Reaction mass of disodium hexatitanium tridecaoxide and disodium trititanium heptaoxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genotoxicity tests demonstrated that uf-TiO 2 was negative in both the bacterial reverse mutation test and in an in vitro mammalian chromosome aberration test with Chinese hamster ovary cells.

Sodium titanates are effectively the sodium salts of the unstable titanic acid (titanium hydroxide).  Titanium hydroxide is hard to isolate without rapid hydrolysis to titanium dioxide and sodium chloride. It is therefore proposed to base environmental and health assessment on these two hydrolysis products. There has been extensive research on similar substances in the ‘titanate’ grouping and these all exhibit similar behaviour in that under acid biological conditions (eg if ingested) or if dispersed in water, there is dissociation of the ions and subsequent hydrolysis / oxidation.

For the purpose of genetic toxicity, the focus has been placed on the titanium element of the hydrolysis expected from this substance.

Sodium ions are naturally present in cells and the structure of sodium chloride does not indicate a genotoxic potential. Therefore, there is no reason to evaluate the potential genotoxicity of the sodium element of the hydrolysis further. Based on the information available for the potential hydrolysis components, no genotoxic effects are expected.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-04-14 to 2010-05-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP guideline study reliable without restrictions

Justification for type of information:

Disodium titanate substance (EC 234-802-9) has the molecular formula Na2TiO3 and its composition is expressed as (Na2O)x(TiO2), where x is ranging from 0.1 to 6 according to the SIP. This substance, Reaction mass of Disodium Hexatitanate and Sodium Metatitanate, has a value of x = 0.21, calculated from XRF results, has been identified as a mixture of two specific types of disodium titanate and is therefore within the scope of the disodium titanate SIP.
It is assessed therefore that disodium titanate is an acceptable read-across substance for Reaction mass of Disodium Hexatitanate and Sodium Metatitanate

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

, 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

, 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2009-03-30

Type of assay:

bacterial reverse mutation assay

Target gene:

the S. typhimurium histidine (his) system

- TA 1537: his C 3076; rfa-; uvrB-
- TA 98: his D 3052, rfa-, uvrB-; R-factor
- TA 1535: his G46; rfa-; uvrB-
- TA 102: his G 428; rfa-, uvrB+; R-factor
- TA 100: his G 46; rfa-, uvrB-, R-factor

The bacterial strains TA 1535, TA 1537, TA 98, TA 100, TA 102 were obtained from Trinova Biochem GmbH (35394 Gießen, Germany).

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 preparation with 34.3 mg/mL (Lot no. R 220110) in the pre-experiment/experiment I and experiment II.

Test concentrations with justification for top dose:

- experiment I: 3, 10, 33, 100, 333, 1000, 2500, and 5000 µg/plate
- experiment II: 3, 10, 33, 100, 333, 1000, 2500, and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: on the day of the experiment, the test item was suspended in deionised water
- Justification for choice of solvent: the solvent was chosen because of its solubility properties

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: without metabolic activation: sodium azide (TA 1535, TA100); 4-nitro-o-phenylene-diamine (TA1537, TA98); methyl methane sulfonate (TA102). With metabolic activation: 2-aminoanthracene (TA1535, TA1537, TA98, TA100, TA102)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Experiment I was performed as a plate incorporation assay. Since a negative result was obtained in this experiment, experiment II was performed as a pre-incubation assay. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate.

Precultures:
From the thawed ampoules of the strains 0.5 mL bacterial suspension was transferred into 250 mL Erlenmeyer flasks containing 20 mL nutrient medium. A solution of 20 μL ampicillin (25 μg/mL) was added to the strains TA 98, TA 100, and TA 102. This nutrient medium contains per litre 8 g Nutrient Broth (MERCK, D-64293 Darmstadt) and 5 g NaCl (MERCK, D-64293 Darmstadt). The bacterial cultures were incubated in a shaking water bath for 4 hours at 37° C. The optical density of the bacteria was determined by absorption measurement and the obtained values indicated that the bacteria were harvested at the late exponential or early stationary phase (10E8-10E9 cells/mL).

