Diiron titanium pentaoxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Diiron titanium pentaoxide is a mixed oxide of titanium dioxide (TiO2) and iron oxide (Fe2O3) , which are both considered not to be mutagenic. Pigments containing diiron titanium pentaoxide have been tested clearly negative in an Ames test (reference 7.6.1-1). Various in vitro genotoxicity studies have been performed with the read-across substances TiO2 and Fe2O3 showing no mutagenic, clastogenic or cell transforming activity.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

July 6, 2005 - August 19, 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

HIS operon (S. thyphimurium), TRP operon (E. coli)

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : liver S9 mix from Aroclor 1254-pretreated rats with standard co-factors
- method of preparation of S9 mix : Male Wistar rats, aged 6-8 weeks were given a single intraperitoneal injection of Aroclor 1254 (500 mg/kg bw) dissolved in Miglyol 812 oil. About 16 h before sacrifice, the rats remained without food. On day 5 or 7, they were sacrified, the livers were removed and collected in ice-cooled sterilized beakers containing 0.15 M KCl.
- concentration or volume of S9 mix and S9 in the final culture medium: 10% and 30% S9 in the S9 mix were used in the 1st and 2nd series
- quality controls of S9: metabolic capability was tested for each batch

Test concentrations with justification for top dose:

1st series: 5.00, 15.8, 50.0, 158, 500, 1580 and 5000 µg per plate (with and without metabolic activation)
2nd series: 50.0, 88.9, 158, 281 and 500 µg per plate (with and without metabolic activation)

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Daunomycin

Remarks:

without S9 mix, TA98

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 mix, TA1537

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without S9 mix, TA1535, WP2 uvrA, TA100

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with S9 mix, TA102

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cumene hydroperoxide

Remarks:

without S9 mix, TA102

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

with S9 mix, TA 98, TA100, TA1535, TA1537, WP2 uvrA

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments : two

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 2 to 3 days at 37°C

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

Evaluation criteria:

The evaluation of the results was performed according to predefined criteria. The definitions of "no", "clear" and "weak" have been defined depending on the historical controls of the laboratory.
A test material is defined as non-mutagenic in this assay if "no" or "weak increases" occur in the first and second series of the main experiment ("Weak increases" randomly occur due to experimental variation.)

A test material is defined as mutagenic in this assay if:

- a dose-related (over at least two test material concentrations) increase in the number of revertants is induced, the maximal effect is a "clear increase", and the effects are reproduced at similar concentration levels in the same test system;

- "clear increases" occur at least at one test material concentration, higher concentrations show strong precipitation or cytotoxicity, and the effects are reproduced at the same concentration level in the same test system.

In all further cases, a third test series with the bacterial strain in question should be performed.
If the criteria for a positive test result are not fulfilled in at least two out of the three series, the test material is defined as being non-mutagenic in this test system.

Statistics:

no

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:

S. typhimurium TA 102

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

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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: Macroscopically visible precipitation of test material on agar plates occured at concentrations ≥500 µg/plate. Presumably, microscopic precipitation occured at all concentrations tested.
STUDY RESULTS
- Signs of toxicity : No toxicity to the bacteria was observed.
- Individual plate counts : see attached tables
- Genotoxicity results : see attached tables

Conclusions:

With and without addition of S9 mix as the external metabolizing system, the test item was not mutagenic under the experimental conditions described.

Executive summary:

The investigations for the mutagenic potential of the test item were performed using according OECD TG 471 using Salmonella typhimurium tester strains TA 98, TA 100, TA 102, TA 1535 and TA 1537, and Escherichia coil WP2 uvrA. The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254-pretreated rats was used. Two independent experimental series were performed. In the two series with S9 mix, 10 or 30 % S9 in the S9 mix were used in the 1st and 2nd series, respectively. The test item was suspended in DMSO and tested at concentrations ranging from 5.00 to 5000 µg/plate. At all concentration levels, the test material was plated as a suspension. Macroscopically visible precipitation of the test material on the agar plates occurred at concentrations >/= 500 µg/plate. Presumably, microscopic precipitation occured at all concentrations tested. Toxicity to the bacteria was not observed.

Daunomycin, N-ethyl-N'-nitro-N-nitrosoguanidine, 9-aminoacridine, and cumene hydroperoxide served as strain specific positive control test materials in the absence of S9 mix. 2-Aminoanthracene and benzo[a]pyrene were used for testing the bacteria and the activity of the S9 mix. Each treatment with the test materials used as positive controls led to a clear increase in revertant colonies, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used. With and without addition of S9 mix as the external metabolizing system, the test item was not mutagenic under the experimental conditions described.

Reference 2

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

accepted calculation method

Qualifier:

no guideline followed

Principles of method if other than guideline:

Genotoxic effects of Fe2O3 particles were determined by analyzing DNA fragmentation using the alkaline comet assay technique (pH 12.7).

