Cells were stored in fixative at 2-8°C for at least 3 hours prior to slide preparation.

- Slide Preparation:
Cells were centrifuged (approximately 1250 g for 2-3 minutes) and re-suspended in a minimal amount of fresh fixative (as required) to give a milky suspension. Several drops of cell suspension were gently spread onto multiple clean, dry microscope slides. Slides were air-dried and stored at room temperature prior to staining. Slides were stained by immersion in 12.5 μg/mL Acridine Orange in PBS, pH 6.8 for approximately 10 minutes followed by a wash with PBS (with agitation) for a few seconds. The quality of the stain was then checked. Slides were air-dried and stored protected from light at room temperature. Immediately prior to analysis 1-2 drops of PBS were added to the slides before mounting with glass coverslips.

- Selection of Concentrations for the Micronucleus Experiment
Slides from the cytotoxicity Range-Finder Experiment were examined, uncoded, for proportions of mono-, bi- and multinucleate cells, to a minimum of 200 cells per concentration. From these data the replication index (RI) was determined.

- Selection of Concentrations for Micronucleus Analysis (Micronucleus Experiment Only):
Slides were examined, uncoded, for RI to a minimum of 500 cells per culture to determine whether chemically induced cell cycle delay or toxicity had occurred. Slides from precipitating cultures were checked to confirm that the presence of precipitate on slides would not preclude accurate analysis of micronuclei.

- Slide Analysis:
Scoring was carried out using fluorescence microscopy. Binucleate cells were only included in the analysis if all of the following criteria were met:
1. The cytoplasm remained essentially intact, and
2. The daughter nuclei were of approximately equal size.

A micronucleus was only recorded if it met the following criteria:
1. The micronucleus had the same staining characteristics and a similar morphology to the main nuclei, and
2. Any micronucleus present was separate in the cytoplasm or only just touching a main nucleus, and
3. Micronuclei were smooth-edged and smaller than approximately one third the diameter of the main nuclei.

For each treatment regime, two vehicle control cultures were analysed for micronuclei. Slides from the positive control treatments were checked to ensure that the system was operating satisfactorily. One concentration from each positive control, which satisfactory responses in terms of quality and quantity of binucleated cells andnumbers of micronuclei, was analysed. This pre-analysis slide check was conducted under non-blinded conditions.
All slides for analysis were coded by an individual not connected with the scoring of the slides, such that analysis was conducted under blind conditions. Labels with onlythe study number, assay type, experiment number, the sex of the donor and the codewere used to cover treatment details on the slides.

One thousand binucleate cells from each culture (2000 per concentration) were analysed for micronuclei. The number of cells containing micronuclei and the number of micronuclei per cell on each slide was recorded.
Nucleoplasmic bridges (NPBs) between nuclei in binucleate cells were recorded during micronucleus analysis to provide an indication of chromosome rearrangement. Micronucleus analysis was not conducted on slides generated from the Range-Finder treatments.

- Acceptance Criteria
The assay was considered valid if the following criteria were met:
1. The binomial dispersion test demonstrated acceptable heterogeneity (in terms of MNBN cell frequency) between replicate cultures, particularly where no positive responses were seen
2. The frequency of MNBN cells in vehicle controls fell within the current 95th percentile of the observed historical vehicle control (normal) ranges (please refer to 'attached background material')
3. The positive control chemicals induced statistically significant increases in the proportion of cells with micronuclei. Both replicate cultures at the positive control concentration analysed under each treatment condition demonstrated MNBN cell frequencies that clearly exceeded the normal range
4. A minimum of 50% of cells had gone through at least one cell division (as measured by binucleate + multinucleate cell counts) in vehicle control cultures at the time of harvest
5. The maximum concentration analysed under each treatment condition met the criteria specified in 'Selection of concentrations for the micronucleus experiment' (see above).

Evaluation criteria:

For valid data, the test article was considered to induce clastogenic and/or aneugenic events if:
1. A statistically significant increase in the frequency of MNBN cells at one or more concentrations was observed
2. An incidence of MNBN cells at such a concentration that exceeded the normal range in both replicates was observed
3. A concentration-related increase in the proportion of MNBN cells was observed (positive trend test).

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

The test article was considered negative in this assay if none of the above criteria were met.

Statistics:

The proportions of MNBN cells in each replicate were used to establish acceptable heterogeneity between replicates by means of a binomial dispersion test (Richardson et al., 1989)*. The proportions of MNBN cells for each treatment condition were compared with the proportion in vehicle controls by using Fisher's exact test (Richardson et al., 1989). A Cochran-Armitage trend test was applied to each treatment condition. Probability values of p≤0.05 were accepted as significant.

*References:
- Richardson C, Williams D A, Allen J A, Amphlett G, Chanter D O and Phillips B (1989). Analysis of data from in vitro cytogenetic assays. In "Statistical Evaluation of Mutagenicity Test Data", (UKEMS Guidelines Sub-committee Report, Part III), Ed D J Kirkland, Cambridge University Press, pp 141-154

Key result

Species / strain:

Chinese hamster Ovary (CHO)

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:

OSMOLALITY AND PH
In the Range-Finder experiment, no marked changes in osmolality (shifts of greater than 50 mOsm/kg) or pH (shifts of greater than 1 pH unit) were observed in post treatment medium samples at the highest three concentrations tested in the Range-Finder (720, 1200 and 2000 μg/mL), compared to the concurrent vehicle controls.

CYTOTOXICITY RANGE-FINDER
A suitable range of concentrations (up to a maximum of 300 μg/mL) was selected for the Micronucleus Experiment based on these toxicity data, also taking post-treatment precipitate in the cultures and appearance of precipitate on slides into account.

VALIDITY OF THE STUDY
The processed data confirm that:
1. The binomial dispersion test demonstrated acceptable heterogeneity (in terms of MNBN cell frequency) between replicate cultures
2. The frequency of MNBN cells in vehicle controls fell within the normal ranges
3. The positive control chemicals induced statistically significant increases in the proportion of MNBN cells. Both replicate cultures at the positive control concentration analysed under each treatment condition demonstrated MNBN cell frequencies that clearly exceeded the normal range
4. A minimum of 50% of cells had gone through at least one cell division (as measured by binucleate + multinucleate cell counts) in vehicle control cultures at the time of harvest
5. The maximum concentration analysed under each treatment condition met the concentration selection criteria specified

MICRONUCLEUS TEST
- Selection of concentrations: The highest concentration selected for micronucleus analysis following the 3+21 hour treatments was the highest concentration tested, 300 μg/mL. A range of concentrations were selected for analysis from both treatment conditions, including precipitating and non-precipitating concentrations where possible. The highest concentration selected for micronucleus analysis following the 24+0 hour treatment condition (75 μg/mL) was the maximum concentration at which the presence of precipitate on the slides did not preclude accurate analysis of micronuclei under this treatment condition. A range of concentrations were selected for analysis. Slides from the highest selected concentration and four lower concentrations were taken for microscopic analysis. The positive control concentrations analysed did not exceed the cytotoxicity limits for the test article concentration selection.