Agar:
The plates with the minimal agar were obtained from E. Merck, D-64293 Darmstadt. The overlay agar contains per litre 6.0 g Agar Agar, 6.0 g NaCl, 10.5 mg L-Histidine x HCl x H2O and 12.2 mg Biotin (MERCK, D-64293 Darmstadt). Sterilisations were performed at 121 °C in an autoclave.

MAMMALIAN MICROSOMAL FRACTION S9 MIX
The bacteria used in this assay do not possess the enzyme systems which, in mammals, are known to convert promutagens into active DNA damaging metabolites. In order to overcome this major drawback an exogenous metabolic system is added in form of mammalian microsome enzyme activation mixture.

S9 (Preparation by Harlan CCR):
Phenobarbital/β-Naphthoflavone induced rat liver S9 is used as the metabolic activation system. The S9 is prepared from 8 - 12 weeks old male Wistar rats (Hsd Cpb: WU, Harlan Laboratories GmbH, 33178 Borchen, Germany), weight approx. 220 - 320 g induced by applications of 80 mg/kg b.w. Phenobarbital i.p. (Desitin; D-22335 Hamburg) and β-Naphthoflavone p.o. (Aldrich, D-89555 Steinheim) each on three consecutive days. The livers are prepared 24 hours after the last treatment. The S9 fractions are produced by dilution of the liver homogenate with a KCl solution (1+3) followed by centrifugation at 9000 g. Aliquots of the supernatant are frozen and stored in ampoules at -80 °C. Small numbers of the ampoules can be kept at -20 °C for up to one week. Each batch of S9 mix is routinely tested with 2-aminoanthracene as well as benzo(a)pyrene. The protein concentration in the S9 preparation was 34.3 mg/mL (lot no. R 220110) in both experiments.

S9 Mix:
Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 co-factor solution. The amount of S9 supernatant was 10% v/v in the S9 mix. Cofactors are added to the S9 mix to reach the following concentrations in the S9 mix:
- 8 mM MgCl2
- 33 mM KCl
- 5 mM Glucose-6-phosphate
- 4 mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.

During the experiment the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al. (1977).

EXPERIMENTAL PERFORMANCE:
For each strain and dose level, including the controls three plates were used. The following materials were mixed in a test tube and poured onto the selective agar plates:
- 100 μL Test solution at each dose level (solvent or reference mutagen solution (positive control)),
- 500 μL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
- 100 μL Bacteria suspension (cf. test system, pre-culture of the strains),
- 2000 μL Overlay agar

In the pre-incubation assay 100 μL test solution (solvent or reference mutagen solution (positive control)), 500 μL S9 mix / S9 mix substitution buffer and 100 μL bacterial suspension were mixed in a test tube and incubated at 37 °C for 60 minutes. After pre-incubation 2.0 mL overlay agar (45 °C) was added to each tube. The mixture was poured on minimal agar plates. After solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark.

DATA RECORDING:
The colonies were counted using the Petri Viewer Mk2 (Perceptive Instruments Ltd, Suffolk CB9 7BN, UK) with the software program Ames Study Manager. The counter was connected to a PC running under Windows XP. The individual and mean values of the plates for each concentration together with standard deviations and enhancement factors as compared to the spontaneous reversion rates (see tables of results). Due to precipitation the plates were partly counted manually.

Evaluation criteria:

The Salmonella typhimurium reverse mutation assay is considered acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of historical data
- the positive control substances should produce a significant increase in mutant colony frequencies

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA98, TA100, and TA102) or thrice (strains TA1535 and TA1537) the colony count of the corresponding solvent control is observed.

A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.

An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.

A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: the test item precipitated in the overlay agar in the test tubes and on the incubated agar plates from 1000 up to 5000 μg/plate in both experiments. The undissolved particles of the test item had no influence on the data recording.