GLP compliance:

not specified

Remarks:

No details on GLP status.

Type of assay:

comet assay

Species / strain / cell type:

mammalian cell line, other: BEAS-2B

Details on mammalian cell type (if applicable):

MEDIA USED
- Type and composition of media: DKSFM supplemented with 0.1% EGF protein and 1% antibiotics
- The cells were cultivated at 36°C and 5% CO2.

Metabolic activation:

without

Test concentrations with justification for top dose:

10, 25, 50, and 250 µg/mL

Vehicle / solvent:

medium

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylnitrosurea

Details on test system and experimental conditions:

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 10E5 cells (BEAS-2B) were plated in Falcon T-25 flasks and preincubated for 24 h
- Test substance added in medium

Postexposure, the cells were washed, trypsinized, suspended in low melting agarose, and cast onto a gel bond film. Following the polymerization of the agarose, the cells were lysed overnight in freshly prepared and precooled cell lysis buffer. Electrophoresis of the lysed
cells was performed at a pH of 12.7 for 10 min (conditions: 300 mA, 1.5 V/cm at 4°C) following which the agarose was treated with neutralization solution for 30 min and dehydrated in absolute CH3COOH for 2 h. These agarose gels were dried in darkness overnight at 4°C and stained with SYBR-Green. Imaging and analysis were performed on a Leica microscope.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Trypan blue assay

Evaluation criteria:

The parameter of Olive tail moment was used for analysis of the DNA damage. The quantitative measurement of all the assays was expressed as mean percentage increase relative to unexposed control ± SD.

Statistics:

The results were statistically analyzed (exposure vs. negative control) using one-way ANOVA followed by Holm-Sidak post hoc test.

Key result

Species / strain:

mammalian cell line, other: BEAS-2B

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>50 µg/mL

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

Cyto- and genotoxicity caused by test item can only be observed at very high concentrations of >50 µg/mL. At occupationally relevant concentrations of < 10 µg/mL no cyto-and genotoxic effects can be observed.

Executive summary:

Genotoxic effects of Fe2O3 particles were determined by analyzing DNA fragmentation using the alkaline comet assay technique (pH 12.7). Significant genotoxic effects were only found at very high particle concentrations (> 50 µg/mL) were high cytotoxicity have been observed. At occupationally relevant concentrations of < 10 µg/mL no cyto-and genotoxic effects can be observed.

Reference 3

Endpoint:

in vitro transformation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

accepted calculation method

Qualifier:

no guideline followed

Principles of method if other than guideline:

Investigations of transformation in the mouse embryo cell line BALB/3T3/A3 1-1-1 using various minerals.

GLP compliance:

no

Type of assay:

in vitro mammalian cell transformation assay

Species / strain / cell type:

mammalian cell line, other: BALB/3T3/A3 1-1-1

Details on mammalian cell type (if applicable):

The cells were cultured at 37°C and 5% C02 in Eagle’s minimal essential medium (MEM), supplemented with 10% fetal bovine serum (FBS, heat inactivated at 56°C for 30 min), 2 mM glutamine, and 50 pg/ml gentamicin (complete medium).

Metabolic activation:

without

Test concentrations with justification for top dose:

0, 12.5, 25, 50, 100 µg/cm2

Vehicle / solvent:

medium

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

not specified

Details on test system and experimental conditions:

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 10E4 cells per 50 mm dish
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 3 days
- Harvest time after the end of treatment: After 3 days exposure, medium was exchanged, which was changed twice weekly until 5 weeks from treatment, when the cultures were fixed and stained

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative colony forming efficiency (CFE)

Evaluation criteria:

Morphologically transformed foci (type III) were scored when they showed densely aggregated or piled up cells, with a criss-cross growth pattern of spindle-shaped and hyperchromatic cells at the periphery of the focus. The number of transformed foci per 10E5 clonal survivors (as determined by the concurrent cytotoxicity assay) was used to estimate the transformation frequencies and their standard error.

Species / strain:

mammalian cell line, other: BALB/3T3/A3 1- 1-1

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

not specified

Conclusions:

Fe2O3 shows no cytotoxicity and no transforming activity in the mouse embryo cell line BALB/3T3/A3 1-1-1.

Executive summary:

The mouse embryo cell line BALB/3T3/A3 1-1-1 was incubated with Fe2O3 for 3 days using exposure concentrations of 0, 12.5, 25, 50, 100 µg/cm2. After 5 weeks from treatment the cultures were fixed and stained. Morphologically transformed foci were scored when they showed densely aggregated or piled up cells, with a criss-cross growth pattern of spindle-shaped and hyperchromatic cells at the periphery of the focus. The number of transformed foci per 10E5 clonal survivors (as determined by the concurrent cytotoxicity assay) was used to estimate the transformation frequencies. Fe2O3 shows no cytotoxicity and no transforming activity in the mouse embryo cell line BALB/3T3/A3 1-1-1.

