Dichlorobis(η-cyclopentadienyl)titanium

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

There are 3 Ames test available for the substance, one is disregarded. In the key study it was mutagenic in Salmonella typhimurium strain TA100 in the absence of exogenous metabolic activation (S9) (33.3 to 3333.3 μg/plate); it was not mutagenic in TA100 with S9, nor was it mutagenic in TA1535, TA1537, or TA98 with or without S9. In a second study (0.3 - 333.3 μg/plate) a positive response was not observed in TA1535, TA1537, TA98, TA100, TA1538 or E. coli WP2 with and without S9.

In a MLA study the substance induced statistically significant and dose related (linear trend) increases that in the mutant frequency at the TK +/- locus in L5178Y cells in the absence of metabolic activation. It is considered to be mutagenic under the conditions of this test.

Based on this information an in vivo mutagenicity study is proposed to come to a final conclusion on the endpoint.

There are two in vitro cytogenicity studies available. In these studies it was shown that the substance does not induce chromosomal aberrations in Chinese hamster ovary cells or sister chromatid exchanges in Chinese hamster ovary cells, with or without S9. No further testing is required for this endpoint.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was published in 1983 and included in this review in 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Testing was performed as reported by Ames et at! (1975) with modifications described in Haworth et al, (1983).

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Substance name: TITANOCENE DICHLORIDE
CAS NO.1271-19-8
Titanocene dichloride was sent to the laboratory as a coded aliquot from Radian Corporation, Austin, TX.

Target gene:

histidine locus

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

metabolic activation enzymes and cofactors from Aroclor 1254-induced male Sprague-Dawley rat or Syrian hamster liver

Test concentrations with justification for top dose:

High dose was limited by toxicity or solubility
0.0, 33.3, 100.0, 333.3, 1000.0 and 3,333.3 µg/plate

Vehicle / solvent:

dimethylsulfoxide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

dimethylsulfoxide

True negative controls:

no

Positive controls:

yes

Remarks:

for all strains

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9-mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

dimethylsulfoxide

True negative controls:

no

Positive controls:

yes

Remarks:

for strain TA98

Positive control substance:

other: 4-nitro-o-phenylenediamine

Remarks:

without S9-mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

dimethylsulfoxide

True negative controls:

no

Positive controls:

yes

Remarks:

for strains TA100 and TA1535

Positive control substance:

sodium azide

Remarks:

without S9-mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

dimethylsulfoxide

True negative controls:

no

Positive controls:

yes

Remarks:

for strain TA1537

Positive control substance:

9-aminoacridine

Remarks:

without S9-mix

Details on test system and experimental conditions:

Testing was performed as reported by Ames et al. (1975) with modifications as listed below and described in greater detail in Haworth et al, (1983). The test chemical was incubated with the Salmonella typhimurium tester strain (TA98,TA100, TA1535, TA1537) either in buffer or S9 mix for 20 minutes at 37ºC prior to the addition of soft agar supplemented with e-histidine and d-biotin, and subsequent plating on minimal glucose agar plates. Incubation was continued for an additional 48 hours.
In this assay, each test consisted of triplicate plates of concurrent positive and negative controls and of at least 5 doses of the test chemical. High dose was limited by toxicity. All positive assays were repeated under the conditions that elicited the positive response.

Evaluation criteria:

A positive response was defined as a reproducible, dose-related increase in histidine-independent (revertant) colonies in any one strain/activation combination. An equivocal response was defined as an increase in revertants that was not dose-related, not reproducible, or of insufficient magnitude to support a determination of mutagenicity. A negative response was obtained when no increase in revertant colonies was observed following chemical treatment.

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

slight toxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

slight toxicity in absence of S9-mix

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

slight toxicity in absence of S9-mix

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

slight toxicity in absence of S9-mix

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Titanocene dichloride was tested at concentrations of up to 3,333 µg/plate for the induction of gene mutations in Salmonella typhimurium strains TA100, TA1535, TA1537, and TA98 both in the presence and in the absence of Aroclor 1254-induced male Sprague-Dawley rat or Syrian hamster liver S9. A positive response was observed only for the base-substitution strain TAl00 tested in the absence of S9; all other strain/activation combinations yielded negative results.