RANGE-FINDING/SCREENING STUDIES:
To evaluate the toxicity of the test item a pre-experiment was performed with all strains used. Eight concentrations were tested for toxicity and mutation induction with each 3 plates. The experimental conditions in this pre-experiment were the same as described for the experiment I (plate incorporation test). Toxicity of the test item can be evident as a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn. The pre-experiment is reported as main experiment I, since the criteria evaluable plates (>0 colonies) at five concentrations or more in all strains used are met.

Remarks on result:

other: Test system: all strains/cell types tested

DISCUSSION OF RESULTS

- The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.

- Toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5), were observed at the following concentrations (μg/plate):

Strain	Experiment I		Experiment II
	without S9 mix	with S9 mix	without S9 mix	with S9 mix
TA 1535	/	2500 - 5000	2500	5000
TA 1537	5000	2500 - 5000	/	5000
TA 98	/	5000	/	5000
TA 100	/	5000	/	5000
TA 102	5000	2500 - 5000	5000	2500 - 5000

/ = no toxic effects evident as a reduction in the number of revertants (below the induction factor of 0.5)

- No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Disodium titanate at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

 

Summary of results of experiment I:

Metabolic activation	Test group	Dose level [µg/plate]	Revertant colony counts (mean ± SD)
			TA 1535	TA 1537	TA 98	TA 100	TA 102
Without activation	Deionised water	-	16±5	11±1	28±6	139±12	382±24
	Untreated	-	14±1	13±2	34±6	146±8	369±34
	Disodium titanate	3µg	13±3	11±3	27±7	131±3	363±26
		10 µg	15±3	13±2	24±2	148±11	398±9
		33 µg	15±1	10±2	30±3	140±5	413±14
		100 µg	15±2	12±2	26±5	124±12	403±22
		333 µg	16±4	10±2	25±5	131±9	373±13
		1000 µg	11±4^P	09±1^P	28±5^P	132±4^P	398±29^P
		2500 µg	12±2^P	6±1^P	23±1^P	123±18^P	238±19^P
		5000 µg	11±1^PM	2±2^PM	24±3^PM	131±4^PM	75±10^PM
	NaN3	10 µg	1935±127	-	-	2012±67	-
	4-NOPD	10 µg	-	-	329±26	-	-
	4-NOPD	50 µg	-	70±5	-	-	-
	MMS	3.0 µL	-	-	-	-	3163±174
With activation	Deionised water	-	22±5	14±4	39±10	131±17	611±24
	Untreated	-	22±2	13±3	51±4	137±7	559±19
	Disodium titanate	3 µg	23±3	17±3	40±5	125±10	568±8
		10 µg	22±6	14±3	41±10	131±5	598±12
		33 µg	23±1	15±1	41±3	128±5	565±9
		100 µg	20±6	14±1	37±4	138±5	537±23
		333 µg	15±1	15±7	43±13	137±8	530±38
		1000 µg	14±1^P	14±3^P	41±10^P	118±13^P	421±37^PM
		2500 µg	8±2^PM	4±1^PM	22±6^PM	108±4^PM	25±4^PM
		5000 µg	2±2^PM	1±1^PM	11±1^PM	38±3^PM	5±2^PM
	2-AA	2.5 µg	483±31	437±42	2941±199	3297±56	-
	2-AA	10.0 µg	-	-	-	-	3252±169

Summary of results of experiment II:

 

