Diiron titanium pentaoxide

Genetic toxicity: in vivo

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1997

Deviations:

no

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

Diiron titanium pentaoxide

EC Number:

234-679-1

EC Name:

Diiron titanium pentaoxide

Cas Number:

12023-27-7

Molecular formula:

Fe2O5Ti

IUPAC Name:

titanium(4+) diiron(3+) pentaoxidandiide

Test material form:

solid: particulate/powder

Test animals

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

Administration / exposure

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.

Doses / concentrations

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.

Examinations

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.

Results and discussion

Test results

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

Any other information on results incl. tables

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)

Applicant's summary and conclusion

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.