Mutagenicity of Titanocene Dichloride in Salmonella typhimurium (a) 

		Revertants/plate (b)
Strain	Dose (µg/plate)	-S9		+10% hamster S9	+10% rat S9
		Trial 1	Trial 2
TA100	0.0	91 ± 0.9	83 ± 7.6	104 ± 2.1	82 ± 5.1
	33.3	166 ± 9.7	102 ± 0.0	110 ± 9.2	93 ± 2.5
	100.0	178 ± 11.0	210 ± 8.5	91 ± 12.3	94 ± 4.6
	333.3	193 ± 9.3	271 ± 4.3	90 ± 8.4	89 ± 5.6
	1000.0	184(c) ± 20.9	153(c) ± 13.8	101 ± 10.0	99 ± 7.2
	3333.3	85(c) ± 11.6	64(c) ± 4.4	100 ± 13.4	93 ± 6.3
Trial summary		Positive	Positive	Negative	Negative
Positive control (d)		352 ± 16.0	486 ± 21.5	1482 ± 54.3	338 ± 9.4
TA1535	0.0	11 ± 2.0		5 ± 0.3	7 ± 1.5
	33.3	18 ± 0.7		5 ± 0.9	7 ± 1.2
	100.0	13 ± 3.5		6 ± 2.0	7 ± 0.0
	333.3	13 ± 1.5		7 ± 0.7	12 ± 2.3
	1000.0	14(c) ± 3.2		7 ± 0.6	15 ± 1.0
	3333.3	4(c) ± 2.6		14 ± 5.0	16 ± 5.2
Trial summary		Negative		Negative	Negative
Positive control (d)		285 ± 7.8		404 ± 18.0	211 ± 12.4
TA1537	0.0	7 ± 0.7		7 ± 1.0	18 ± 0.3
	33.3	6 ± 0.7		5 ± 0.6	14 ± 2.3
	100.0	6 ± 1.8		6 ± 1.5	18 ± 1.2
	333.3	7 ± 1.2		4 ± 0.6	19 ± 1.0
	1000.0	7(c) ± 1.5		5 ± 0.6	16 ± 2.0
	3333.3	5(c) ± 0.7		4 ± 1.5	13 ± 1.9
Trial summary		Negative		Negative	Negative
Positive control (d)		312 ± 24.1		466 ± 35.1	83 ± 6.9
TA98	0.0	24 ± 4.2		33 ± 4.1	36 ± 3.1
	33.3	33 ± 4.6		27 ± 3.5	30 ± 1.2
	100.0	38 ± 1.5		25 ± 5.4	24 ± 2.0
	333.3	30 ± 1.2		32 ± 2.6	26 ± 1.2
	1000.0	29(c) ± 5.6		29 ± 1.9	28 ± 5.4
	3333.3	11(c) ± 3.2		26 ± 3.6	21 ± 4.1
Trial summary		Equivocal		Negative	Negative
Positive control (d)		529 ± 11.8		1222 ± 59.4	153 ± 12.2

(a) Study performed at SRI International. Cells and the test chemical or solvent (dimethylsulfoxide) were incubated in the absence of exogenous metabolic activation or with Aroclor 1254-induced S9 from male Syrian hamster liver or male Sprague-Dawley rat liver. High dose was limited by toxicity or solubility, but did not exceed 10 mg/plate; 0 µg/plate dose is the solvent control.

(b) Revertants are presented as mean ± the standard error from 3 plates.

(c) Slight toxicity

(d) 2-aminoanthracene was used for all strains in the presence of S9. In the absence of metabolic activation, 4-nitro-o-phenylenediamine was tested on TA98, sodium azide was tested on TAlOO and TA1535, and 9-aminoacridine was tested on TA1537.