Metabolic activation	Test group	Dose level [µg/plate]	Revertant colony counts (mean ± SD)
			TA 1535	TA 1537	TA 98	TA 100	TA 102
Without activation	Deionised water	-	15±2	25±1	29±1	145±10	409±9
	Untreated	-	18±2	24±0	36±8	144±7	398±35
	Disodium titanate	3	16±6	28±3	26±3	139±10	385±6
		10	17±2	27±3	30±4	147±16	386±6
		33	16±4	24±9	30±3	137±4	394±4
		100	19±2	26±1	30±2	150±4	370±9
		333	14±6	25±3	27±4	134±10	371±3
		1000	9±3^P	24±10^P	29±1^P	141±17^P	360±14^P
		2500	5±3^P	21±7^P	25±2^P	118±14^P	334±10^P
		5000	9±2^PM	18±4^PM	22±4^PM	124±10^P	53±9^PM
	NaN3	10	1757±92	-	-	1997±58	-
	4-NOPD	10	-	-	305±2	-	-
	4-NOPD	50	-	80±9	-	-	-
	MMS	3.0 µL	-	-	-	-	3617±110
With activation	Deionised water	-	23±4	24±3	39±2	152±3	603±10
	Untreated	-	21±4	27±2	46±1	154±19	517±60
	Disodium titanate	3	23±7	22±3	38±3	155±10	598±16
		10	23±4	25±4	40±4	156±11	600±14
		33	20±2	30±3	41±7	162±7	636±54
		100	24±5	29±1	43±5	168±14	588±77
		333	20±5	21±8	39±6	149±20	562±65
		1000	16±4^P	19±4^P	38±6^P	141±7^P	427±20^PM
		2500	14±4^P	13±0^P	31±10^P	97±16^P	63±9^PM
		5000	3±1^PM	1±1^PM	16±4^PM	66±10^PM	6±2^PM
	2-AA	2.5	427±28	387±3	2644±148	2542±4	-
	2-AA	10.0	-	-	-	-	2491±7

NaN3	sodium azide	P	Precipitate
2-AA	2-aminoanthracene	M	Manual count
MMS	methyl methane sulfonate
4-NOPD	4-nitro-o-phenylene-diamine

Conclusions:

Interpretation of results: negative

Disodium titanate is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Executive summary:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Sodium titanates are effectively the sodium salts of the unstable titanic acid (titanium hydroxide). Titanium hydroxide is hard to isolate without rapid hydrolysis to titanium dioxide and sodium chloride. It is therefore proposed to base environmental and health assessment on these two hydrolysis products. There has been extensive research on similar substances in the ‘titanate’ grouping and these all exhibit similar behaviour in that under acid biological conditions (eg if ingested) or if dispersed in water, there is dissociation of the ions and subsequent hydrolysis / oxidation. Read-across justification for the use of TiO2 data is available in section 13.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

GLP compliance:

not specified

Remarks:

The paper does not state whether GLP conditions were used

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Ultrafine TiO2 particles were used in this test.
The test substance was prepared in Milli-Q water as this vehicle was determined to be the solvent of choice based on the solubility of the test substance and compatibility with the target cells. The test substance formed a white suspension in the vehicle at approximately 50 mg/mL, the highest stock concentration prepared on the study.

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Test concentrations with justification for top dose:

In the preliminary toxicity assay, the highest concentration tested was 5000 g/mL, the OECD 473 guideline limit dose for this test system.
Based on the observed test substance precipitation (generally regarded as an overload dose) in the preliminary toxicity assay, the highest concentration initially chosen for the chromosome aberration assay was 2500 g/mL, for all three test conditions.

Vehicle / solvent:

Water

Negative solvent / vehicle controls:

yes

Remarks:

water

Details on test system and experimental conditions:

Osmolality, pH, and test substance precipitation were taken into account for dose level setting, in addition to cell count and mitotic activity. Cytogenetic evaluations of structural aberrations were conducted in 200 cells in metaphase at 750, 1250, and 2500 g/mL for the 4 h nonactivated test condition; at 62.5, 125, and 250 g/mL, for the 4 h activated test condition, and at 25, 50, and 100 g/mL for the 20h non-activated test condition. Numerical aberrations were recorded as well. Appropriate positive controls were included in the test.
However, for the 4h activated and the 20h non-activated test conditions, substantial test substancerelated inhibition of the mitotic activity was observed at >750 g/mL in the 4 h activated test condition, and all concentration levels in the 20 h non-activated test condition. Therefore, cytogenetic evaluations were conducted at 750, 1250, and 2500 g/mL for the 4 h non-activated test condition, at 62.5, 125, and 250 g/mL, for the 4h activated test condition, and at 25, 50, and 100 g/mL for the 20 h non-activated test condition.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

