Mill scale (ferrous metal)

Specific investigations: other studies

Link to relevant study record(s)

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

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.

Description of key information

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.