-Treatment of cells with Iron Oxide Sicovit® Yellow 10 E172 for 3+21 hours in the absence and presence of S-9 and for 24+0 hours in the absence of S-9 resulted in frequencies of MNBN cells that were generally similar to and not significantly different (at the p≤0.05 level), compared to those observed in the concurrent vehicle controls (Table 3; please refer to 'any other information on results incl. tables' ), at all concentrations analysed under each treatment condition. The MNBN cell frequencies in treated cultures fell within the normal ranges (please refer to 'attached background material') at all concentrations analysed under each treatment condition. A weak but statistically significant linear trend (p≤0.05) was observed for the 3+21 hour treatment in the absence of S-9. However, in the absence of any marked increases in MNBN cell frequencies which exceeded the normal range at any concentration analysed, this isolated observation was considered not biologically relevant.

- No test article related increases in cells with NPBs were observed.

Table 3. Summary - Results of the Micronucleus Experiments.

Treatment	Concentration (µg/mL)	Cytotoxicity (%)	Mean MNBN cell frequency (%)	Historical control data range (%)^#	Statistical significance
3+21 h -S-9	Vehicle^a	-	0.45	0.30 to 1.41	-
	0.5859	3	0.30		NS
	1.172	0	0.50		NS
	37.50	0	0.70		NS
	75.0	0	0.50		NS
	300.0	4	0.75		NS
	MMC, 0.12^$	5	3.25		p≤0.001
3+21 h +S-9	Vehicle^a	-	0.40	0.10 to 1.81	-
	2.344	0	0.65		NS
	9.375	0	0.80		NS
	37.50	0	0.60		NS
	75.0	0	0.80		NS
	300.0	0	0.55		NS
	CPA, 6.0	4	3.80		p≤0.001
24+0 h -S-9	Vehicle^a	-	0.60	0.21 to 1.49	-
	2.344	3	0.55		NS
	9.375	0	0.65		NS
	18.75	0	0.65		NS
	37.50	4	0.70		NS
	75.0	8	0.65		NS
	NOS, 2.0	5	25.60		p≤0.001

Conclusions:

In the main study, one thousand binucleate cells from each culture (2000 per concentration) were analysed for micronuclei (MNBN cells). Sterile McCoys’s 5A medium was used as vehicle. Vehicle and positive (mitomycin C, cyclophosphamide, noscapine) controls were run concurrently. Treatment of cells with Iron Oxide Sicovit® Yellow 10 E172 for 3+21 hours in the absence and presence of S-9 and for 24+0 hours in the absence of S-9 resulted in frequencies of MNBN cells that were similar to and not significantly different (at the p≤0.05 level), compared to those observed in the concurrent vehicle controls, at all concentrations analysed under each treatment condition. The MNBN cell frequencies in treated cultures fell within the normal ranges at all concentrations analysed under each treatment condition. A weak but statistically significant linear trend (p≤0.05) was observed for the 3+21 hour treatments in the absence of S-9. However, in the absence of any marked increases in MNBN cell frequencies which exceeded the normal range at any concentration analysed, this isolated observation was considered not biologically relevant. The frequency of MNBN cells in vehicle controls fell within the normal ranges. The positive control chemicals induced statistically significant increases in the proportion of MNBN cells.
Based on the study results, it is concluded that Iron Oxide Sicovit® Yellow 10 E172 did not induce micronuclei when tested up to and including precipitating concentrations (which did not preclude accurate analysis of micronuclei) for 3+21 hours in the absence and presence of a rat liver metabolic activation system (S-9) and for 24+0 hours in the absence of S-9 under the experimental conditions described.

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study - a study on clastogenicity in mammalian cells in vitro was performed with Fe3O4 as a representative for the iron oxide group - see Category approach for Fe2O3, Fe3O4, FeOOH, ZnFe2O4 and (Fe,Mn)2O3

Justification for type of information:

see attachment "Iron oxide category read-across concept-HH " in IUCLID section 13.2.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

0, 6.25, 12.5 and 25 µg/ml

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: mitomycin C and cyclophosphamide

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

only with S9 mix at 25 µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: strain/cell type: V79

Remarks:

Migrated from field 'Test system'.

None of the cultures treated with Bayferrox 306 (Fe3O4) in the absence and in the presence of S9 mix showed biologically relevant or statistically significant increased numbers of aberrant metaphases

Conclusions:

Interpretation of results (migrated information):
negative

Executive summary:

Method: chromosome aberration test with chinese hamster V79 cells

Result: negative (with and without metabolic activation)

Reference 5

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: guideline study - a study on gene mutation in mammalian cells (HPRT test) was performed with Fe3O4 as a representative for the iron oxide group

Justification for type of information:

see attachment "Iron oxide category read-across concept-HH " in IUCLID section 13.2.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

6. 9. 12, 18, 24, 36 µg/ml

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: ethyl sulfonate and dimethylbenzanthracene

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: strain/cell type: V79

Remarks:

Migrated from field 'Test system'.

Without and with S9 mix there was no biologically relevant increase in mutant frequency above that of the negative controls

Conclusions:

Interpretation of results (migrated information):
negative

Executive summary:

Method: V79/HPRT test according OECD 476

Result: negative

Reference 6

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:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: well documented and scientifically acceptable

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Test concentrations with justification for top dose:

up to 5000 µg/plate

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, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Interpretation of results (migrated information):
negative

Executive summary:

Bayferrox 3950 was investigated using the Salmonella/microsome test for point mutagenic effects in doses up to 5000 µg per plate on four Salmonella typimurium LT2 mutants. These comprised the histidine-auxotropic strains TA 1535, TA 100, TA 1537 and TA 98. Doses up to and including 5000 µg per plate did not cause any bacteriotoxic effects.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The in vivo genetic toxicity of Fe3O4 and Fe2O3 was assessed in two in vivo comet assays after oral administration, returning a negative result, thus Fe3O4 and Fe2O3 are non-genotoxic in vivo.

Details on the category justification are given in the read-across document attached in IUCLID section 13.2.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

12 July 2019 - 28 February 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

see attachment "Iron oxide category read-across concept-HH " in IUCLID section 13.2.