The percentage of cells with structural or numerical aberrations in the test substance-treated groups was not significantly increased above that of the vehicle control at any concentration

Conclusions:

The percentage of cells with structural or numerical aberrations in the test substance-treated groups was not significantly increased above that of the vehicle control at any concentration ( p < 0.05, Fisher’s exact test). Uf-C TiO 2 particles did not induce structural or numerical chromosome aberrations in this study
Genotoxicity tests demonstrated that uf-TiO 2 was negative in an in vitro mammalian chromosome aberration test with Chinese hamster ovary cells.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

All six TiO2 test substances were negative for in vivo genotoxicity effects; (however, it is noted that the exposure to target tissues was likely negligible). One pigment grade and one ultrafine material each were evaluated for potential systemic exposure/uptake from the gastrointestinal tract by analysis of TiO 2 into blood and liver. No signifi cant increases in TiO2 over controls were measured in blood (48 or 72 h) or liver (72 h) following exposures to 2000 mg/kg bw TiO 2 . These data indicate that there was no absorption of the test material from the gastrointestinal tract into the blood circulation and the lack of genotoxic effects is therefore attributed to a lack of exposure due to the inability of the test material to migrate from the gastrointestinal tract into the blood and then into target tissues.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Sodium titanates are effectively the sodium salts of the unstable titanic acid (titanium hydroxide). Titanium hydroxide is hard to isolate without rapid hydrolysis to titanium dioxide and sodium chloride. It is therefore proposed to base environmental and health assessment on these two hydrolysis products. There has been extensive research on similar substances in the ‘titanate’ grouping and these all exhibit similar behaviour in that under acid biological conditions (eg if ingested) or if dispersed in water, there is dissociation of the ions and subsequent hydrolysis / oxidation. Read-across justification for the use of TiO2 data is available in section 13.

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

Mammalian Erythrocyte Micronucleus Test (1997).

Principles of method if other than guideline:

Six pigment-grade (pg) or ultrafi ne (uf)/nanoscale (anatase and/or rutile) titanium dioxide (TiO 2 ) particulates were evaluated for in vivo genotoxicity (OECD 474 Guidelines) in male and female rats by two different laboratories. All test materials were robustly characterized. The BET surface areas of the pg and uf samples ranged from 7 to 17 m 2 /g and 50 to 82 m 2 /g respectively. The materials were assessed for induction of micronuclei and toxicity in bone marrow by analyzing peripheral blood reticulocytes (RETs) byfl ow cytometry. Single oral gavage doses of 500, 1000 or 2000 mg/kg body weight (bw) of each material were implemented with concurrent negative (water) and positive controls (cyclophosphamide).

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

3 pigment-grade, and 3 ultrafi ne titanium dioxide (TiO 2 ) forms were used in this study. The series of samples analyzed spanned the range from test particles containing both anatase and rutile crystallites to both ultrafi ne and pigmentary materials composed of primarily rutile or anatase crystallites.

Species:

rat

Strain:

other: Crl:CD(SD) rats and Wistar Crl:WI (Han) rats

Sex:

male/female

Details on test animals or test system and environmental conditions:

Crl:CD(SD) rats were obtained from Charles River Breeding Laboratories, Raleigh, North Carolina, USA. Wistar Crl:WI (Han) rats were obtained from Charles River, Sulzfeld, Germany. Animals were housed and maintained in facilities accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International in accordance with the principles described in the Guide to Care and Use of Laboratory Animals . Tap water and pelleted chow (Certifi ed diet from PMI ® Nutrition International, LLC, Harlan or Purina) or Atromin 1324 were available ad libitum throughout the study.

