Titanium tetraisopropanolate

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study performed according to an approved guideline

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

The maximum concentration tested in this study was 5495mg/plate, which exceeded the maximum concentration recommended by the guidelines which this assay follows of 5000 mg/plate.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Titanium tetraisopropanolate

EC Number:

208-909-6

EC Name:

Titanium tetraisopropanolate

Cas Number:

546-68-9

Molecular formula:

C12H28O4Ti

IUPAC Name:

titanium tetraisopropanolate

Details on test material:

2-Propanol, titanium (4+) salt (TIPT), (also known as Vertec TIPT, titanium tetraisopropanolate, EC number 208-909-6), batch number V0303339, was a yellow liquid. It was received into Covance Laboratories Ltd on 1 April 2010 and stored at 15 to 25ºC under desiccant in the dark and from 20 April 2010 it was also stored under nitrogen. Purity was initially stated as ≥90% and the expiry date was given as 7 April 2011. Subsequent to this, following completion of the study, information was received from the Sponsor detailing the following purity profile for the batch: 2-propanol, titanium (4+) salt >99%, Heptanes (naphtha) 0.56%, Iso-propanol <0.5%.

Method

Target gene:

histidine locus in selected strains

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 102

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S-9 fraction

Test concentrations with justification for top dose:

Range-Finder Experiment, Mutation Experiment 1 and additional Mutation Experiment 1 treatments of strain TA102 -S-9:
1.758, 8.792, 43.96, 219.8, 1099 and 5495 microgram/plate TIPT
Mutation experiment 2: 171.8, 343.4, 686.9, 1374, 2748 and 5495 microgram/plate TIPT

Vehicle / solvent:

DMSO
Justification for vehicle: preliminary solubility data indicated that TIPT was soluble in anhydrous analytical grade DMSO at concentrations of at least 100 mg/mL.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Strain: TA98

Migrated to IUCLID6: 2NF

Controls 2

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Strain: TA100 and TA1535

Migrated to IUCLID6: NaN3

Controls 3

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Strain: TA1537

Migrated to IUCLID6: AAC

Controls 4

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Strain: TA102

Migrated to IUCLID6: MMC

Controls 5

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

Strain: TA98

Migrated to IUCLID6: B[a]P

Controls 6

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (AAN)

Remarks:

Strain: TA100, TA1535 and TA1537

Details on test system and experimental conditions:

Bacteria
Five strains of Salmonella typhimurium bacteria (TA98, TA100, TA1535, TA1537 and TA102) were used in this study. All the tester strains, with the exception of strain TA102, were originally obtained from the UK NCTC. Strain TA102 was derived from a culture obtained from Glaxo Group Research Limited. For all assays, bacteria were cultured at 37±1°C for 10 hours in nutrient broth, containing ampicillin (TA98, TA100) or ampicillin and tetracycline (TA102) as appropriate. Incubation was carried out with shaking in an anhydric incubator, set to turn on using a timer switch. All treatments were completed within 6 hours of the end of the incubation period.
The inocula were taken from master plates or vials of frozen cultures, which had been checked for strain characteristics (histidine dependence, rfa character, uvrB character and resistance to ampicillin or ampicillin plus tetracycline). Checks were carried out according to Maron and Ames and De Serres and Shelby.

Maron D M and Ames B N (1983) Revised methods for the Salmonella mutagenicity test. Mutation Research 113, 173-215
De Serres F J and Shelby M D (1979) Recommendations on data production and analysis using the Salmonella/microsome mutagenicity assay. Mutation Research 64, 159-165

Evaluation criteria:

Evaluation criteria
For valid data, the test article was considered to be mutagenic if:
1. Dunnett's test gave a significant response (p<= 0.01) which was concentration related
2. the positive trend/effects described above were reproducible.
The test article was considered as positive in this assay if all of the above criteria were met.
The test article was considered as negative in this assay if none of the above criteria were met.
Results which only partially satisfied the above criteria were dealt with on a case by case basis. Biological relevance was taken into account, for example consistency of response within and between concentrations and (where applicable) between experiments.

Statistics:

Dunnett's test was used to compare the counts at each concentration with the control. The presence or otherwise of a concentration response was checked by non-statistical analysis, up to limiting levels (for example toxicity, precipitation or 5000 microgram/plate).

Results and discussion

Test results

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

Remarks:

evidence of toxicity seen in a reduction of revertants at 5495 microgram/plate in strains TA98 and TA102 in absence of S9.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Precipitation was observed on the test plates at the highest one or two concentrations (1099 and 5495 µg/plate) in the Range-Finder and Experiment 1. Precipitation was observed on the test plates at the highest two concentrations in the absence of S-9 (2748 and 5495 µg/plate) and the highest three concentrations in the presence of S-9 (1374 to 5495 µg/plate) in Experiment 2.