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

2016-07-29

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored at 15-25°C, protected from light.
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Consequently, all formulations were stored at 15-25°C, protected from light, and used within 2 hours of preparation. The recovery rates for iron in the formulation samples/suspensions provided information about the homogeneity of the test article in the formulations and were in the range of 45% to 147%. It is noticeable that the iron contents of the formulation samples are varying in a relatively wide range

Species:

rat

Strain:

other: Sprague Dawley Crl:CD(SD)

Remarks:

out-bred

Details on species / strain selection:

The rat was selected as there is a large volume of background data in this species. As no gender differences in toxicity, metabolism or bioavailability have been previously identified, and gender-specific human exposure was not expected, the study was conducted solely in male animals.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd. (Margate, UK)
- Age at study initiation: 7-8 weeks
- Weight at study initiation: 242-282 g (DRF) and 234-276 g (Main Experiment)
- Assigned to test groups randomly: yes. Checks were made to ensure the weight variation of Main Experiment animals prior to dosing was minimal and did not exceed ±20% of the mean weight.
- Fasting period before study: Animals were not fasted prior to test material administration.
- Housing:
- Diet: 5LF2 EU Rodent Diet (analysed for specific constituents and contaminants); ad libitum with the exception of the Main Experiment where the animals underwent a period of fasting (approximately 21 hours) from the evening on Day 1 following dosing until animal necropsy on Day 2
- Water: Mains water (periodically analysed for specific contaminants); ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-25°C
- Humidity: 40-70%
- Air changes: 15-20 air changes/hour
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 30 July 2019 To: 18 December 2019

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: hydroxypropyl methylcellulose (medium viscosity) 0.5% (w/v)
- Justification for choice of solvent/vehicle: The vehicle was selected because the test article is not soluble in water or organic solvents.
- Concentration of test material in vehicle: 50, 100, and 200 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL/kg

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations were freshly prepared prior to each dosing occasion by formulating Red Ferroxide 212P in HPMC 0.5% (w/v). To ensure homogeneity, and to avoid any interaction of the nanoparticles with the magnetic stirrer, dose formulations were gently inverted prior to dosing.

RATIONALE FOR ROUTE OF ADMINISTRATION:
All treatments were given via oral gavage as this is the intended route of human exposure.

Duration of treatment / exposure:

24 hours

Frequency of treatment:

Two administrations at 0 and 21 (Main Experiment) or 23 hours (DRF)

Post exposure period:

1 (DRF) or 3 hours (Main Experiment)

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

DRF and Main Experiment

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

DRF and Main Experiment

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

DRF and Main Experiment

No. of animals per sex per dose:

6 male rats per dose (Main experiment and 1. DRF); 3 male rats per dose (2. DRF)

Control animals:

yes, concurrent vehicle

Positive control(s):

Ethyl methanesulfonate (EMS, Sigma-Aldrich Chemical Co; Poole, UK)
- Route of administration: single oral administration
- Doses / concentrations: 200 mg/kg (20 mg/mL)

Tissues and cell types examined:

single cells of the stomach and duodenum (site of contact tissues)

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Based on the lack of adverse effects in a sub-chronic (90 day) oral repeated dose toxicity study in rats up to the limit dose of 1000 mg/kg bw/day, the limit dose of 2000 mg/kg bw/day was selected for this in vivo comet assay, as recommended by the current test guideline. Based on this information, the first Range-Finding Experiment was conducted at 2000 mg/kg/day. However, the nanoparticles were granular and difficult to remove from the tissue at the point of processing impacting on the quality of the comet slides. It was therefore decided to perform additional Range-Finding work at 500, 1000 or 2000 mg/kg/day, along with a concurrent vehicle control group as a comparator in order to further refine the tissue processing and allow comet scoring to take place in order to obtain further data.

TREATMENT AND SAMPLING TIMES:
The test article was administered as two administrations, at 0 and 23 hours in the Range-Finder Experiment. In the Range-Finding Experiments the animals were not fasted. However, the bolus of food and test article present within the animals’ digestive system at the time of necropsy made removing the nanoparticles and processing the tissues challenging. Therefore, the time was extended between the final dose on Day 2 and necropsy for the Main Experiment animals to 0 and 21 hours (i.e from 1 hour from final dose to necropsy, to 3 hours). The EMS positive control was administered once only at 21 hours. All animals were sampled at 24 hours. In order to further reduce nanoparticle contamination of the tissues and subsequent cell suspensions, during the Main Experiment the animals underwent a period of fasting (approximately 21 hours) from the evening on Day 1 following dosing until animal necropsy on Day 2.

DETAILS OF SLIDE PREPARATION:
- Histopathology: Preserved stomach and duodenum samples were embedded in wax blocks and sectioned at 5 μm nominal. Slides were stained with haematoxylin and eosin.

- Comet assay:
Preparation of Cell Suspension:
The comet stomach samples were washed in Merchants solution and then incubated on ice for 15 minutes, covered in fresh Merchants solution. After incubation the stomach samples were removed and placed in 200 μL of fresh Merchants solution. In order to remove as many of the particles as possible, stomach samples from the Range-Finder Experiment and Main Experiment were placed in a honey pot containing 20 mL of fresh Merchants solution and vortex mixed for approximately 15 seconds. Cells were gently scraped from the inside surface of the stomach using the back of a scalpel blade to produce single cell suspensions. Three independently coded slides were prepared per single cell suspension per tissue. Slides were dipped in molten normal melting point agarose (NMA) such that all of the clear area of the slide and at least part of the frosted area was coated. The underside of the slides was wiped clean and the slides allowed to dry. 40 μL of each single cell suspension was added to 400 μL of 0.7% low melting point agarose (LMA) at approximately 37°C. 100 μL of cell suspension/agarose mix was placed on to each slide. The slides were then coverslipped and allowed to gel on ice.

Cell Lysis:
Once gelled the coverslips were removed and all slides placed in lysis buffer (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, pH adjusted to pH 10 with NaOH, 1% Triton X-100, 10% DMSO) overnight at 2-8°C, protected from light.

Unwinding and Electrophoresis:
Following lysis, slides were washed in purified water for 5 minutes, transferred to electrophoresis buffer (300 mM NaOH, 1 mM EDTA, pH>13) at 2-8°C and the DNA unwound for 20 minutes. At the end of the unwinding period the slides were electrophoresed in the same buffer at 0.7 V/cm for 20 minutes. As not all slides could be processed at the same time a block design was employed for the unwinding and electrophoretic steps in order to avoid excessive variation across the groups for each electrophoretic run; i.e. for all animals the same number of triplicate slides was processed at a time.

Neutralisation:
At the end of the electrophoresis period, slides were neutralised in 0.4 M Tris, pH 7.0 (3 x 5 minute washes). After neutralisation the slides were dried and stored at room temperature prior to scoring.

Staining:
Prior to scoring, the slides were stained with 100 μL of 2 μg/mL ethidium bromide and coverslipped.

METHOD OF ANALYSIS:
- Comet:
Slides from the first Range-Finder Experiment were visually assessed to confirm that there were no anomalies which could confound comet scoring in the Main Experiment. However, no formal scoring was conducted. It was noted that extensive background debris was present on the slides. In the following Range-Finder Experiments (2RF-5RF) and the Main Experiment, scoring was carried out using fluorescence microscopy at an appropriate magnification and with suitable filters. All slides were allocated a random code and analysed by an individual not connected with the dosing phase of the study.
In the Main Experiment, scoring was carried out using fluorescence microscopy. A slide from a vehicle and positive control animal were checked for quality and/or response prior to analysis. All slides were allocated a random code and analysed by an individual not connected with soring of the study. All available animals per group were analysed. Measurements of tail intensity (%DNA in tail) and tail moment were obtained from 150 cells/animal/tissue. In general this was evenly split over two or three slides. The number of ‘hedgehogs’ (a morphology indicative of highly damaged cells often associated with severe cytotoxicity, necrosis or apoptosis) observed during comet scoring was recorded for each slide. Each slide was scanned starting to the left of the centre of the slide. After completion of microscopic analysis and decoding of the data the percentage tail intensity (i.e. %DNA in the tail) and Olive tail moment were calculated.