Route of administration:

oral: gavage

Vehicle:

The test materials, vehicle, and the positive control were administered as single doses by oral gavage, based on the potential and relevant route for human exposure to TiO 2 in food or dietary supplements. This exposure strategy was considered suitable and most appropriate for the purpose of these studies, mimicking the likely human exposure route for any food or dietary supplement application incorporating TiO2 particles. Sterile water was used as the vehicle in all cases

Details on exposure:

Based on range-fi nding results, doses of 500, 1000, and 2000 mg/kg body weight (bw) of each test material were selected for the micronucleus tests. Concurrent control groups were administered sterile water, as the vehicle (negative) control, or 10 mg/kg bw of cyclophosphamide (positive control).

Duration of treatment / exposure:

On the day of dosing, all study animals were between 7 and 8 weeks of age, with body weights within ± 20% of the mean. Five animals per sex per dose group (seven in the highest dose group) were dosed by oral gavage (dose volume 10 mL/kg bw) as follows: 1) vehicle control (0 mg/kg bw); 2) low dose (500 mg/kg bw); 3) intermediate dose (1000 mg/kg bw); 4) high dose (2000 mg/kg bw); and 5) positive (cyclophosphamide) control (10 mg/kg bw).

Frequency of treatment:

Single dose

Post exposure period:

Approximately 48 and 72 h after exposure, blood samples were collected and 20,000 RETs per animal were analyzed.

Dose / conc.:

0 mg/kg bw (total dose)

Dose / conc.:

500 mg/kg bw (total dose)

Dose / conc.:

1 000 mg/kg bw (total dose)

Dose / conc.:

2 000 mg/kg bw (total dose)

No. of animals per sex per dose:

Five animals per sex per dose group (seven in the highest dose group)

Control animals:

yes, concurrent vehicle

Positive control(s):

positive controls (cyclophosphamide)

Tissues and cell types examined:

The materials were assessed for induction of micronuclei and toxicity in bone marrow by analyzing peripheral blood reticulocytes (RETs) by flow cytometry.

Evaluation criteria:

The objective of these six studies was to evaluate the potential of the test materials, namely 3 pigment-grade, and 3 ultrafi ne titanium dioxide (TiO2 ) forms, to induce micronuclei in rat bone marrow by analyzing micronucleated reticulocytes (MN-RETs) in rat peripheral blood cells. An increase in the frequency of MN-RETs relative to negative controls would indicate that a test material induces chromosomal and/or cell spindle damage in erythroblasts. Possible toxic effects (inhibition of erythropoiesis) may be indicated by a signifi cant decrease in the frequency of reticulocytes (RETs) among the total erythrocytes [RETs plus normochromatic erythrocytes (NCEs)].

Statistics:

A test was considered valid if the range of MN-RET values in the vehicle control animals were within reasonable limits of the laboratory historical control range, and if the positive control induced a signifi cant increase in the frequency of MN-RETs as compared to the vehicle control group, judged by biological and/or statistical signifi cance (p 0.05).

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Titanium was present in blood and liver of untreated (control) rats. Tissue concentrations of rats treated with 500, 1000, or 2000 mg TiO2 /kg/bw did not show a dose-dependent increase in titanium content. Single oral intubation resulted in no discernible dose-dependent increases of TiO2 in the blood and liver of treated rats relative to control rats.

Conclusions:

Under the conditions of these six studies, uf-1, uf-2, uf-3, pg-1, pg-2, and pg-3 titanium dioxide did not induce biologically relevant increases in micronucleated RETs in rat peripheral blood. Accordingly, all of the test materials were concluded to be essentially negative in the in vivo Mammalian Erythrocyte Micronucleus Test specifi c to oral administration, noting that the result can at least in part be attributed to the lack of achieving exposure of the test substance to the bone marrow even at the OECD 474 limit dose of 2000 mg/kg bw, when dosed by oral administration. With the exception of demonstrated target cell exposure, all other criteria for valid studies were met.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)