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Principles of method if other than guideline:

Induction of sister chromatid exchange (SCE) and Chromosome Aberration (CA) in Chinese hamster ovary (CHO) cells in vitro.

GLP compliance:

no

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

0, 15, 20, 25 µg/mL (Chomosome Aberration test)
0, 2.5, 8.3, 25 µg/mL (SCE assay)

Vehicle / solvent:

medium

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9 mix

Details on test system and experimental conditions:

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium

METHOD CHROMOSOMAL ABERRATION ASSAY
- Exposure duration/duration of treatment: without activation, cells were exposed to the test chemical for 8 hr, afterwards, test chemical was washed off, and the cells were treated with 0.1 µg/mL Colcemid for 2-2.5 hr
- Exposure duration/duration of treatment: with metabolic activation, cells were exposed to the test chemical plus the metabolic activation mixture for 2 hr, washed, incubated for 8 hr, and then treated with Colcemid for 2-2.5 hr
- Air-dried slides were coded and stained with Giemsa. One hundred to 200 cells from each of the three highest scorable doses were analyzed. All aberrations were individually classified (e.g., chromatid breaks, chromosome breaks, triradials), Gaps and endoreduplications were recorded

METHOD SCE ASSAY
- Exposure duration/duration of treatment: without metabolic activation, cells were exposed for approximately 25 hr; for the trials with metabolic activation the exposure was for 2 hr
- 10 µM bromodeoxyuridine was added 2 hr after dosing and cells were continuously exposed to BrdUrd up until the time of harvest with 0.1 µg/mL Colcemid present for the last 2-2.5 hr of incubation
- in cultures without activation the cells were washed to remove the test chemical prior to Colcemid addition (25 hr)
- In cultures with metabolic activation cells were washed to remove test chemical and the metabolic activation components 2 hr after the initial exposure
- mitotic cells were treated with 0.075 M KCl and fixed in 3:1 methanol:glacial acetic acid
- In order to evaluate cell cycle kinetics, slides from the highest doses were stained with Hoechst 33258 (0.5 µg/mL) and examined by fluorescence microscopy
- fifty cells were scored per dose in the initial trial, and, generally 25 were scored in the repeat trials. In those instances where the number of SCE/cell analyzed for a particular dose consistently exceeded the mean value of the high-dose positive control, only five to ten cells were scored

Evaluation criteria:

A significant increase in the aberration assay was based on a binomial sampling assumption. An increase of 20% or greater increase in SCE per chromosome over the solvent control was considered significant.

Statistics:

Dunnett’s method

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

Titanium dioxide did not induce SCE or chromosomal aberrations in vitro.

Executive summary:

The in vitro genotoxicity of titanium dioxide was determined using the Sister-Chromatid Exchange (SCE) and Chromosome Aberration (CA) assay in Chinese hamster ovary cells (CHO). In the CA Assay the following concentrations have been tested: 0, 15, 20, and 25 µg/mL. In the SCE Assay 0, 2.5, 8.3 and 25 µg/mL have been used as test concentrations. The tests have been performed with and without exogenous metabolic activation (S9 mix). Titanium dioxide did not induce SCE or chromosomal aberrations; the high dose was limited by solubility. In the first aberration trial with activation, there was a positive response at 20 µg/mL; this response did not repeat in the subsequent trial, and the chemical was, therefore, judged to be negative.

Reference 5

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

Mutagenicity studies on various metal compounds.

GLP compliance:

no

Type of assay:

Bacillus subtilis recombination assay

Species / strain / cell type:

bacteria, other: B. subtilis H17 and M45

Details on mammalian cell type (if applicable):

Medium: B-2 broth (meat extract 10 g, polypeptone powder 10 g and NaCl 5 g in 1L, pH adjusted to 7.0)

Additional strain / cell type characteristics:

other: H17: (Rec+, arg- try-) M45: (Rec-, arg- try-)

Metabolic activation:

without

Test concentrations with justification for top dose:

0.05 mL-portions of metal solutions (0.005-0.5 M) were applied

Vehicle / solvent:

distilled water

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

not specified

Details on test system and experimental conditions:

On the day of the experiments, each stock of B. subtilis was melted and streaked radially from small pipettes onto B-2 agar. A 0.05-mL portion of test item was dropped onto a filter paper disk (diameter 10 mm), and the disk was placed on the starting point of the streak. The plates were kept at 4°C for 24 h and then incubated at 37°C overnight.

Key result

Species / strain:

bacteria, other: B.subtilis

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

other: Strong positive rec effects were noted with compounds of arsenic and chromium.

Conclusions:

TiO2 gave a negative result in the rec-assay.