Data were treated as follows:
1. The median value per slide was calculated
2. The mean of the slide medians was calculated to give the mean animal value
3. The mean of the animal means and standard error of the mean was calculated for each group.

- Scoring Criteria for Comet Assay:
The following criteria were used for analysis of slides:
1. Only clearly defined non overlapping cells were scored
2. Hedgehogs were not scored
3. Cells with unusual staining artefacts were not scored.

OTHER:
- Clinical signs and body weight: All animals were examined at the beginning and the end (nominal) of the working day to ensure that they were in good health and displayed no signs of overt toxicity. Individual body weights were recorded on a daily base during the dose phase. Clinical chemistry parameters (aspartate aminotransferase, creatinine, alkaline phosphatase, alanine aminotransferase, potassium, sodium, inorganic phosphorus, calcium, total protein, albumin, globulin, albumin/globulin ratio, total cholesterol, glucose, urea, total bilirubin, and chloride) were assessed from plasma derived from terminal blood samples.

Evaluation criteria:

For valid data, the test article was considered to induce DNA damage if:
1. A least one of the test doses exhibited a statistically significant increase in tail intensity, in any tissue, compared with the concurrent vehicle control
2. The increase was dose-related in any tissue
3. The increase exceeded the laboratory’s historical control data for that tissue.

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 and target tissue exposure was confirmed.

Results which only partially satisfied the criteria were dealt with on a case-by-case basis. Biological relevance was taken into account, for example comparison of the response against the historical control data and consistency of response within and between dose levels.

Statistics:

In the second DRF and the Main Experiment, tail intensity data for each slide were supplied for statistical analysis. The median of the log-transformed tail intensities from each slide was averaged to give an animal summary statistic. Where the median value on a slide was zero, a small constant (0.0001) was added before taking the logarithm and calculating the average for the animal. This animal average was used in the statistical analysis.

Data was analysed using one-way analysis of variance (ANOVA) with the fixed factor for treatment group. The positive control group was excluded from this analysis. Levene’s test was used to test for equality of variances among groups. This showed no evidence of heterogeneity (P>0.01). Comparisons between each treated group and control were made using Dunnett’s test. The test was one-sided looking for
an increase in response with increasing dose. The back-transformed difference and p-value are reported. In addition, (where appropriate), a linear contrast was used to test for an increasing dose response.

The positive control group was compared to the control group using a two-sample t-test. Levene’s test was used to test for equality of variances between the groups. This showed no evidence of heterogeneity (P>0.01). The test was one-sided looking for an increase in response with increasing dose. The back-transformed difference and p-value are reported.

Sex:

male

Genotoxicity:

negative

Remarks:

stomach

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Sex:

male

Genotoxicity:

negative

Remarks:

duodenum

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg bw/day (1. DRF) and 500, 1000, and 2000 mg/kg bw/day (2. DRF)
- Observations and signs of toxicity: No clinical signs of toxicity and no losses in animal bodyweight were evident. However, it was noted during the necropsy that the nanoparticles were granular and difficult to remove from the tissue at the point of processing. Subsequent visual assessment of the comet slides demonstrated that the quality of the comet slides were suboptimal and difficult to evaluate due to background debris. It was therefore decided to perform additional Range-Finding work (designated 2RF to 5RF) in groups of 3 male animals at 500, 1000 or 2000 mg/kg/day, along with a concurrent vehicle control group as a comparator in order to further refine tissue processing and allow the subsequent slides generated from these animals to be scored.
- Comet analysis: There were no marked, dose-related increases in %hedgehogs in the stomach or duodenum, thus demonstrating that treatment with Red Ferroxide 212P did not cause excessive DNA damage that could have interfered with comet analysis. However, it was noted that the group mean animal tail intensity values at 2000 mg/kg/day in stomach and at 500, 1000 and 2000 mg/kg/day in duodenum were slightly elevated. This was considered to be due to either mechanical damage as a result of over processing of these tissues or artifacts due to residual particulates remaining within the tissue.
Therefore, in order to further reduce nanoparticle contamination of the tissues and subsequent cell suspensions, during the Main Experiment the following experimental design changes were also implemented:
• The time was extended between the final dose on Day 2 and necropsy from 23 hours to 21 hours (i.e from 1 hour from final dose to necropsy, to 3 hours)
• Animals were allowed to undergo a period of fasting (approximately 21 hours) from the evening on Day 1 following dosing until animal necropsy on Day 2 (Groups 1-4 only).
On this basis, the Main Experiment was conducted at doses of 2000, 1000 and 500 mg/kg/day.

RESULTS OF DEFINITIVE STUDY
- Comet Assay:
In the stomach and duodenum, animals treated with Red Ferroxide 212P at all doses exhibited group mean and individual animal tail intensity (0.55-0.84% and 0.13-0.24%, respectively) and tail moment (0.06-0.08 and 0.04-0.06, respectively) values that were similar to the concurrent vehicle control group (stomach: 0.54% and 0.05, respectively; duodenum: 0.66% and 0.06, respectively) and all tail intensity values fell within the laboratory’s historical vehicle control 95% reference range (stomach: 0.16-7.18%; duodenum: 0.18-7.60%). There were no statistically significant increases in tail intensity for any of the groups receiving the test article, compared to the concurrent vehicle control group and no evidence of a dose-response.
Hedgehog occurrence: There were no marked, dose-related increases in %hedgehogs in stomach or duodenum thus demonstrating that treatment with Red Ferroxide 212P did not cause excessive DNA damage that could have interfered with comet analysis.

- Observations and signs of toxicity: No clinical signs of toxicity were observed in any animal following treatments Red Ferroxide 212P. No direct effect of Red Ferroxide 212P treatment on animal body weights was observed. No clinical chemistry changes considered an effect of Red Ferroxide 212P treatment were recorded.

- Histopathology: On macroscopic examination, there were no changes which were considered related to Red Ferroxide 212P. On microscopic examination, dark, finely granular material was noted along the mucosal surface of the lumen of the stomach and duodenum which was considered to be test article. However, there were no microscopic changes which were considered to be related to treatment with Red Ferroxide 212P.

- Assay validity:
The data generated in this study confirm that:
1. The vehicle control data were comparable to laboratory historical control data for each tissue; 2. The positive control induced responses that were comparable with the laboratory’s historical positive control data and produced a statistically significant increase in TI compared to the concurrent vehicle control; 3. Adequate numbers of cells and doses were analysed; 4. The high dose was considered to be the maximum recommended dose.
With respect to target tissue exposure, as the route of administration was oral, direct exposure to the duodenum and stomach was assured. The histopathology data confirmed presence of the test article. The assay data were therefore considered valid.