Conclusions:

Titanocene dichloride was mutagenic in Salmonella typhimurium strain TA100 in the absence of exogenous metabolic activation (S9); it was not mutagenic in TA100 with S9, nor was it mutagenic in TA1535, TA1537, or TA98 with or without S9.

Executive summary:

Titanocene dichloride, tested at concentrations of 33 to 3,333 µg/plate, induced gene mutations in the S. typhimurium base pair substitution strain TA100 in the absence of S9, but was negative in several frameshift strains, with and without S9 activation

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The studies were published in 1985 and 1987 and included in this review in 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

Testing was performed as reported by Galloway et al. (1985, 1987)

GLP compliance:

no

Type of assay:

other: chromosome aberration assay in mammalian cells

Specific details on test material used for the study:

Substance name: TITANOCENE DICHLORIDE
CAS NO.1271-19-8
Titanocene dichloride was sent to the laboratory as a coded aliquot from Radian Corporation, Austin, TX.

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced male Sprague-Dawley rat liver S9 and cofactor mix

Test concentrations with justification for top dose:

The high dose was limited by toxicity

Without S9-mix: 35, 75, 162 and 349 µg/ml
With S9-mix: 162, 346 and 750 µg/ml

Vehicle / solvent:

dimethylsulphoxide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

dimethylsulfoxide

True negative controls:

no

Positive controls:

yes

Remarks:

0.4 µg/ml

Positive control substance:

mitomycin C

Remarks:

Without S9-mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

dimethylsulfoxide

True negative controls:

no

Positive controls:

yes

Remarks:

20 µg/ml

Positive control substance:

cyclophosphamide

Remarks:

With S9-mix

Details on test system and experimental conditions:

The substance was tested in cultured Chinese hamster ovary (CHO) cells for induction of sister chromatid exchanges and chromosomal aberrations both in the presence and the absence of Aroclor 1254-induced male Sprague-Dawley rat liver S9 and cofactor mix. Cultures were handled under gold lights to prevent photolysis of bromodeoxyuridine-substituted DNA. Each test consisted of concurrent solvent and positive controls and of at least 3 doses of the test chemical.

In the chromosomal aberration (Abs) test without S9, cells were incubated in McCoy's 5A medium with the test chemical for 16 hours; Colcemid was added and incubation continued for 2 hours. Because of significant chemical-induced cell cycle delay, incubation time prior to addition of Colcemid was lengthened to 16 hours to allow suftlcient metaphases at harvest. The cells were then harvested by mitotic shake-off, fixed, and stained with Giemsa. For the Abs test with S9, cells were treated with the test chemical and S9 for 2 hours at 37ºC, after which the treatment medium was removed and the cells were incubated for 11 hours in fresh medium, with Colcemid present for the final 2 hours. Cells were harvested in the same manner as for the treatment without S9. The harvest time for the Ab test was based on the cell cycle information obtained in the SCE test. If cell cycle delay was anticipated, the incubation period was extended approximately 5 hours.
Cells were selected for scoring on the basis of good morphology and completeness of karyotype (21 ± 2 chromosomes). All slides were scored blind and those from a single test were read by the same person. 200 first-division metaphase cells were scored at each dose level for the Abs test. Classes of aberrations included simple (breaks and terminal deletions), complex (rearrangements and translocations), and other (pulverized cells, despiralized chromosomes, and cells containing 10 or more aberrations).

Statistics:

Statistical analyses were conducted on the slopes of the dose-response curves and on the individual dose pints. Chromosomal aberration data are presented as percentage of cells with aberrations. As with SCE, both the dose-response curve and individual dose points were statistically analyzed.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at the 349 µg/ml without S9-mix

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

In cytogenetic tests with Chinese hamster ovary cells, titanocene dichloride was negative for chromosomal aberrations, with and without metabolic activation. In the chromosomal aberration test without S9, doses ranged from 35 to 348 µg/ml and a delayed harvest protocol was used to allow sufficient metaphase cells to accumulate for analysis. In the presence of S9, doses of 162 to 750 µg/mL were tested, a precipitate formed at the two highest concentrations (349 and 750 pglmL).