Mutation
Following treatments of all the test strains in the absence and presence of S-9, only Experiment 1 treatments of strain TA102 in the absence of S-9 at 1.758, 8.792 and 219.8 ug/plate resulted in increases in revertant numbers that were statistically significant when the data were analysed at the 1% level using Dunnett’s test. The increases were small in magnitude and occurred at intermediate concentrations with no indication of a concentration relationship and were not reproducible in two further experiments. Accordingly, these increases were considered to have been due to normal biological variability and not evidence of mutagenic activity.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Raw plate counts and calculated mutagenicity data Experiment 1

Experiment 1, TA98 +S-9

Compound	Concentration (μg/plate)	Revertant numbers/plate	Mean	N	Fold Increase	Standard Deviation	Dunnett’s t‑value	Significance
DMSO		31, 29, 34, 34, 31	31.8	5		2.2
TIPT	1.758	31, 25, 29	28.3	3	0.9	3.1	-1.08	NS
TIPT	8.792	30, 26, 23	26.3	3	0.8	3.5	-1.74	NS
TIPT	43.96	29, 34, 19	27.3	3	0.9	7.6	-1.51	NS
TIPT	219.8	33, 29, 26	29.3	3	0.9	3.5	-0.77	NS
TIPT	1099	28, 23, 34	28.3	3	0.9	5.5	-1.12	NS
TIPT	5495	28 P M, 25 P M, 22 P M	25.0	3	0.8	3.0	-2.18	NS
B[a]P	10	328, 376, 355	353.0	3	11.1	24.1

 Experiment 1,TA98 –S-9

Compound	Concentration (μg/plate)	Revertant numbers/plate	Mean	N	Fold Increase	Standard Deviation	Dunnett’s t‑value	Significance
DMSO		25, 24, 29, 23, 21	24.4	5		3.0
TIPT	1.758	20, 28, 23	23.7	3	1.0	4.0	-0.27	NS
TIPT	8.792	19, 19, 28	22.0	3	0.9	5.2	-0.90	NS
TIPT	43.96	19, 16, 20	18.3	3	0.8	2.1	-2.24	NS
TIPT	219.8	23, 19, 21	21.0	3	0.9	2.0	-1.21	NS
TIPT	1099	23, 15, 15	17.7	3	0.7	4.6	-2.57	NS
TIPT	5495	13 P M, 16 P M, 9 P M	12.7	3	0.5	3.5	-4.78	NS
2NF	5	1299, 1116, 1347	1254.0	3	51.4	121.9

 Experiment 1, TA100 -S-9

Compound	Concentration (μg/plate)	Revertant numbers/plate	Mean	N	Fold Increase	Standard Deviation	Dunnett’s t‑value	Significance
DMSO		108, 110, 122, 123, 125	117.6	5		8.0
TIPT	1.758	130, 124, 130	128.0	3	1.1	3.5	1.18	NS
TIPT	8.792	125, 124, 107	118.7	3	1.0	10.1	0.12	NS
TIPT	43.96	142, 124, 155	140.3	3	1.2	15.6	2.47	NS
TIPT	219.8	128, 134, 127	129.7	3	1.1	3.8	1.36	NS
TIPT	1099	155, 144, 118	139.0	3	1.2	19.0	2.31	NS
TIPT	5495	141 P M, 107 P M, 107 P M	118.3	3	1.0	19.6	0.04	NS
NaN3	2	915, 783, 752	816.7	3	6.9	86.6

Experiment 1, TA100 +S-9

Compound	Concentration (μg/plate)	Revertant numbers/plate	Mean	N	Fold Increase	Standard Deviation	Dunnett’s t‑value	Significance
DMSO		122, 139, 135, 163, 149	141.6	5		15.4
TIPT	1.758	152, 159, 134	148.3	3	1.0	12.9	0.78	NS
TIPT	8.792	142, 115, 113	123.3	3	0.9	16.2	-2.18	NS
TIPT	43.96	124, 130, 127	127.0	3	0.9	3.0	-1.69	NS
TIPT	219.8	137, 154, 140	143.7	3	1.0	9.1	0.27	NS
TIPT	1099	132, 129, 148	136.3	3	1.0	10.2	-0.59	NS
TIPT	5495	128 P M, 126 P M, 123 P M	125.7	3	0.9	2.5	-1.85	NS
AAN	5	1481, 1784, 1581	1615.3	3	11.4	154.4

Experiment 1, TA1535 -S-9

Compound	Concentration (μg/plate)	Revertant numbers/plate	Mean	N	Fold Increase	Standard Deviation	Dunnett’s t‑value	Significance
DMSO		10, 15, 9, 18, 18	14.0	5		4.3
TIPT	1.758	11, 14, 23	16.0	3	1.1	6.2	0.52	NS
TIPT	8.792	10, 21, 11	14.0	3	1.0	6.1	-0.04	NS
TIPT	43.96	14, 15, 21	16.7	3	1.2	3.8	0.76	NS
TIPT	219.8	19, 20, 15	18.0	3	1.3	2.6	1.12	NS
TIPT	1099	11, 24, 11	15.3	3	1.1	7.5	0.29	NS
TIPT	5495	19 P M, 11 P M, 11 P M	13.7	3	1.0	4.6	-0.08	NS
NaN3	2	677, 618, 640	645.0	3	46.1	29.8

