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Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information
Several in vitro genotoxicity tests are available. Out of 10 references two indicator tests (SCE) and two micronucleus assays show ambiguous or positive results. In addition three in vivo tests with negative results are included. All tests have been conducted with TiO2.
Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
no data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Meets generally accepted scientific standards, well documented and acceptable for assessment
Reason / purpose for cross-reference:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
In this publication the induction of chromosomal aberration and micronuclei in bone marrow cells of mice by 65 chemicals, among them TiO2, was elucidated.
GLP compliance:
no
Type of assay:
micronucleus assay
Species:
mouse
Strain:
B6C3F1
Sex:
male
Details on test animals or test system and environmental conditions:
no data
Route of administration:
intraperitoneal
Vehicle:
corn oil
Details on exposure:
The three exposure mouse bone marrow micronucleus test protocol is presented by Shelby et al. 1993.

TiO2 was tested for induction of micronuclei in mouse bone marrow cells using two different sacrifice times.

Groups of 5 male B6C3F1 mice were injected intraperitoneally three times at 24 h intervals with the test substance dissolved in corn oil. The total dosing volume per mouse was 0.4 ml.

Duration of treatment / exposure:
24 h
Frequency of treatment:
Three times at 24 h intervals
Post exposure period:
24 h
Remarks:
Doses / Concentrations:
0 mg/kg
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
250 mg/kg
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
500 mg/kg
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
1000 mg/kg
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
1500 mg/kg
Basis:
nominal conc.
No. of animals per sex per dose:
8 animals per dose group
Control animals:
yes, concurrent vehicle
Positive control(s):
It is reported that concurrent positive control groups were run for each test but the data are not presented.
Tissues and cell types examined:
24 h after the final injection smears of the bone marrow cells from femurs were prepared.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: not specified; based on a preliminary dose range study and another publication by Shelby et al. 1993.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): 24 h after the final injection smears of the bone marrow cells from femurs were prepared.

DETAILS OF SLIDE PREPARATION: Air-dried smears were fixed and stained with acridine orange;

METHOD OF ANALYSIS: 2000 polychromatic erythrocytes PCE were scored per animal for frequency of micronucleated cells. The percentage of the PCEs among the total erythrocyte population in the bone marrow was scored for each dose group as a measure of toxicity. The results were tabulated as the mean of the pooled results from all animals within a treatment group, plus or minus the standard error mean.
Statistics:
Data were analysed using the Micronucleus Assay Data Management and Statistical software package (version 1.4) that employed a one-tailed Cochran-Armitage trend test across exposure groups and pairwise comparison between exposure group and concurrent control. The level of significance was set at an alpha level of 0.05.
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
other: not reported
Additional information on results:
RESULTS OF RANGE-FINDING STUDY:
a preliminary dose-determination experiment was carried out. Details on the preliminary test are not reported

RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): the test substance did not induice structural aberrations
- Induction of micronuclei (for Micronucleus assay): yes; see Table 1
- Ratio of PCE/NCE (for Micronucleus assay): not reported
- Appropriateness of dose levels and route: no data
- Statistical evaluation: Data were analysed using the Micronucleus Assay Data Management and Statistical software package (version 1.4) that employed a one-tailed Cochran-Armitage trend test across exposure groups and pairwise comparison between exposure group and concurrent control. The level of significance was set at an alpha level of 0.05.

The MN data on TiO2 described here were originally published by Shelby et al. (1993).
The initial MN experiment on TiO2 (0, 250, 500, 1000 mg/kg bw) gave a significant trend with the effect at the highest dose (1000 mg/kg bw) significantly elevated; however, the effects observed were small.

In a second trial on TiO2 (0, 500, 1000, 1500 mg/kg bw) , a single dose group (1000 mg/kg bw) was significantly elevated. However this result was only seen in a single dose, was not concentration dependent and only of minor significance. Therefore this effect is judged as irrelevant biological fluctuation.

Table1: TiO2 Micronucleus Test (solvent corn oil; bone marrow cells)

Dose (mg/kg bw)

MN-PCE/1,000

Trend P value

Survival (No. scored)

1sttrial

0

1.7±0.26

0.016*

(5/5)

250

3.00± 0.47

(5/5)

500

2.60± 0.49

(5/5)

1000

3.50± 0.45*

(5/5)

2ndtrial

0

1.50± 0.27

0.128 (0.002)c,*

(5/5)

500

2.60± 0.37

(5/5)

1000

3.60± 0.78*

(5/5)

1500

2.00± 0.35

(5/5)

* Significant positive effect

c Trend test P value after dropping high dose group 



Furthermore the chromosome aberration test also reported in the same reference, no clastogenic effects could be observed at all doses (0, 625, 1250, 2500 mg/kg bw). Thus it can be concluded that TiO2 has no clastogenic effect in vivo.
Conclusions:
Interpretation of results (migrated information): negative
Under the reported experimental conditions TiO2 did not induce significant elevated levels of micronuclei in the bone marrow cells of the mouse. Therefore TiO2, is considered to be non-mutagenic in this micronucleus assay.
Executive summary:

In a B6C3F1 mouse bone marrow micronucleus test, 5 male mice per dose were treated intraperitoneally with Titanium dioxide at doses of 0, 250, 500, 1000 and 1500 mg/kg bw. The animals were injected intraperitoneally three times at 24 h intervals. The vehicle was corn oil.

The initial micronucleus test gave a significant trend with the effect at the highest dose significantly elevated; the effects observed were small. In a second trial, effects of a similar magnitude were observed. However this result was only seen in a single dose, was not concentration dependent and only of minor significance. Therefore this effect is judged as irrelevant biological fluctuation.

Therefore, Titanium Dioxide is not considered to be mutagenic according to the results of the in vivo micronucleus test reported here.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

In vitro genetic toxicity tests:

Titanium dioxide did not induce effects in most of the reported in vitro genotoxicity assays (bacterial mutagenicity (Zeiger, 1988, Kanematsu, 1984; mouse lymphoma assay (Myhr, 1991), micronucleus assay (Miller, 1995); Sister Chromatide Exchange & Chromosome aberration assay (Invett, 1989)). Contradicting results have been reported in CHO cells by Lu (1998) (positive Sister Chromatide & micronucleus assay). Positive results have also been reported by Türkez (2007) in human erythrocytes (Sister Chromatide Exchange & micronucleus assay). However, in the latter report the activities of antioxidant enzymes in erythrocytes showed significant decreases with increasing doses of TiO2. Thus, it seems to be a secondary genotoxic effect caused by an increase in reactive oxygen species.

In vivo genetic toxicity tests:

Titanium dioxide does not induce micronuclei or chromosome aberration in the bone marrow of male B6C3F1 mice following a single intraperitoneal injection of 1500 and 2500 mg titanium dioxide /kg bw, respectively (Shelby, 1995). In addition TiO2 is negative in a Drosophila sex-linked recessive lethal (SLRL) assay (Foureman, 1994).

From the available data it can be concluded, that titanium dioxide is not genotoxic. These data are used for read-across to Titanium carbide.


Justification for selection of genetic toxicity endpoint
valid in vivo micro nucleus test

Justification for classification or non-classification

The majority of in vitro genotoxicity studies, among them a bacterial reverse mutation assays, mammalian cell gene mutation tests (TK assay) and mammalian cell chromosome aberration are negative, supporting the negative findings in three in vivo tests as cited above. Therefore, Titanium dioxide is not considered to be mutagenic. These data are used for read-across to Titanium carbide.