Iron manganese trioxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Iron manganese trioxide was tested for mutagenicity in a bacterial reverse mutation assay using Salmonella typhimurium, both in the absence and presence of S9 metabolism (Herbold, 2008). Iron manganese trioxide was assayed in tester strains TA98, TA100, TA102, 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 from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: as reported in source record

Justification for type of information:

see attachment "Endpoint-specific read-across justification for the iron oxide category" in IUCLID section 13.2.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

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:

key study

Justification for type of information:

see attachment "Endpoint-specific read-across justification for the iron oxide category" in IUCLID section 13.2.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

see attachment "Endpoint-specific read-across justification for the iron oxide category" in IUCLID section 13.2.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

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

Endpoint:

genetic toxicity in vivo

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

see attachment "Endpoint-specific read-across justification for the iron oxide category" in IUCLID section 13.2.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

duodenum

Toxicity:

no effects

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

stomach

Toxicity:

no effects

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 (2008) tested Iron manganese trioxide 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, TA100 and TA102 when tested up to the limit concentration of 5000 µg/plate. Based on the results of this test, Iron manganese trioxide 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.