Executive summary:

A recombination assay (rec-Assay) was carried out with 127 metal compounds including titanium dioxide using Bacillus subtilis to check their DNA-damaging capacity and mutagenicity. 0.05 mL-portions of metal solutions (0.005-0.5 M) were applied.TiO2 gave a negative result in the rec-assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Pigments containing diiron titanium pentaoxide have been tested clearly negative in an in vivo micronucleus assay in rats (reference 7.6.2-1). Furthermore, no mutagenic or clastogenic effects have been reported for both read-across substances TiO2 and Fe2O3 in vivo in the comet assay, the micronucleus test or the chromosome aberration assay.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 September 2000 - 2 October 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Species:

rat

Strain:

Wistar

Remarks:

HsdCpb:WU

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Wistar rats
- Age at study initiation: 8-10 weeks
- Weight at study initiation: 264 - 299 g
- Housing: individually in Makrolon cages type 3
- Diet: ad libitum, Standard Diet from Eberle Nafag AG, Gossau, Switzerland
- Water: ad libitum, tap water
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 - 23.5
- Humidity (%): 57 - 62
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

0.25% aqueous Methocel K4M Premium.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test material group was dosed once with a limit dose of 2000 mg/kg bw.
Dosing volume: 10 mL/kg bw
Rats of the control group received the vehicle only.

Duration of treatment / exposure:

The control and positive control were killed 24 hours after treatment.
The two test material groups were killed 24 and 48 hours after treatment.

Frequency of treatment:

Single oral administration.

Dose / conc.:

2 000 mg/kg bw (total dose)

No. of animals per sex per dose:

5 males /vehicle control group
5 males/ positive control group
10 males/ test material group

Control animals:

yes, concurrent vehicle

Positive control(s):

The animals of the positive control were orally treated with cyclophosphamide at a dose of 16.5 mg/kg bw.

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
The highest dose given in the present study was selected to produce signs of toxicity but no mortality. Since the internationally valid guidelines recommend a maximum dose of 2000 mg/kg bw for this assay, 6 male and 6 female rats were treated orally with 2000 mg test item in preliminary range-finding experiments. No toxic effects were seen. For this reason, the dose of 2000 mg/kg body weight was selected as the highest, limit dose for male rats only in the main study of this investigation. The rats of the negative control group were treated orally with 10 mL of a 0.25 % aqueous MethocelR K4M premium solution. Animals of the positive control group received an oral dose of 16.5 mg cyclophosphamide/kg body weight With this dose a statistically significant increase in the number of polychromatic erythrocytes with micronuclei, as compared to the negative control, was to be expected.

TREATMENT AND SAMPLING TIMES
The control and positive control were killed 24 hours after treatment. The two test material groups were killed 24 and 48 hours after treatment.

DETAILS OF SLIDE PREPARATION
Immediately after the rats had been killed, one femur of each animal was dissected and cleaned from adherent muscles. The epiphyses were cut off and bone marrow cells were flushed out with fetal calf serum using a syringe, and suspended in the serum. This suspension was filtered through cellulose and centrifuged for 5 min at 150 x g. The sediment was resuspended in fetal calf serum and bone marrow smears were prepared from the resulting cell suspension. After 3 hours of drying, the slides were stained using Giemsa's solution with Weise buffer solution and mounted in Entellan.

METHOD OF ANALYSIS
A total of 2000 polychromatic erythrocytes per animal were scored for micronuclei using Zeiss light microscopes with plane optics (magnification: 1250x). Round particles with about 1/20 - 1/5 the diameter of an erythrocyte that stained violet, like nucleic
material, were scored as micronuclei. They were differentiated from granules by thorough examination at different optical levels. Only erythrocytes with a distinct bluish touch were evaluated as polychromatic. For determination of the quotient of normochromatic to polychromatic erythrocytes, both erythrocyte stages were screened for micronuclei and counted separately up to a total of 1000 erythrocytes per animal. Then counting was limited to polychromatic erythrocytes.

Evaluation criteria:

A total of 2000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei.
A positive effect in this test system is defined by the occurence of mean micronucleated PCEwhich are statistically significantly higher than those of the actual negative control, taking into account the historical negative controls of the laboratory.

Statistics:

Exact Mann-Whitney-test.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF DEFINITIVE STUDY
No clinical findings were onserved in treated animals. No relevant treatment-related decrease in body weight was observed.
No statistically significant or biologically relevant increase in the number of polychromatic erythrocytes with micronuclei (MN-PCE) was observed. For the number of normochromatic cells with micronuclei, calculated per 1000 normochromatic erythrocytes by means of the above mentioned quotient, no increase was observed.
Quotient normochromatic : polychromatic erythrocytes:
No relevant treatment-related increase was observed for the test item

Table 1: Results

Test material	Dose [mg/kg]	Preparation time [h]	MN-PCE [‰]^a Males
Solvent	—	24	0.40
Test item	2000	24	0.40
Test item	2000	48	0.80
Cyclophosphamide	16.5	24	16.5*

a: Mean numbers of polychromatic erythrocytes with micronuclei per 1000 PCE
*: Statistically significant (p<0.01)

Conclusions:

The test material was not mutagenic in the micronucleus assay in male rats under conditions where the positive control exerted potent mutagenic effects.

