Reaction mass of disodium hexatitanium tridecaoxide and disodium trititanium heptaoxide

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

, 1997

Deviations:

no

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

Disodium titanate

EC Number:

234-802-9

EC Name:

Disodium titanate

Cas Number:

12034-34-3

Molecular formula:

Na2TiO3

IUPAC Name:

disodium titanate

Details on test material:

- Name of test material (as cited in study report): Disodium titanate
- Physical state: light yellow powder
- Storage condition of test material: At room temperature, in tightly closed container

No further information on the test material was stated.

Method

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

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

Controls

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.

Results and discussion

Test results

Test results 1

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

Test results 2

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

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.