Conclusions:

Titanocene dichloride did not induce chromosomal aberrations in Chinese hamster ovary cells, with or without S9.

Executive summary:

The mutagenicity data available for titanocene dichloride indicate that the chemical has no clastogenic activity as measured by the induction of chromosomal aberrations in Chinese hamster ovary cells

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Starting Date: 09 September 2016 Experimental Completion Date: 10 October 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ``Kanpoan No. 287 - - Environment Protection Agency`` • ``Eisei No. 127 - - Ministry of Health and Welfare`` • ``Heisei 09/10/31 Kikyoku No. 2 - - Ministry of International Trade & Industry``

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: In vitro mammalian cell gene mutation assay

Specific details on test material used for the study:

Identification: Dichlorobis(η-cyclopentadienyl)titanium (CAS# 1271-19-8)
Product name: TDC (titanocene dichloride)
Batch Number: 0708501022
Product Number: 65662
CAS Number: 1271-19-8
Chemical Name: bis(cyclopentadienyl) titanium dichloride
EC Name: dichlorobis(η-cyclopentadienyl)
Purity: 99.3% REACH analytics
Expiry: 04 March 2019

Target gene:

Thymidine kinase TK +/- locus of the L5178Y mouse lymphoma cell line

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Cell Culture
The stocks of cells are stored in liquid nitrogen at approximately -196 °C. Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 µg/mL), Sodium pyruvate (1 mM), Amphotericin B (2.5 µg/mL) and 10% donor horse serum (giving R10 media) at 37 °C with 5% CO2 in air. The cells have a generation time of approximately 12 hours and were subcultured accordingly. RPMI 1640 with 20% donor horse serum (R20) and without serum (R0) are used during the course of the study. Master stocks of cells were tested and found to be free of mycoplasma.


Cell Cleansing
The TK +/- heterozygote cells grown in suspension spontaneously mutate at a low but significant rate. Before the stocks of cells were frozen they were cleansed of homozygous (TK -/-) mutants by culturing in THMG medium for 24 hours. This medium contained Thymidine (9 µg/mL), Hypoxanthine (15 µg/mL), Methotrexate (0.3 µg/mL) and Glycine (22.5 µg/mL). For the following 24 hours the cells were cultured in THG medium (i.e. THMG without Methotrexate) before being returned to R10 medium.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

The S9-mix was prepared prior to the dosing of the test cultures and contained the S9 fraction (20% (v/v)), MgCl2 (8mM), KCl (33mM), sodium orthophosphate buffer pH 7.4 (100mM), glucose-6-phosphate (5mM) and NADP (5mM).

Test concentrations with justification for top dose:

Preliminary toxicity test: 0, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000, 2000 µg/mL

Main Experiment:(4 hours, minus S9): 3.75, 7.5, 20, 40, 60 µg/mL plated out
7.5, 20, 40, 60, 80, 120, µg/mL (4 hours. plus S9)
0.94, 1.88, 3.75, 7.5, 15 µg/mL (24 hours, minus S9)

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

400 µg/mL and 150 µg/mL for 4-hour and 24-hour exposures respectively, was used as the positive control in the absence of metabolic activation.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

1.5 µg/mL was used as the positive control in the presence of metabolic activation.

Details on test system and experimental conditions:

Test Item Preparation
Following solubility checks performed in-house, the test item was accurately weighed and formulated in DMSO prior to serial dilutions being prepared. The test item was a molecular weight of 248.96 and therefore the maximum dose level in the solubility test was set at 2000 µg/mL, with a correction for purity (98.5%). There was no marked change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm (Scott et al. 1991). The pH and osmolality readings are in the following table:


µg/mL 0 7.81 15.63 31.25 62.5 125 250 500 1000 2000
pH 7.47 7.41 7.43 7.45 7.41 7.40 7.35 7.26 7.14 6.84
mOsm 449 460 457 456 - - - 457 458 444
- = not determined

No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. The test item was formulated within two hours of it being applied to the test system. It is assumed that the formulation was stable for this duration. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Control Preparation
Vehicle and positive controls were used in parallel with the test item. Solvent (DMSO) treatment groups were used as the vehicle controls. Ethylmethanesulphonate (EMS) Sigma batch BCBQ0451V (CAS No.62-50-0 Expiry 12.02.17) at 400 µg/mL and 150 µg/mL for 4-hour and 24-hour exposures respectively, was used as the positive control in the absence of metabolic activation. Cyclophosphamide (CP) Acros batch A0355340 (CAS No. 6055-19-2 Expiry 01.01.20 Purity 97%) at 1.5 µg/mL was used as the positive control in the presence of metabolic activation.