- Formulation analysis: It was noted that mean %recovery for Groups 2, 3 and 4 (50, 100 and 200 mg/mL) on Day 1 and Groups 2 and 3 (50 and 100 mg/mL) on Day 2 fell outside protocol specification (85-115% nominal). It was also noted that there was some variation (%RSD >10%) between the samples in all groups with the exception of Group 3 (100 mg/mL) on Day 1. No iron was detected in the vehicle control samples.

Conclusions:

It is concluded that Red Ferroxide 212P did not induce DNA strand breaks in the stomach or duodenum in male animals when tested up to 2000 mg/kg/day (the
regulatory maximum dose level).

Reference 2

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

weight of evidence

Study period:

25 June 2019 - 26 February 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

see attachment "Iron oxide category read-across concept-HH " in IUCLID section 13.2.

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

2016-07-29

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored at 15-25°C, protected from light.
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: : All formulations were stored at 15-25°C, protected from light, and used within 2 hours of preparation. The recovery rates, determined by the formulation analysis, for iron in the formulation samples/suspensions provided information about the homogeneity of the test article in the formulations and were in the range of 60% to 99% except for the last-named samples with a nominal content of 200 mg/mL.

Species:

rat

Strain:

other: Sprague Dawley Crl:CD(SD)

Remarks:

out-bred

Details on species / strain selection:

The rat was selected as there is a large volume of background data in this species. As no gender differences in toxicity, metabolism or bioavailability have been previously identified, and gender-specific human exposure was not expected, the study was conducted solely in male animals.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd. (Margate, UK)
- Age at study initiation: 7-8 weeks
- Weight at study initiation: 242-282 g (DRF) and 234-276 g (Main Experiment)
- Assigned to test groups randomly: yes. Checks were made to ensure the weight variation of Main Experiment animals prior to dosing was minimal and did not exceed ±20% of the mean weight.
- Fasting period before study: Animals were not fasted prior to test material administration.
- Housing: Animals were housed in wire-topped, solid-bottomed cages, with three animals per cage. Bedding was provided on a weekly basis to each cage by use of clean European softwood bedding (Datesand Ltd., Manchester, UK). In order to enrich both the environment and the welfare of the animals, they were provided with wooden Aspen chew blocks and rodent retreats.
- Diet: 5LF2 EU Rodent Diet (analysed for specific constituents and contaminants); ad libitum with the exception of the Main Experiment where the animals underwent a period of fasting (approximately 21 hours) from the evening on Day 1 following dosing until animal necropsy on Day 2
- Water: Mains water (periodically analysed for specific contaminants); ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-25°C
- Humidity: 40-70%
- Air changes: 15-20 air changes/hour
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 30 July 2019 To: 18 December 2019

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: hydroxypropyl methylcellulose (medium viscosity) 0.5% (w/v)
- Justification for choice of solvent/vehicle: The vehicle was selected because the test article is not soluble in water or organic solvents.
- Concentration of test material in vehicle: 50, 100, and 200 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL/kg

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations were freshly prepared prior to each dosing occasion by formulating Ferroxide Black 86 in HPMC 0.5% (w/v). To ensure homogeneity dose formulations were gently inverted prior to dosing.

RATIONALE FOR ROUTE OF ADMINISTRATION:
All treatments were given via oral gavage as this is the intended route of human exposure.

Duration of treatment / exposure:

24 hours

Frequency of treatment:

Two oral administrations at 0 and 23 hours

Post exposure period:

1 hour (please refer to "Details of tissue and slide preparation" for details and justification)

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Main Experiment

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Main Experiment

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

Main Experiment and DRF

No. of animals per sex per dose:

6 male rats per dose (Main experiment and DRF)

Control animals:

yes, concurrent vehicle

Positive control(s):

Ethyl methanesulfonate (EMS)
- Route of administration: single oral administration
- Doses / concentrations: 150 mg/kg (15 mg/mL)

Tissues and cell types examined:

single cells of the stomach and duodenum (site of contact tissues)

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Based on the lack of adverse effects in a sub-chronic (90 day) oral repeated dose toxicity study in rats up to the limit dose of 1000 mg/kg bw/day, the limit dose of 2000 mg/kg bw/day was selected for this in vivo comet assay, as recommended by the current test guideline.

TREATMENT AND SAMPLING TIMES:
The test article and vehicle control were given as two administrations, at 0 (Day 1) and 23 hours (Day 2); the positive control was administered once only at 21 hours (Day 2). All animals were sampled at 24 hours (Day 2). The stomach and duodenum were removed from each control (vehicle and positive) and test article-treated animal. The samples were collected after 1 hour based on gastric and intestinal transit times of test material in rats after oral treatment (Purdon and Bass, 1973)*.

DETAILS OF SLIDE PREPARATION:
- Histopathology: Preserved stomach and duodenum samples were embedded in wax blocks and sectioned at 5 μm nominal. Slides were stained with haematoxylin and eosin.

- Comet assay:
Preparation of Cell Suspension:
The comet stomach samples were washed in Merchants solution and then incubated on ice for 15 minutes, covered in fresh Merchants solution. After incubation the stomach samples were removed and placed in 200 μL of fresh Merchants solution. In order to remove as many of the particles as possible, stomach samples from the Range-Finder Experiment and Main Experiment were placed in a honey pot containing 20 mL of fresh Merchants solution and vortex mixed for approximately 15 seconds. Cells were gently scraped from the inside surface of the stomach using the back of a scalpel blade to produce single cell suspensions. Three independently coded slides were prepared per single cell suspension per tissue. Slides were dipped in molten normal melting point agarose (NMA) such that all of the clear area of the slide and at least part of the frosted area was coated. The underside of the slides was wiped clean and the slides allowed to dry. 40 μL of each single cell suspension was added to 400 μL of 0.7% low melting point agarose (LMA) at approximately 37°C. 100 μL of cell suspension/agarose mix was placed on to each slide. The slides were then coverslipped and allowed to gel on ice.

Cell Lysis:
Once gelled the coverslips were removed and all slides placed in lysis buffer (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, pH adjusted to pH 10 with NaOH, 1% Triton X-100, 10% DMSO) overnight at 2-8°C, protected from light.

Unwinding and Electrophoresis:
Following lysis, slides were washed in purified water for 5 minutes, transferred to electrophoresis buffer (300 mM NaOH, 1 mM EDTA, pH>13) at 2-8°C and the DNA unwound for 20 minutes. At the end of the unwinding period the slides were electrophoresed in the same buffer at 0.7 V/cm for 20 minutes. As not all slides could be processed at the same time a block design was employed for the unwinding and electrophoretic steps in order to avoid excessive variation across the groups for each electrophoretic run; i.e. for all animals the same number of triplicate slides was processed at a time.

Neutralisation:
At the end of the electrophoresis period, slides were neutralised in 0.4 M Tris, pH 7.0 (3 x 5 minute washes). After neutralisation the slides were dried and stored at room temperature prior to scoring.

Staining:
Prior to scoring, the slides were stained with 100 μL of 2 μg/mL ethidium bromide and coverslipped.

