Iron hydroxide oxide yellow

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 November 2018 - 22 March 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

cell culture study

Remarks:

Genetic toxicity

Type of information:

experimental study

Adequacy of study:

other information

Study period:

not specified

Reliability:

other: Not rated according to Klimisch et al.

Rationale for reliability incl. deficiencies:

other: Non-guideline mechanistic study on in vitro cellular uptake of test material, which is in accordance with generally accepeted scientific standards.

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

no guideline followed

Principles of method if other than guideline:

Evans, S.J. et al. (2019) investigated on the in vitro cellular uptake of Iron Oxide Sicovit® Yellow 10 E172 in two different cell lines. The exposed cells were obtained from genotoxicity experiments (cf. cross-referenced studies). Adherent Chinese hamster ovary (CHO) cells were analysed after exposure to 0.1465, 2.344, and 75 µg/mL for 3 and 24 h. The non-adherent mouse lymphoma L5178Y cells were treated for 3 and 24 h at concentrations of 0.1955, 3.120, and 100.1 µg/mL. Both cell lines were examined for uptake of the test material using transmission electron microscopy (TEM). The identity of internalised particles was analysed via energy dispersive x-ray spectroscopy (EDX).

GLP compliance:

no

Remarks:

non-guideline mechanistic study

Type of method:

in vitro

Endpoint addressed:

genetic toxicity

Dose / conc.:

0.146 other: µg/mL

Remarks:

CHO cells

Dose / conc.:

2.344 other: µg/mL

Remarks:

CHO cells

Dose / conc.:

75 other: µg/mL

Remarks:

CHO cells

Dose / conc.:

0.196 other: µg/mL

Remarks:

L5178Y cells

Dose / conc.:

3.12 other: µg/mL

Remarks:

L5178Y cells

Dose / conc.:

100.1 other: µg/mL

Remarks:

L5178Y cells

Details on study design:

CELL PREPARATION
All cell exposures to the test agent were undertaken by Covance Laboratories (cf. cross-referenced studies), where L5178Y and CHO cells were treated with the test agent for 3 and 24 h respectively. On receipt of treated cells from Covance Laboratories, 1x106 cells of each exposed cell population were placed into 0.5 mL Eppendorf tubes and centrifuged at 230 g for 5 min. Cell pellets were subsequently resuspended in 100 mM phosphate buffered 2.5% glutaraldehyde fixative (Agar Scientific, UK) for 15 min at 37°C; cells were then pelleted (230 g for 10 min) and resuspended in fresh 2.5% glutaraldehyde fixative for 4 h at 4°C. Fixative was aspirated and 0.5 mL of maintenance buffer (0.1 M sucrose, 200 mM disodium hydrogen orthophosphate dihyrdrate and 200 mM sodium dihydrogen orthophosphate monohydrate in double distilled H2O) placed over cell pellets, samples were then left overnight at 4°C.
Following overnight incubation, cells were pelleted (230 g for 10 min) and washed once in maintenance buffer. Cells were then post fixed in 1% osmium tetroxide fixative (2.26% sodium dihydrogen orthophosphate, 2.52% sodium hydroxide, 5.4% glucose and 1% osmium tetroxide) for 1.5 h at 4°C in the dark on a rocker. After secondary fixation cells were re-pelleted at 230 g for 10 min and the fixative aspirated off. At this stage the TAAB Premix resin kit (TAAB Laboratory and Microscopy Reagents, UK) was prepared by the addition of the hardener to the resin and placed on a roller for 1 h and the accelerator component was added. Prior to adding resin to cell pellets dehydration stages were undertaken whereby cells were placed in 10% ethanol for 10 min, 70% ethanol for 30 min and then twice in 100% ethanol for 20 min; all dehydration stages were undertaken under gentle agitation. Cell samples were subsequently placed into 100% propylene oxide for 20 min (twice), then placed in 1:1 ratio of resin and propylene oxide for 90 min, finally cells were placed into 100% resin overnight at 4°C. Resin was pre-warmed at room temperature on a roller for 1 h, then the cell sample resin replaced with 100% fresh resin). Cell samples were placed in an oven at 60°C for 24 h with the caps left open (to allow any residual propylene oxide to evaporate).

CELL SECTIONING
The resin blocks were cut free from the 0.5 mL Eppendorf and trimmed with glass knifes using an EM-UC7 ultramicrotome (Leica Microsystems, UK) at 100 mm/sec approach distance set at 150 nm until the blocks had a flat face edge that was encompassing the cell pellet. From this block face a raised mesa was cut with the dimensions 750 μm x 750 μm and 50 μm deep at 100 mm/sec and approach set at 100 nm. Sections, 70 nm thick, were then cut from this mesa using an Ultra 45° diamond knife (Diatome, Switzerland), the cutting speed was set at 1 mm/sec and the approach distance was set at 70 nm. Sections were floated out onto a water bath (part of the diamond knife component) and picked up onto 150 mesh copper grids (Agar Scientific, UK) held in 0.07 mm tipped self-closing tweezers (Agar Scientific, UK). Sample grids were carbon coated (~3.5 nm coat) using a Q150-TE carbon coater (Quorum Technologies, UK).

TEM IMAGING
Samples were examined using a FEI Tecnai TF20 at Leeds University equipped with a field emission gun operated at 200 kV accelerating voltage. An Oxford Instruments INCA 350 EDX system/80 mm X-Max SDD detector was used to measure the Energy-dispersive X-ray (EDX) spectra and the images were captured on a Gatan Orius SC600A CCD camera. Where the presence of the test material was suspected within the tissue sections, elemental analysis was undertaken using EDX to provide an elemental spectrum of the observed object. A comparison of this spectra was then made using a parallel background region of the sample, selected from an adjacent area in the cell where there was no visual presence of the test material.

Details on results:

UPTAKE
L5178Y cell line:
- No cellular uptake of the test material was observed in L5178Y cells across the dose range analysed.
- In cells exposed to 3.120 and 100.1 µg/mL, the test material was associated with the cell surface

CHO cell line:
- Uptake of the test material was observed at all treatment concentrations in a dose-dependent manner (0.1465, 2.344 and 75 µg/mL)
- Only a small percentage of CHO cells treated with the lowest dose of 0.1465 µg/mL test material demonstrated internalisation, likely due to low availability of the material.
- CHO samples treated with 2.344 and 75 µg/mL test material showed uptake in most of the cells visualised, where it was localised within both the cytoplasm and/or in membrane bound vesicles. The 75 µg/mL treatment demonstrated very heavy loading of the test material into the CHO cells.
- EDX analysis of the internalised particles showed presence of iron and oxygen, confirming that the test material was taken up.

Conclusions:

Evans, S.J. et al. (2019) investigated on the ability of Iron Oxide Sicovit® Yellow 10 E172 to be internalised by the L5178Y and CHO cell lines by TEM imaging. The TEM image analyses revealed that the L5178Y did not internalise Iron Oxide Sicovit® Yellow 10 E172 at all concentrations tested. The cells, however, showed particles adhering to 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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 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

Test material

Test material information

Constituent 1

Reference substance name:

Iron hydroxide oxide yellow

EC Number:

257-098-5

EC Name:

Iron hydroxide oxide yellow

Cas Number:

51274-00-1

Molecular formula:

Fe(OH)O

IUPAC Name:

Iron hydroxide oxide

Test material form:

solid: nanoform, no surface treatment

Details on test material:

Appearance: yellow powder

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Test article stock 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.

Method

Target gene:

Hprt

Species / strain

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.

Controls

Controls 1

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

Controls 2

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

Results and discussion

Test results

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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.