Microsomal Enzyme Fraction
Lot No. PB/NF S9 25/08/16 was used in the main test, and was pre-prepared in-house (outside the confines of the study) following standard procedures. Prior to use, each batch of S9 is tested for its capability to activate known mutagens in the Ames test and a certificate of S9 efficacy is presented in Appendix 2.

The S9-mix was prepared prior to the dosing of the test cultures and contained the S9 fraction (20% (v/v)), MgCl2 (8mM), KCl (33mM), sodium orthophosphate buffer pH 7.4 (100mM), glucose-6-phosphate (5mM) and NADP (5mM). The final concentration of S9, when dosed at a 10% volume of S9-mix into culture media, was 2%.


Test Procedure
Preliminary Toxicity Test
A preliminary toxicity test was performed on cell cultures at 5 x 105 cells/mL, using a 4 hour exposure period both with and without metabolic activation (S9), and at 1.5 x 105 cells/mL using a 24-hour exposure period without S9. The dose range used in the preliminary toxicity test was 7.81 to 2000 µg/mL for all three of the exposure groups. Following the exposure period the cells were washed twice with R10, resuspended in R20 medium, counted using a Coulter counter and then serially diluted to 2 x 105 cells/mL.

The cultures were incubated at 37 °C with 5% CO2 in air and sub-cultured after 24 hours by counting and diluting to 2 x 105 cells/mL. After a further 24 hours the cultures were counted and then discarded. The cell counts were then used to calculate Suspension Growth (SG) values. The SG values were then adjusted to account for immediate post treatment toxicity, and a comparison of each treatment SG value to the concurrent vehicle control performed to give a percentage Relative Suspension Growth (%RSG) value.

Results from the preliminary toxicity test were used to set the test item dose levels for the mutagenicity experiments. Maximum dose levels were selected using the following criteria:

i) Maximum recommended dose level, 2000 µg/mL or 10 mM.

ii) The presence of excessive precipitate where no test item-induced toxicity was observed.

iii) Test item-induced toxicity, where the maximum dose level used should produce approximately 10 to 20% survival (the maximum level of toxicity required). This optimum upper level of toxicity was confirmed by an IWGT meeting in New Orleans, USA (Moore et al 2002).


Mutagenicity Test
Main Experiment
Several days before starting the experiment, an exponentially growing stock culture of cells was set up so as to provide an excess of cells on the morning of the experiment. For the 4-hour exposure groups both with and without metabolic activation, the cells were counted and processed to give 1 x 106 cells/mL in 10 mL aliquots in R10 medium in sterile plastic universals. In the 24-hour exposure in absence of metabolic activation the cells were processed to give 0.3 x 106 cells/mL in 10 mL cultures established in 25 cm2 tissue culture flasks. The treatments were performed in duplicate (A + B), at up to 8 dose levels of the test item (3.75 to 120 µg/mL in the 4-hour exposure in the absence of metabolic activation and 3.75 to 240 µg/mL in the 4-hour exposure in the presence of metabolic activation and 0.94 to 40 µg/mL in the 24 hour exposure), vehicle and positive controls. To each universal was added 2 mL of S9-mix (2%) if required, 0.2 mL of the treatment dilutions, (0.15 or 0.2 mL for the positive control) and sufficient R0 medium to bring the total volume to 20 mL (R10 is used for the 24-hour exposure group).
The treatment vessels were incubated at 37 °C for 4 or 24 hours with continuous shaking using an orbital shaker within an incubated hood.