METHOD OF ANALYSIS:
- Comet:
Slides from the Range-Finder Experiment were visually assessed to confirm that there were no anomalies which could confound comet scoring in the Main Experiment. However, no formal scoring was conducted.
In the Main Experiment, scoring was carried out using fluorescence microscopy. A slide from a vehicle and positive control animal were checked for quality and/or response prior to analysis. All slides were allocated a random code and analysed by an individual not connected with soring of the study. All available animals per group were analysed. Measurements of tail intensity (%DNA in tail) and tail moment were obtained from 150 cells/animal/tissue. In general this was evenly split over two or three slides. The number of ‘hedgehogs’ (a morphology indicative of highly damaged cells often associated with severe cytotoxicity, necrosis or apoptosis) observed during comet scoring was recorded for each slide. Each slide was scanned starting to the left of the centre of the slide. After completion of microscopic analysis and decoding of the data the percentage tail intensity (i.e. %DNA in the tail) and Olive tail moment were calculated.

Data were treated as follows:
1. The median value per slide was calculated
2. The mean of the slide medians was calculated to give the mean animal value
3. The mean of the animal means and standard error of the mean was calculated for each group.

- Scoring Criteria for Comet Assay:
The following criteria were used for analysis of slides:
1. Only clearly defined non overlapping cells were scored
2. Hedgehogs were not scored
3. Cells with unusual staining artefacts were not scored.

OTHER:
- Clinical signs and body weight: All animals were examined at the beginning and the end (nominal) of the working day to ensure that they were in good health and displayed no signs of overt toxicity. Individual body weights were recorded on a daily base during the dose phase. Clinical chemistry parameters (aspartate aminotransferase, creatinine, alkaline phosphatase, alanine aminotransferase, potassium, sodium, inorganic phosphorus, calcium,
total protein, albumin, globulin, albumin/globulin ratio, total cholesterol, glucose, urea, total bilirubin, and chloride) were assessed from plasma derived from terminal blood samples.

*References:
- Purdon, R.A.; Bass, P., 1973. Gastric and intestinal transit in rats measured by a radioactive test meal. Gastroenterology 64, 968-976

Evaluation criteria:

For valid data, the test article was considered to induce DNA damage if:
1. A least one of the test doses exhibited a statistically significant increase in tail intensity, in any tissue, compared with the concurrent vehicle control
2. The increase was dose-related in any tissue
3. The increase exceeded the laboratory’s historical control data for that tissue.

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 and target tissue exposure was confirmed.

Results which only partially satisfied the criteria were dealt with on a case-by-case basis. Biological relevance was taken into account, for example comparison of the response against the historical control data and consistency of response within and between dose levels.

Statistics:

Tail intensity data for each slide were supplied for statistical analysis. The median of the log-transformed tail intensities from each slide was averaged to give an animal summary statistic. Where the median value on a slide was zero, a small constant (0.0001) was added before taking the logarithm and calculating the average for the animal. This animal average was used in the statistical analysis.

Data was analysed using one-way analysis of variance (ANOVA) with the fixed factor for treatment group. The positive control group was excluded from this
analysis. Levene’s test was used to test for equality of variances among groups. This showed no evidence of heterogeneity (P>0.01) in duodenum, but heterogeneity was seen in stomach. Comparisons between each treated group and control were made using Dunnett’s test. The test was one-sided looking for an increase in response with increasing dose. The back-transformed difference and p-value are reported. In addition, a linear contrast was used to test for an increasing dose response.

The positive control group was compared to the control group using a two-sample t-test. Levene’s test was used to test for equality of variances between the groups. This showed no evidence of heterogeneity (P>0.01). The test was one-sided looking for an increase in response with increasing dose. The back-transformed difference and p-value are reported.

Sex:

male

Genotoxicity:

negative

Remarks:

stomach

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Sex:

male

Genotoxicity:

negative

Remarks:

duodenum

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg bw/day
- Clinical signs of toxicity in test animals: The maximum dose of 2000 mg/kg/day was shown to be well tolerated with no clinical signs of toxicity and no losses in animal bodyweight.
- Macroscopy: Macroscopic observations taken at necropsy noted dark contents in the stomach, small intestines, large intestines and caecum. This was considered to be the presence of the test article.

RESULTS OF DEFINITIVE STUDY
- Comet Assay:
Stomach:
In the stomach, animals treated with Ferroxide Black 86 at 500 and 1000 mg/kg/day exhibited group mean and individual animal tail intensity and tail moment values that were similar to the concurrent vehicle control group and which fell within the laboratory’s historical vehicle control 95% reference range (tail intensity: 0.16-7.18%). At 2000 mg/kg/day, there was a statistically significant increase (P≥0.05) in group mean tail intensity which also contributed to a significant linear trend (please refer to ‘attached background material’: Summary Comet Data). The increase was primarily due to two animals within the group showing elevated tail intensity values (R0305 TI of 9.58 and R0306 TI of 11.23; please refer to ‘attached background material’: Animal Comet Data_Stomach) which were close to or exceeded the historical vehicle control observed maximum tail intensity of 10.69 (please refer to ‘attached background material: historical control data). Although there were no corresponding pathology findings to suggest target tissue toxicity or inflammation, the increases were concomitant with some small increases in %hedgehogs (highly damaged cells). Given the known challenges of working with small particles on site of contact tissues (Elespuru et al; 2018) and that additional technical steps were included in order to ensure the tissues were visually free of the particles at the time of tissue processing, it is likely that these increases in tail intensity were due to either mechanical damage due to over processing of these tissues or artifacts due to residual particulates remaining within the tissue (the histopathology data demonstrated residual particles present within the tissue) rather than a true genotoxic effect and therefore the biological relevance is considered to be unlikely.

Duodenum:
In the duodenum, animals treated with Ferroxide Black 86 at all doses exhibited group mean and individual animal tail intensity and tail moment values that were similar to the concurrent vehicle control group and all tail intensity values fell within the laboratory’s historical vehicle control 95% reference range (0.18-7.60%). There were no statistically significant increases in tail intensity for any of the groups receiving the test article, compared to the concurrent vehicle control group and no evidence of a dose-response.

Hedgehog occurrence:
There were no dose-related increases in %hedgehogs in stomach or duodenum thus demonstrating that treatment with Ferroxide Black 86 did not cause excessive DNA damage that could have interfered with Comet analysis.

- Clinical signs of toxicity in test animals: No clinical signs of toxicity were observed in any animal following treatments with vehicle, Ferroxide Black 86 (at 500, 1000 or 2000 mg/kg/day) or the positive control (EMS). No clinical chemistry changes considered an effect of Ferroxide Black 86 were recorded.

- Histopathology: On macroscopic examination, dark contents were noted in the stomach, jejunum, ileum, cecum, and colon of animals administered 2000 mg/kg/day; the stomach, jejunum, and ileum of animals administered 1000 mg/kg/day; and the stomach of one animal administered 500 mg/kg/day. On microscopic examination, in the stomach and duodenum, dark material (which was considered to be test article), was noted in the lumen of all animals administered Ferroxide Black 86.

