Triiron tetraoxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

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.

Ames tests are also available for FeOOH, Fe2O3, ZnFe2O4 and (Fe,Mn)3O4. The results of all available Ames tests were negative.

The in vitro cytotoxicity and genotoxicity of commercially available nano-sized and powder Fe3O4, and Fe2O3 particles were compared in Syrian hamster embryo (SHE) cells. No significant increase in DNA damage was detected from nano-sized and powder iron oxides. None of the samples tested showed significant induction of micronuclei formation after 24 h of exposure. These findings were supported by the study of Karlsson, were no clear difference between nano-sized and micro-sized particles was found.

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 cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: guideline study

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 S9 mix)

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: guideline study

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

Test concentrations with justification for top dose:

6, 9, 12, 18, 24, 36 µg/ml

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 3

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: only 4 strains have been used; full report available positive control produced the expected effects

Principles of method if other than guideline:

according to Ames BN (1975), Mutation Res. 31, 347-364

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

8 - 40 - 200 - 1000 - 5000 µg/plate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: Endoxan, Trypaflavin, 2-aminoanthracen

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

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

Executive summary:

method: Ames test

result: negative (with and without metabolic activation)

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The in vivo genetic toxicity of Fe3O4 was assessed in an in vivo comet assays after oral administration, returning a negative result, thus Fe3O4 is 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

Endpoint:

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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 June 2019 - 26 February 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

Several Ames tests, a study on clastogenicity in mammalian cells in vitro as well as a study on gene mutation in mammalian cells (HPTR) was performed with Fe3O4 powder particles as a representative for the iron oxide group.

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.

 

Additionally the in vitro cytotoxicity and genotoxicity of commercially available nano-sized and powder Fe3O4, and Fe2O3 particles were compared in Syrian hamster embryo (SHE) cells. No significant increase in DNA damage was detected from nano-sized and powder iron oxides. None of the samples tested showed significant induction of micronuclei formation after 24 h of exposure. This findings were supported by the study of Karlsson, were no clear difference between nano-sized and micro-sized particles was found.

 

in vivo

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.