Evaluation criteria:

Please refer to "Any other information on materials an methods"

Statistics:

Please refer to "Any other information on materials an methods"

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Mutagenicity Test
A summary of the results from the test is presented in Table 1 - please refer to the attached for tables


4-hour exposure in the absence and presence of metabolic activation
The results of the microtitre plate counts and their analysis are presented in Tables 2 to 7.

There was evidence of toxicity following exposure to the test item in both the absence and presence of metabolic activation, as indicated by the %RSG and RTG values (Tables 3 and 6). There was evidence of marked reductions in viability (%V); therefore indicating that residual toxicity had occurred. Optimum levels of toxicity were considered to have been achieved in both 4-hour exposure groups. In the 4-hour exposure in the absence of metabolic activation, at 40 µg/mL, the dose level has not been discarded as the %RSG is within the acceptable range even though the RTG is marginally too toxic. Including this dose level shows the full extent of the dose related response. The excessive toxicity observed at and above 80 µg/mL in the absence of metabolic activation and at 240 µg/mL in the presence of metabolic activation resulted in these dose levels not being plated for viability or 5-TFT resistance. Acceptable levels of toxicity were seen with both positive control substances (Tables 3 and 6).

Neither of the vehicle control mutant frequency values were outside the normal in-house range of 50 to 170 x 10-6 viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional (Tables 3 and 6).

The test item induced both statistically significant and dose related (linear-trend) increases in the mutant frequency in the 4-hour exposure in the absence of metabolic activation. The GEF value was exceeded at 20 and 40 µg/mL (Table 3). In the presence of metabolic activation a small but statistically significant and linear trend response can be observed. However all of the mutant frequency values were bordering on the acceptable range for a vehicle control culture and the GEF value was not exceeded. Therefore the response is considered to be artefactual and of no toxicological significance (Table 6)

The numbers of small and large colonies and their analysis are presented in Tables 4 and 7. The colony formation in the absence of metabolic activation at 40 µg/mL was predominantly small indicative of a possible contribution to a clastogenic effect. In the 4-hour exposure in the presence of metabolic activation, the colony formation is relatively even.



24-hour exposure in the absence of metabolic activation
The results of the microtitre plate counts and their analysis are presented in Tables 8 to 10.

There was evidence of toxicity following exposure to the test item as indicated by the %RSG and RTG values (Table 9). There was evidence of moderate reductions in viability (%V), therefore indicating that residual toxicity occurred in this exposure group. Optimum levels of toxicity were achieved based on the RTG value. The excessive toxicity observed at and above 20 µg/mL resulted in these dose levels not being plated for viability or 5-TFT resistance. Acceptable levels of toxicity were seen with the positive control substance (Table 9).

The 24-hour exposure without metabolic activation (S9) treatment, demonstrated that the extended time point had a marked effect on the toxicity of the test item. The vehicle control mutant frequency was within the normal range of 50 to 170 x 10-6 viable cells. The positive control produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily (Table 9).

The test item induced both statistically significant and dose related (linear-trend) increases in the mutant frequency in the 4-hour exposure in the absence of metabolic activation. The GEF value was exceeded at 7.5 µg/mL (Table 9).

The numbers of small and large colonies and their analysis are presented in Table 10. The colony formation at 7.5 µg/mL in the 24-hour exposure group was predominantly small indicative of a possible contribution to a clastogenic effect.

Preliminary Cytotoxicity Test

The dose range of the test item used in the preliminary toxicity test was 7.81 to 2000 µg/mL. The results for the Relative Suspension Growth (%RSG) were as follows:

 

Dose (mg/mL)	% RSG (-S9) 4-Hour Exposure	% RSG (+S9) 4-Hour Exposure	% RSG (-S9) 24-Hour Exposure
0	100	100	100
7.81	81	91	49
15.63	61	90	17
31.25	49	77	6
62.5	24	47	1
125	4	15	0
250	2p	4p	0
500	1p	1p	0p
1000	0p	0p	0p
2000	0p	0p	0p

p = precipitate

 