- Assay validity: The vehicle control data were within the laboratory’s historical vehicle control data ranges. The positive control induced statistically significant increases in tail intensity in the stomach and duodenum (over the current vehicle control group) that were comparable with the laboratory’s historical positive control data. The assay was therefore accepted as valid.

- Formulation analysis: It was noted that mean %recovery for Group 3 (100 mg/mL) on Day 1 and Groups 2 and 3 (50 and 100 mg/mL) on Day 2 fell outside protocol specification (85-115% nominal). It was also noted that there was some variation (%RSD >10%) between the samples in Group 3 (100 mg/mL) on Day 1 and Groups 3 and 4 (100 and 200 mg/mL) on Day 2. No iron was detected in the vehicle control samples.

REFERENCES:
- Elespuru R, Pfuhler S, Aardema M J, Chen T, Doak S H, Doherty A, Farabaugh CS, Kenny J, Manjanatha M, Mahadevan B, Moore MM, Ouedraogo G, Stankowski LF Jr, Tanir JY (2018). Genotoxicity Assessment of Nanoparticles: Recommendations on Best Practices and Methods. Toxicological Sciences 164(2), 391-416.

ADDITIONAL INFORMATION ON FORMULATION ANALYSIS

It was noted that the mean % recovery for the 100 mg/mL, Day 1 samples were slightly low at approximately 83% nominal. In addition, the Day 2 mean % recovery for the 50 and 100 mg/mL formulations were approximately 72 and 82% nominal, respectively. It was also noted that there was some variation (mean %RSD >10%) between samples at 100 mg/mL on Day 1 and 100 and 200 mg/mL on Day 2). However, given the type of analysis employed and the test article was in the form of nanoparticles, this is not unexpected. Therefore, all concentrations have been reported as nominal.

Conclusions:

It is concluded that Ferroxide Black 86 did not induce DNA strand breaks in the duodenum in male animals when tested up to 2000 mg/kg/day (the regulatory maximum dose level). In the same animals, a statistically significant increase in tail intensity in the stomach was observed at 2000 mg/kg/day. Although there were no corresponding pathology findings to suggest target tissue toxicity or inflammation, the increases were concomitant with some small increases in %hedgehogs. Given the known challenges of working with nanoparticles on site of contact tissues, it is likely that these increases in tail intensity were due to either mechanical damage due to over processing of these tissues or artifacts due to residual particulates remaining within the tissue rather than a true genotoxic effect and therefore the biological relevance is considered to be unlikely.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Introductory remark on read-across:

In this dossier, the endpoint genetic toxicity is not addressed by substance-specific information, but instead by a weight of evidence approach based on collected information for all substances of the iron oxide category. The predominant characteristic of the iron oxide category substances is the inertness being a cause of their chemical stability and very poor reactivity. This is shown by a very low dissolution in water and artificial physiological fluids as well as a very low in vivo bioavailability after oral administration. This very low reactivity, solubility and bioavailability leads to a complete lack of systemic toxicity after acute, sub-acute or sub-chronic oral or inhalation exposure up to the limit dose of the maximum tolerated concentration of the respective test. Similarly, a lack of systemic effects for the endpoints mutagenicity and reproductive toxicity are anticipated. Further information on the read-across approach is given in the report attached to IUCLID section 13.2.

 

in vitro:

Herbold (1990) tested zinc ferrite in a bacterial reverse mutation assay (according to OECD 471) both in absence and presence of metabolic activation. No increase in mutation frequency was observed in S. typhimurium TA 1535, TA1537, TA98 and TA100 when tested up to the limit concentration of 5000 µg/plate. Based on the results of this test, zinc ferrite is non-mutagenic for bacterial reverse mutation.

Hartmann (2019) investigated the mutagenic potential of Iron Oxide Sicovit® Yellow 10 E172 in a bacterial reverse mutation assay according to OECD TG 471 (1997) under GLP. The test was performed in S. typhimurium TA 98, TA 100, TA 1535, and TA 1537 as well as E. coliWP2uvrA pKM101 in two independent experiments in triplicate cultures. The cell cultures were treated according to the plate incorporation and pre-incubation method up to the maximum recommended concentration of 5000 µg/plate. However, 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. Treatment with Iron Oxide Sicovit® Yellow 10 E172 did not result in a mutagenic response at all concentrations tested both in absence and presence of S9 metabolic activation. Appropriate positive controls demonstrated the sensitivity of the test system.

Herbold (1982) tested Fe3O4 (Bayferrox AC 5110 M) in a bacterial reverse mutation assay (similar to OECD 471) both in absence and presence of metabolic activation. No increase in mutation frequency was observed in S. typhimurium TA 1535, TA1537, TA98 and TA100 when tested up to the limit concentration of 5000 µg/plate – precipitate was observed as of 1000µg/plate. Based on the results of this test, Fe3O4 is non-mutagenic for bacterial reverse mutation.

Thum (2008) tested Fe3O4 (Bayferrox 306) in an in vitro chromosome aberration assay (OECD 473) both in absence and presence of metabolic activation. After 4 hours treatment of Chinese hamster V79 cells with Fe3O4 concentrations of 6.25, 12.5 and 25 µg/mL were used without and with S9 mix for assessment of the clastogenic potential of Fe3O4. In addition, cells were evaluated for chromosomal aberrations after 18 hours treatment with Fe3O4 concentrations of 6.25, 12.5 and 25 µg/mL without S9 mix. None of these cultures treated with Fe3O4 both with and without metabolic activation showed statistically significant or biologically relevant increases of numbers of metaphases with aberrations. The positive controls induced clear clastogenic effects and demonstrated the sensitivity of the test system and the activity of the S9 mix used. Based on the results of this test, Fe3O4 is considered to be non-clastogenic for mammalian cells in vitro.

Entian (2008) Fe3O4 (Bayferrox 306) in the HPRT test (OECD 476) at concentrations ranging from 6-36 µg/mL without and with S9 mix. Under both activation conditions, no relevant cytotoxic effects were induced. However, the test material was tested up to and over the limits of solubility in the medium. Fe3O4 induced no biological relevant or biological statistically significant increases in the mutant frequency. The positive control EMS and DBA had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant frequencies as compared to the corresponding untreated controls and thus demonstrated the sensitivity of the test system and the activity of the S9 mix. Based on these results, Fe3O4 is considered to be non-mutagenic in the mammalian cell gene mutation assay, both with and without metabolic activation.

 

Presence of test material precipitates is a known confounder of in vitro genotoxicity testing, especially in test systems using adherent mammalian cells. Thus, an accurate determination of the presence of precipitates is indispensable in order to obtain valid and reliable results. The in vitro studies with FeOOH presented below included accurate analyses of the presence of test material precipitates including both analysis via the unaided eye and via microscopy.