Executive summary:

The test item was tested in an in vivo micronucleus assay in the rat. The test item was administered once orally by gavage to 10 male rats at the limit dose of 2000 mg/kg bw. A control group (5 animals) received the vehicle (0.25% aqueous Methocel®K4M premium) only. Cyclophosphamide was used as the positive substance (5 animals). Bone marrow smears were prepared from one femur of each animal and stained with Giemsa’s solution. For the test material group, preparation took place at two different times, i.e. 24 and 48 hours after administration of the test material. For the positive and negative control groups, the preparation time was 24 hours after start of the treatment. The number of polychromatic erythrocytes with micronuclei per 2000 polychromatic erythrocytes per animal was determined. The positive control showed the expected significant increase in the number of polychromatic erythrocytes with micronuclei. No statistically significant or biologically relevant increase in the number of polychromatic erythrocytes with micronuclei was observed in any of the pigment-treated groups. In conclusion, the test pigment item was not mutagenic in the micronucleus test in male rats under conditions of this study.

Reference 2

Endpoint:

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

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

The genotoxicity was evaluated at 6, 24, 48 and 72 h by the comet assay in leucocytes, 48 and 72 h by micronucleus test (MNT) in peripheral blood cells, 18 and 24 h by chromosomal aberration (CA) assay and 24 and 48 h by MNT in bonemarrow cells.

GLP compliance:

not specified

Type of assay:

mammalian comet assay

Species:

mouse

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: from National Institute of Nutrition,Hyderabad, India
- Age at study initiation: 6–8 weeks
- Weight at study initiation: 80–120 g
- Housing: animals were housed in groups of five in standard polypropylene cages with stainless steel top grill
- Diet: ad libitum, commercial pellet diet
- Water: ad libitum
- Acclimation period: one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3
- Humidity (%): 55-65
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

Milli Q water

Duration of treatment / exposure:

single treatment

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide (40 mg/kg bw), given intraperitonially (i.p.) and the volume injected was 0.01 mL/g bw

Tissues and cell types examined:

peripheral blood

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Doses based on acute oral toxicity test.

TREATMENT AND SAMPLING TIMES: The peripheral blood was collected at 6, 24, 48 and 72 h after the dosing.

DETAILS OF SLIDE PREPARATION: Three slides were prepared for each experimental point. Cell viability was determined by trypan blue exclusion assay. The microscope slides were coated with 0.75% normal-melting-point agarose (NMPA) in PBS, 20 μL of heparinised peripheral blood wasmixed with 110 μL of 0.5% low-melting-point agarose (LMPA) in phosphate buffered saline (PBS) and applied to slides. The slides were coveredwith a cover slip and refrigerated for 5 min to solidified the gel. The cover slips were removed and slides were immersed for at least 1 h in ice-cold alkaline lysing solution
[2.5 M NaCl, 10 mM Tris, 100 mM ethylenediaminetetraacetic acid (EDTA), 10% dimethyl sulphoxide, 1% Triton X-100] at final pH 10.0.

METHOD OF ANALYSIS: electrophoresis at 25 V : 300 mA (1.25 V/cm) for 25 min
After electrophoresis, slides were neutralized with neutralizing buffer (Tris 0.4 M, pH 7.5) and then stained with ethidium bromide (20 μg/mL). One hundred cells per rat (50 cells analysed in each slide) were scored at 400× using a fluorescence microscope (Olympus-Japan) with a blue (488 nm) excitation filter and yellow (515 nm) emission (barrier) filter.

Statistics:

The statistical significant change in genotoxicity assays between treated and control groups were analyzed by one-way ANOVA. Multiple comparisons were performed by Tukey's test.

Key result

Sex:

female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF DEFINITIVE STUDY
No statistically significant damage was observed at 6, 24, 48, 72 h sampling time in comparison to control (p>0.05). An intraperitoneal injection of CP (40 mg/kg) induced DNA damage in rat peripheral blood leukocytes. The mean % tail DNA was significantly (p<0.001) higher compared to control.

Table 1

Mean % tail DNA in peripheral blood leucocytes    

	mg/kg bw/day	6 h	24 h	48 h	72 h
Control		3.82 ±0.77	5.44 ±0.81	4.17 ±0.80	4.36±0.92
Fe203	500	3.77 ±0.47	4.81 ±59	4.08 ±0.67	3.46 ±0.47
	1000	3.99 ±0.81	5.28 ±0.70	4.30 ±0.75	4.15 ±0.85
	2000	4.01 ±0.69	5.61 ±0.86	4.59 ±0.88	4.29 ±0.77
CP (b)	40	43.94 ±6.50 (c)	54.56 ± 6.3 (c)	25.39±4.65 (c)	16.10±2.21 (c)

a Cyclophosphamide (positive control), Data represented as mean±S.E., of 3 replicated experiments, 150 cells per

animal, n —5 animals, significantly different from control at Significantly different from control at c=p<0.001.