In the 4-hour exposures, both in the absence and presence of metabolic activation (S9), there was evidence of marked reductions in the relative suspension growth (%RSG) of cells treated with the test item when compared to the concurrent vehicle controls. In the 24-hour exposure in the absence of S9 there was evidence of marked reductions of %RSG values of cells treated with test item. In both 4-hour exposures a precipitate of the test item was observed at and above 250 µg/mL at the end of exposure. In the 24-hour exposure in the absence of metabolic activation a precipitate was observed at and above 500 µg/mL at the end of exposure. In the subsequent mutagenicity experiments the maximum dose was limited by test item-induced toxicity in the absence of metabolic activation and a combination of test item induced toxicity and precipitate in the presence of metabolic activation.

Conclusions:

The test item, Dichlorobis(η-cyclopentadienyl)titanium (CAS# 1271-19-8), induced statistically significant and dose related (linear trend) increases that in the mutant frequency at the TK +/- locus in L5178Y cells in the absence of metabolic activation. The GEF value is exceeded at several concentrations subsequently is considered to be mutagenic under the conditions of the test.

Executive summary:

Introduction

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No 490 "In VitroMammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene" adopted 28 July 2015, Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA OPPTS 870.5300 Guideline, and in alignment with the Japanese MITI/MHW guidelines for testing of new chemical substances.

 

 

Methods…….

One main experiment was performed. In the main experiment, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at up to 8 dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4‑hour exposure groups both in the absence and presence of metabolic activation (2% S9) and a 24-hour exposure in the absence of metabolic activation.

 

The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The dose levels plated out for expression of mutant colonies were as follows:

 

Main Experiment

Group	Concentration of Dichlorobis(η-cyclopentadienyl)titanium (CAS# 1271-19-8) (µg/mL)  plated for mutant frequency
4-hour without S9	3.75, 7.5, 20, 40, 60
4-hour with S9 (2%)	7.5, 20, 40, 60, 80, 120
24-hour without S9	0.94, 1.88, 3.75, 7.5, 15

 

 

Results……..

The maximum dose level used was limited by test item-induced toxicity in the absence of metabolic activation, and a combination of test item induced toxicity, and precipitate in the presence of metabolic activation. A precipitate of the test item was observed at 240 µg/mL in the 4-hour exposure in presence of metabolic activation only. The vehicle (solvent) controls had acceptable mutant frequency values that were within the acceptable range for the L5178Y cell line at the TK +/- locus. The positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolizing system.

 

The test item induced statistically significant and dose related (linear-trend) increases in both 4-hour and 24-hour exposures without metabolic activation. The GEF value was exceeded at several dose levels and the colony formation was predominantly small indicative of possible contribution to a clastogenic effect.

 

Conclusion

The test item, Dichlorobis(η-cyclopentadienyl)titanium (CAS# 1271-19-8), induced statistically significant and dose related (linear trend) increases in the mutant frequency at the TK +/- locus in L5178Y cells in the absence of metabolic activation. The GEF value is exceeded at several concentrations subsequently is considered to be mutagenic under the conditions of the test.

Reference 4

Endpoint:

genetic toxicity in vitro, other

Remarks:

in vitro cytogenicity / sister chromatid exchange study

Type of information:

other: Review of experimental studies

Adequacy of study:

key study

Study period:

The studies were published in 1985 and 1987 and included in this review in 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 479 (Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells)

Version / remarks:

Testing was performed as reported by Galloway et al. (1985, 1987)

GLP compliance:

no

Type of assay:

sister chromatid exchange assay in mammalian cells

Specific details on test material used for the study:

Substance name: TITANOCENE DICHLORIDE
CAS NO.1271-19-8
Titanocene dichloride was sent to the laboratory as a coded aliquot from Radian Corporation, Austin, TX.

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced male Sprague-Dawley rat liver S9 and cofactor mix.

Test concentrations with justification for top dose:

The high dose was limited by toxicity.