Experiment 1, TA1535 +S-9

Compound	Concentration (μg/plate)	Revertant numbers/plate	Mean	N	Fold Increase	Standard Deviation	Dunnett’s t‑value	Significance
DMSO		14, 9, 16, 11, 16	13.2	5		3.1
TIPT	1.758	8, 15, 23	15.3	3	1.2	7.5	0.47	NS
TIPT	8.792	25, 14, 9	16.0	3	1.2	8.2	0.64	NS
TIPT	43.96	14, 20, 15	16.3	3	1.2	3.2	0.89	NS
TIPT	219.8	16, 26, 16	19.3	3	1.5	5.8	1.61	NS
TIPT	1099	18, 16, 11	15.0	3	1.1	3.6	0.52	NS
TIPT	5495	10 P M, 15 P M, 13 P M	12.7	3	1.0	2.5	-0.14	NS
AAN	5	128, 160, 119	135.7	3	10.3	21.5

Experiment 1, TA1537 -S-9

Compound	Concentration (μg/plate)	Revertant numbers/plate	Mean	N	Fold Increase	Standard Deviation	Dunnett’s t‑value	Significance
DMSO		18, 13, 13, 13, 13	14.0	5		2.2
TIPT	1.758	14, 9, 8	10.3	3	0.7	3.2	-1.54	NS
TIPT	8.792	14, 9, 23	15.3	3	1.1	7.1	0.32	NS
TIPT	43.96	14, 16, 15	15.0	3	1.1	1.0	0.39	NS
TIPT	219.8	19, 16, 18	17.7	3	1.3	1.5	1.33	NS
TIPT	1099	10, 10, 19	13.0	3	0.9	5.2	-0.49	NS
TIPT	5495	9 P M, 11 P M, 7 P M	9.0	3	0.6	2.0	-2.12	NS
AAC	50	71, 80, 76	75.7	3	5.4	4.5

 Experiment 1, TA1537 +S-9

Compound	Concentration (μg/plate)	Revertant numbers/plate	Mean	N	Fold Increase	Standard Deviation	Dunnett’s t‑value	Significance
DMSO		16, 16, 21, 20, 14	17.4	5		3.0
TIPT	1.758	17, 15, 20	17.3	3	1.0	2.5	-0.01	NS
TIPT	8.792	24, 17, 14	18.3	3	1.1	5.1	0.31	NS
TIPT	43.96	24, 27, 24	25.0	3	1.4	1.7	2.72	*
TIPT	219.8	17, 27, 21 M B	21.7	3	1.2	5.0	1.54	NS
TIPT	1099	22, 24, 16	20.7	3	1.2	4.2	1.20	NS
TIPT	5495	13 P M, 8 P M, 13 P M	11.3	3	0.7	2.9	-2.63	NS
AAN	5	62, 44, 51	52.3	3	3.0	9.1

 Experiment 1, TA102 -S-9

Compound	Concentration (μg/plate)	Revertant numbers/plate	Mean	N	Fold Increase	Standard Deviation	Dunnett’s t‑value	Significance
DMSO		242, 216, 219, 210, 214	220.2	5		12.6
TIPT	1.758	299, 279, 269	282.3	3	1.3	15.3	3.55	**
TIPT	8.792	317, 325, 292	311.3	3	1.4	17.2	5.07	**
TIPT	43.96	222, 212, 266	233.3	3	1.1	28.7	0.76	NS
TIPT	219.8	241, 345, 312	299.3	3	1.4	53.1	4.37	**
TIPT	1099	234, 243, 258	245.0	3	1.1	12.1	1.47	NS
TIPT	5495	99 P M, 118 P M, 96 P M	104.3	3	0.5	11.9	-8.37	NS
MMC	0.2	838, 680, 764	760.7	3	3.5	79.1

Experiment 1, TA102 +S-9

Compound	Concentration (μg/plate)	Revertant numbers/plate	Mean	N	Fold Increase	Standard Deviation	Dunnett’s t‑value	Significance
DMSO		192, 192, 195, 293, 243	223.0	5		44.7
TIPT	1.758	231, 219, 219	223.0	3	1.0	6.9	0.06	NS
TIPT	8.792	217, 194, 247	219.3	3	1.0	26.6	-0.09	NS
TIPT	43.96	207, 198, 207	204.0	3	0.9	5.2	-0.62	NS
TIPT	219.8	251, 222, 214	229.0	3	1.0	19.5	0.26	NS
TIPT	1099	272, 229, 125	208.7	3	0.9	75.6	-0.63	NS
TIPT	5495	196 P M, 184 P M, 232 P M	204.0	3	0.9	25.0	-0.64	NS
AAN	20	1817, 1141, 996	1318.0	3	5.9	438.2

P - precipitation of test article observed, M plate counted manually, B bubbles or split in agar, NS not significant, * p<0.05, ** p<0.01