Hargreaves (2019) examined on the capacity of FeOOH (Iron Oxide Sicovit® Yellow 10 E172) to induce gene mutations at the Hprt locus in mouse lymphoma L5178Y cells. The assay was performed according to OECD TG 476 (2016) and under GLP. The cell cultures were treated with the test material for 3 hours up to precipitating concentrations (≤ 100.1 µg/mL). Due to precipitation of the test item, the highest test material concentrations analysed were 6.255 and 3.128 µg/mL in the absence and presence of S9 metabolic activation, respectively. The top concentrations selected showed precipitation of the test item (particulates visible to the unaided eye) both at the time of treatment and at the end of the treatment incubation period. No biological meaningful increases in the mutant frequency were observed following a 3-hour treatment up to precipitating FeOOH concentrations both in absence and presence of a metabolic activation system. The positive control used demonstrated the sensitivity of the test system. 

Lloyd (2019) assessed the clastogenic and aneugenic activity of FeOOH (Iron Oxide Sicovit® Yellow 10 E172) in an in vitro mammalian cell micronucleus test according to OECD TG 487 (2016) and under GLP. Chinese hamster ovary (CHO) cells were treated either in absence of S9 metabolic activation for 3+21 and 24+0 hours or in presence of S9 metabolic activation for 3 hours. The micronucleus experiment was performed at test material concentrations ranging from 0.1465 to 300.0 µg/mL. The highest concentrations selected for analysis, based on precipitation, were 300 and 75 µg/mL for short-term and long-treatment, respectively. The concentration ranges analysed for micronucleus occurrence included several concentrations with and without visible precipitates (determined both via the unaided eye and microscopy). Treatment of cells with FeOOH for 3+21 hours in the absence and presence of S-9 and for 24+0 hours in the absence of S9 metabolic activation did not induce any clastogenic or aneugenic effects up to and including precipitating concentrations. The positive control substances demonstrated the sensitivity of the test system.

Evans, S.J. et al. (2019) investigated on the ability of FeOOH (Iron Oxide Sicovit® Yellow 10 E172) to be internalised by the L5178Y and CHO cells obtained from the experiments conducted Lloyd (2019) and Hargreaves (2019). No cellular uptake could be seen in the TEM analysis of the L5178Y cells at all concentrations tested, however, images taken of the cells exposed to 3.120 and 100.1 µg/ml demonstrated the test material was associated with the cell surface. In contrast, CHO cells showed a dose-dependent internalisation of the test material being localised within both the cytoplasm and/or in membrane bound vesicles. The internalised particles were analysed via EDX and verified as iron and oxygen containing particles.

 

in vivo

Fe2O3 (Red Ferroxide 212P) was tested for its potential to induce DNA strand breaks in the stomach and duodenum of treated rats (Keig-Shevlin, 2020). Fe2O3 (purity 99%, Fe content 69.8%) was administered to male Sprague Dawley rats (6 animals/sex/group, 3 for the positive control group) via gavage at nominal doses of 500, 1000 and 2000 mg/kg bw/day in two administrations at 0 (Day 1) and 21 hours (Day 2). A positive control group receiving Ethyl methanesulfonate 150 mg/kg via single oral administration at 21 hours (Day 2) was run concurrently. There were no marked, dose-related increases in %hedgehogs in the stomach or duodenum, thus demonstrating that treatment with Fe2O3 did not cause excessive DNA damage that could have interfered with comet analysis. In the stomach and duodenum, animals treated with Fe2O3 at all doses exhibited group mean and individual animal tail intensity and tail moment values that were similar to the concurrent vehicle control group and all tail intensity values fell within the laboratory’s historical vehicle control 95% reference range. There were no statistically significant increases in tail intensity for any of the groups receiving the test article, compared to the concurrent vehicle control group and no evidence of a dose-response. It is concluded that diiron trioxide did not induce DNA strand breaks in the stomach or duodenum in male animals when tested up to 2000 mg/kg/day (the regulatory maximum dose level).

Keig-Shevlin (2020) tested Fe3O4 (Ferroxide Black 86) for its potential to induce DNA strand breaks in the stomach and duodenum of treated rats. Fe3O4 (purity 99%, Fe content 72%) was administered to male Sprague Dawley rats (6 animals/sex/group, 3 for the positive control group) via gavage at nominal doses of 500, 1000 and 2000 mg/kg bw/day in two administrations at 0 (Day 1) and 23 hours (Day 2). A positive control group receiving Ethyl methanesulfonate 150 mg/kg via single oral administration at 21 hours (Day 2) was run concurrently. The Range-Finder Experiment was conducted in a single group of 6 male animals at the regulatory maximum dose of 2000 mg/kg/day. This was shown to be well tolerated with no clinical signs of toxicity and no losses in animal bodyweight. Macroscopic observations taken at necropsy noted dark contents in the stomach, small intestines, large intestines and caecum. This was considered to be the presence of the test article. From these results 2000 mg/kg/day was considered to be an appropriate maximum dose for the Main Experiment. Two lower doses of 1000 and 500 mg/kg/day were also selected. There were no dose-related increases in %hedgehogs in stomach or duodenum thus demonstrating that treatment with Fe3O4 did not cause excessive DNA damage that could have interfered with Comet analysis. In the stomach, animals treated with Fe3O4 at 500 and 1000 mg/kg/day exhibited group mean and individual animal tail intensity and tail moment values that were similar to the concurrent vehicle control group and which fell within the laboratory’s historical vehicle control 95% reference range. At 2000 mg/kg/day, there was a statistically significant increase (P≥0.05) in group mean tail intensity which also contributed to a significant linear trend. The increase was primarily due to two animals within the group showing elevated tail intensity values (R0305 TI of 9.58 and R0306 TI of 11.23) which were close to or exceeded the historical vehicle control observed maximum tail intensity of 10.69. Although there were no corresponding pathology findings to suggest target tissue toxicity or inflammation, the increases were concomitant with some small increases in %hedgehogs (highly damaged cells). Given the known challenges of working with particles on site of contact tissues (Elespuru et al; 2018) and that additional technical steps were included in order to ensure the tissues were visually free of the particles at the time of tissue processing, it is likely that these increases in tail intensity were due to either mechanical damage due to over processing of these tissues or artifacts due to residual particulates remaining within the tissue (the histopathology data demonstrated residual particles present within the tissue) rather than a true genotoxic effect and therefore the biological relevance is considered to be unlikely. In the duodenum, animals treated with Fe3O4 at all doses exhibited group mean and individual animal tail intensity and tail moment values that were similar to the concurrent vehicle control group and all tail intensity values fell within the laboratory’s historical vehicle control 95% reference range. There were no statistically significant increases in tail intensity for any of the groups receiving the test article, compared to the concurrent vehicle control group and no evidence of a dose-response. It is concluded that black iron oxide did not induce DNA strand breaks in the duodenum in male animals when tested up to 2000 mg/kg/day (the regulatory maximum dose level) and that the isolated findings in the stomach is likely that these increases in tail intensity were due to either mechanical damage due to over processing of these tissues or artifacts due to residual particulates remaining within the tissue rather than a true genotoxic effect.

Justification for classification or non-classification

Based on the results of in vitro bacterial gene mutation study, in vitro gene mutation study in mamalian cells and in vitro micronucleus study, no classification is proposed for genotoxicity according to the criteria of CLP regulation 1272/2008.