Conclusions:

Fe2O3 was not genotoxic at the doses tested in an in vivo Comet Assay (peripheral blood).

Executive summary:

An in vivo Comet Assay in leucocytes was conducted to assess the genotoxicity of Fe2O3 in female Wistar rats. The rats were treated orally with the single doses of 500, 1000, 2000 mg/kg bw and the genotoxicity was evaluated at 6, 24, 48 and 72 h by the comet assay in leucocytes.

No statistically significant damage was observed at 6, 24, 48, 72 h sampling time in comparison to control (p>0.05). An intraperitoneal injection of the positive control Cyclophosphamide (40 mg/kg) induced DNA damage in rat peripheral blood leukocytes. 

Reference 3

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Principles of method if other than guideline:

The genotoxicity was evaluated at 18 and 24 h by chromosomal aberration (CA) assay.

GLP compliance:

not specified

Type of assay:

mammalian bone marrow chromosome aberration test

Species:

mouse

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: from National Institute of Nutrition,Hyderabad, India
- Age at study initiation: 6–8 weeks
- Weight at study initiation: 80–120 g
- Housing: animals were housed in groups of five in standard polypropylene cages with stainless steel top grill
- Diet: ad libitum, commercial pellet diet
- Water: ad libitum
- Acclimation period: one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3
- Humidity (%): 55-65
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

Milli Q water

Duration of treatment / exposure:

single treatment

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide (40 mg/kg bw), given intraperitonially (i.p.) and the volume injected was 0.01 mL/g bw

Tissues and cell types examined:

rat's bone marrow cells (femur and tibia)

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Doses based on acute oral toxicity test.

TREATMENT AND SAMPLING TIMES: The analysis was carried out at two sampling of 18 and 24 h.

DETAILS OF SLIDE PREPARATION: The bone marrow was collected and centrifuged. Cells were then fixed through several changes of ice-cold methanol/glacial acetic acid (3:1, v/v) until the pellets were clean. After refrigeration for at least 24 h, cells were centrifuged and resuspended in fresh fixative, dropped onto slides, dried and stained with Geimsa. Three slides for each animal were made by the flame-dried technique.

METHOD OF ANALYSIS: on the basis of criteria established by the OECD Guideline 475
Five hundred well spread metaphases were selected to detect the presence of CAs; while the mitotic index (MI) was determined with 1000 or more cells at both the sampling times.

Statistics:

The statistical significant change in genotoxicity assays between treated and control groups were analyzed by one-way ANOVA. Multiple comparisons were performed by Tukey's test.

Key result

Sex:

female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF DEFINITIVE STUDY
Fe2O3 did not induce a dose dependent effect on the structural (gaps, breaks, minute, acentric fragment and reciprocal translocation) and numerical (aneuploidy and polyploidy) CAs as well as percentage (%) of aberrant cells at all the doses and treatment times. The total cytogenetic changes (numerical+structural CAs), total aberrations (structural aberrations) including and excluding gaps frequencies in Fe2O3 were well within normal control range, and were not significantly different (p>0.05) from concurrent controls.

Conclusions:

Fe2O3 was not genotoxic at the doses tested in an in vivo Chromosomal Aberration Assay.

Executive summary:

An in vivo Chromosom Aberration Assay was conducted to assess the genotoxicity of Fe2O3 in female Wistar rats. The rats were treated orally with the single doses of 500, 1000, 2000 mg/kg bw and the genotoxicity was evaluated at 18 and 24 hours by chromosomal aberration assay. No statistically significant damage was observed comparison to control (p>0.05). An intraperitoneal injection of the positive control Cyclophosphamide (40 mg/kg) induced DNA damage. 

Reference 4

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Principles of method if other than guideline:

The genotoxicity was evaluated at 48 and 72 h by micronucleus test (MNT) in peripheral blood cells and at 24 and 48 h by MNT in bonemarrow cells.

GLP compliance:

not specified

Type of assay:

mammalian bone marrow chromosome aberration test

Species:

mouse

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: from National Institute of Nutrition,Hyderabad, India
- Age at study initiation: 6–8 weeks
- Weight at study initiation: 80–120 g
- Housing: animals were housed in groups of five in standard polypropylene cages with stainless steel top grill
- Diet: ad libitum, commercial pellet diet
- Water: ad libitum
- Acclimation period: one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3
- Humidity (%): 55-65
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

Milli Q water

Duration of treatment / exposure:

single treatment

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide (40 mg/kg bw), given intraperitonially (i.p.) and the volume injected was 0.01 mL/g bw

Tissues and cell types examined:

bonemarrow cells extracted from thigh bone

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Doses based on acute oral toxicity test.