Without S9-mix: 10.0, 33.0, 100.0 and 333.0 µg/ml
With S9-mix: 33.0, 100.0, 333.0 and 1,000.0 µg/ml

Vehicle / solvent:

dimethylsulfoxide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

dimethylsulfoxide

True negative controls:

no

Positive controls:

yes

Remarks:

0.001 and 0.005 µg/ml

Positive control substance:

mitomycin C

Remarks:

Without S9-mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

dimethylsulfoxide

True negative controls:

no

Positive controls:

yes

Remarks:

0.125 and 0.5 µg/ml

Positive control substance:

cyclophosphamide

Remarks:

With S9-mix

Details on test system and experimental conditions:

The substance was tested in cultured Chinese hamster ovary (CHO) cells for induction of sister chromatid exchanges and chromosomal aberrations both in the presence and the absence of Aroclor 1254-induced male Sprague-Dawley rat liver S9 and cofactor mix. Cultures were handled under gold lights to prevent photolysis of bromodeoxyuridine-substituted DNA. Each test consisted of concurrent solvent and positive controls and of at least 3 doses of the test chemical; the high dose was limited by toxicity.

In the sister chromatid exchange (SCE) test without S9, CHO cells were incubated for 26 hours at 37 ºC with the study chemical in McCoy's 5A medium supplemented with 10% fetal bovine serum, e-glutamine (2mM), and antibiotics. Bromodeoxyuridine (BrdU) was added 2 hours after culture initiation. After 26 hours, the medium containing the test chemical was removed and replaced with fresh medium plus BrdU and Colcemid, and incubation was continued for 2 more hours. Cells were then harvested by mitotic shake-off, fixed, and stained with Hoechst 33258 and Giemsa. In the SCE test with S9, cells were incubated at 37 ºC with the test chemical, serum-free medium, and S9 for 2 hours. The medium was then removed and replaced with medium containing BrdU and no test chemical and incubation proceeded for an additional 26 hours, with Colcemid present for the final 2 hours. Harvesting and staining procedures were the same as for cells treated without S9.

Cells were selected for scoring on the basis of good morphology and completeness of karyotype (21 ± 2 chromosomes). All slides were scored blind and those from a single test were read by the same person. For the SCE test, usually 50 second division metaphase cells were scored for frequency of SCE per cell from each dose level.

Statistics:

Statistical analyses were conducted on the slopes of the dose-response curves and on the individual dose pints. A sister chromatid exchange frequency 20% above the concurrent solvent control value was chosen as a statistically conservative positive response. The probability of this level of difference occurring by chance at one dose point is less than 0.01; the probability for such a chance occurrence at two dose points is less than 0.001.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at highest doses tested

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

In cytogenetic tests with Chinese hamster ovary cells, titanocene dichloride was negative for the induction of sister chromatid exchanges and chromosomal aberrations, with and without metabolic activation. An increase in sister chromatid exchange frequency was observed at three of the four concentrations tested without S9, but these increases were not of sufficient magnitude to be considered a positive response and the results of the trend test were not significant.
Doses in the sister chromatid exchange test ranged from 10 to 333 µg/mL without S9 and 33 to 1,000 µg/mL with S9.

Conclusions:

Titanocene dichloride did not induce sister chromatid exchanges in Chinese hamster ovary cells, with or without S9.

Executive summary:

The mutagenicity data available for titanocene dichloride indicate that the chemical has no clastogenic activity as measured by the induction of sister chromatid exchanges in Chinese hamster ovary cells. An increase in SCE frequency was observed at three of the four concentrations tested without S9, but these increases were not of sufficient magnitude to be considered a positive response and the results of the trend test were not significant. Doses in the SCE test ranged from 10 to 333 µg/mL without S9 and 33 to 1,000 µg/mL with S9.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Based on the in vitro mutagenicity data an in vivo mutagenicity study is proposed to come to a final conclusion on the endpoint. The proposed study is a Comet Assay, OECD 489.

Endpoint conclusion

Endpoint conclusion:

no study available (further information necessary)

Justification for classification or non-classification

Based on the available information the substance is not classified for this endpoint.