TREATMENT AND SAMPLING TIMES: The study was done at 24 and 48 h of after treatment according to OECD Guideline 474

DETAILS OF SLIDE PREPARATION: The femurs were removed, the bone marrow was collected, centrifuged, spread on the slides and allowed to dry in humidified air overnight. The slides were fixed with methanol and stained with Giemsa (Sigma Chemical Co., St. Louis, MO) solution in phosphate buffer saline for the assessment of the micronuclei (MN) occurrence.

METHOD OF ANALYSIS: Three slidesweremade for each animal; the slideswere microscopically analyzed at 1000×magnification. Per animal, 2000 PCEs were randomly selected from three slides and scored for the presence of MN.

Statistics:

The statistical significant change in genotoxicity assays between treated and control groups were analyzed by one-way ANOVA. Multiple comparisons were performed by Tukey's test.

Key result

Sex:

female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF DEFINITIVE STUDY
The MN-PCEs frequencies in the Fe2O3 treated groups were very similar to those in controls and were not significantly different (p>0.05). On the other hand, CP (40 mg/kg bw) treated positive control groups induced a substantially significant (p<0.001) effect on MN-PCEs frequency. In the MNT with varied doses of test item exhibited no statistically difference in % PCEs with the negative control at 24 and 48 h after treatment, demonstrating the absence of bone marrow cytotoxicity.

The results from blood MNT: The % of PCEs in Fe2O3-treated groups of animals were not reduced at various doses (500, 1000, 2000 mg/kg bw). The data reveals that the compounds did not cause cytotoxicity. The MN-PCEs calculated after treatment with all the three doses did not show statistically significant differences (p>0.05) as compared to control at both the sampling times suggesting lack of clastogenicity. CP treated group showed significant difference (p<0.001) with respect to the control group.

Conclusions:

Fe2O3 was not genotoxic at the doses tested in an in vivo Micronucleus Test (peripheral blood and bonemarrow).

Executive summary:

An in vivo Micronucleus assay was conducted to assess the genotoxicity of Fe2O3 in female Wistar rats. The rats were treated orally with the single doses of 500, 1000, 2000 mg/kg bw and the genotoxicity was evaluated at 48 and 72 h by micronucleus test (MNT) in peripheral blood cells and 24 and 48 h by MNT in bonemarrow cells. No statistically significant damage was observed in comparison to control (p>0.05). An intraperitoneal injection of the positive control Cyclophosphamide (40 mg/kg) induced DNA damage.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

A WoE Approach was used to investigate the genotoxicity of diiron titanium pentaoxide using data from pigments which contain diiron titanium pentaoxide (up to 37%) and data from the oxides titanium dioxide (TiO2) and iron oxide (Fe2O3). There are no data available for diiron titanium pentaoxide.

In vitro

The investigations for the mutagenic potential of the pigment (8-37% diiron titanium pentaoxide) were performed according OECD TG 471 using Salmonella typhimurium tester strains TA 98, TA 100, TA 102, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA. The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254-pretreated rats was used. Two independent experimental series were performed. In the two series with S9 mix, 10 or 30 % S9 in the S9 mix were used in the 1st and 2nd series, respectively. The test item was suspended in DMSO and tested at concentrations ranging from 5.00 to 5000 µg/plate. At all concentration levels, the test material was plated as a suspension. Macroscopically visible precipitation of the test material on the agar plates occurred at concentrations >/= 500 µg/plate. Presumably, microscopic precipitation occured at all concentrations tested. Toxicity to the bacteria was not observed. Daunomycin, N-ethyl-N'-nitro-N-nitrosoguanidine, 9-aminoacridine, and cumene hydroperoxide served as strain specific positive control test materials in the absence of S9 mix. 2-Aminoanthracene and benzo[a]pyrene were used for testing the bacteria and the activity of the S9 mix. Each treatment with the test materials used as positive controls led to a clear increase in revertant colonies, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used. With and without addition of S9 mix as the external metabolizing system, the test item was not mutagenic under the experimental conditions described.

Furthermore, the in vitro genotoxicity of the read-across substance titanium dioxide was determined using the Sister-Chromatid Exchange (SCE) and Chromosome Aberration (CA) assay in Chinese hamster ovary cells (CHO). In the CA Assay the following concentrations have been tested: 0, 15, 20, and 25 µg/mL. In the SCE Assay 0, 2.5, 8.3 and 25 µg/mL have been used as test concentrations. The tests have been performed with and without exogenous metabolic activation (S9 mix). Titanium dioxide did not induce SCE or chromosomal aberrations; the high dose was limited by solubility. In the first aberration trial with activation, there was a positive response at 20 µg/mL; this response did not repeat in the subsequent trial, and the chemical was, therefore, judged to be negative.

A recombination assay (rec-Assay) was carried out with 127 metal compounds including titanium dioxide using Bacillus subtilis to check their DNA-damaging capacity and mutagenicity. 0.05 mL-portions of metal solutions (0.005-0.5 M) were applied.TiO2 gave a negative result in the rec-assay.

Genotoxic effects of Fe2O3 particles were determined by analyzing DNA fragmentation using the alkaline comet assay technique (pH 12.7). Significant genotoxic effects were only found at very high particle concentrations (> 50 µg/mL) were high cytotoxicity has been observed. At occupationally relevant concentrations of < 10 µg/mL no cyto-and genotoxic effects was be observed.

The mouse embryo cell line BALB/3T3/A3 1-1-1 was incubated with Fe2O3 for 3 days using exposure concentrations of 0, 12.5, 25, 50, 100 µg/cm2. After 5 weeks from treatment the cultures were fixed and stained. Morphologically transformed foci were scored when they showed densely aggregated or piled up cells, with a criss-cross growth pattern of spindle-shaped and hyperchromatic cells at the periphery of the focus. The number of transformed foci per 10E5 clonal survivors (as determined by the concurrent cytotoxicity assay) was used to estimate the transformation frequencies. Fe2O3 shows no cytotoxicity and no transforming activity in the mouse embryo cell line BALB/3T3/A3 1-1-1. In the same study TiO2 was tested and showed no transforming activity.

In vivo

The pigment containing 8 -37% diiron titanium pentaoxide was tested in an in vivo micronucleus assay according to OECD TG 474 in the rat. The test item was administered once orally by gavage to 10 male rats at the limit dose of 2000 mg/kg bw. A control group (5 animals) received the vehicle (0.25% aqueous Methocel®K4M premium) only. Cyclophosphamide was used as the positive substance (5 animals). Bone marrow smears were prepared from one femur of each animal and stained with Giemsa’s solution. For the test material group, preparation took place at two different times, i.e. 24 and 48 hours after administration of the test material. For the positive and negative control groups, the preparation time was 24 hours after start of the treatment. The number of polychromatic erythrocytes with micronuclei per 2000 polychromatic erythrocytes per animal was determined. The positive control showed the expected significant increase in the number of polychromatic erythrocytes with micronuclei. No statistically significant or biologically relevant increase in the number of polychromatic erythrocytes with micronuclei was observed in any of the pigment-treated groups. In conclusion, the test pigment item was not mutagenic in the micronucleus test in male rats under conditions of this study.

An in vivo Comet Assay in leucocytes was conducted to assess the genotoxicity of the read-across substance Fe2O3 in female Wistar rats. The rats were treated orally with the single doses of 500, 1000, 2000 mg/kg bw and the genotoxicity was evaluated at 6, 24, 48 and 72 h by the comet assay in leucocytes. No statistically significant damage was observed at 6, 24, 48, 72 h sampling time in comparison to control (p>0.05). An intraperitoneal injection of the positive control Cyclophosphamide (40 mg/kg) induced DNA damage in rat peripheral blood leukocytes. In the same study, an in vivo Chromosome Aberration Assay was conducted to assess the genotoxicity of Fe2O3 in female Wistar rats. The rats were treated orally with the single doses of 500, 1000, 2000 mg/kg bw and the genotoxicity was evaluated at 18 and 24 hours by chromosomal aberration assay. No statistically significant damage was observed comparison to control (p>0.05). An intraperitoneal injection of the positive control Cyclophosphamide (40 mg/kg) induced DNA damage. 

An in vivo Micronucleus assay was conducted to assess the genotoxicity of Fe2O3 in female Wistar rats. The rats were treated orally with the single doses of 500, 1000, 2000 mg/kg bw and the genotoxicity was evaluated at 48 and 72 h by micronucleus test (MNT) in peripheral blood cells and 24 and 48 h by MNT in bonemarrow cells. No statistically significant damage was observed in comparison to control (p>0.05). An intraperitoneal injection of the positive control Cyclophosphamide (40 mg/kg) induced DNA damage.

Furthermore, male Sprague-Dawley rats were treated with Fe2O3 by intratracheal instillation of a suspension of iron oxide. The control group was treated with the vehicle (saline) only. 24h after treatment rats were sacrificed and cells were isolated (alveolar macrophages, lung cells, peripheral lymphocytes, hepatocytes. Alkaline single cell electrophoresis (comet assay) was performed. No statistically significant damage was observed in alveolar macrophages, lung cells, peripheral lymphocytes and hepatocytes from endotracheally treated rats sacrificed 24 h after treatment with test item compared with the control groups.

Therefore, based on the composition of the substance and the results obtained with TiO2, Fe2O3 and the pigment, diiron titanium pentaoxide is evaluated as not mutagenic.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The available test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Thus, the test item is considered not to be classified for genotoxicity under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EU) No 2019/521.