Tin titanium zinc oxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- in vitro gene mutation (bacteria): negative (OECD 471)
- in vitro chromosome aberration (mammalian cells): negative (OECD 473)
- in vitro gene mutation (mammalian cells): negative (OECD 476)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

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:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Experimental Toxicology and Ecology, BASF SE

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not applicable

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM (minimal essential medium with Earle's salts) containing a L-glutamine source supplemented with 10% (v/v) fetal calf serum (FCS), 1% (v/v) penicillin/streptomycin (10 000 IU / 10 000 μg/mL), 1% (v/v) amphotericin B (250 μg/mL). During exposure to the test substance (only 4-hour treatment), MEM medium was used without FCS supplementation.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S-9 mix

Test concentrations with justification for top dose:

0.5, 1, 2, 4, 8 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO; 1% (v/v)
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in the commonly used solvents, a homogeneous dispersion in DMSO was used in this study. It had been demonstrated that the vehicle DMSO is suitable in several mutagenicity test methods including the V79 in vitro cytogenetic test and for which historical control data are available.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation Migrated to IUCLID6: 500 μg/ml dissolved in MEM without FCS (2.5 mg/mL)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation Migrated to IUCLID6: 0.5 μg/ml dissolved in MEM without FCS (2.5 μg/mL)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4, 18 h
- Expression time (cells in growth medium): 10, 14, 24 h
- Fixation time (start of exposure up to fixation or harvest of cells): 2 - 3 hours

SPINDLE INHIBITOR (cytogenetic assays): 100 μL colcemid
STAIN (for cytogenetic assays): 7.5% (v/v) Giemsa/Titrisol solution pH 7.2 for 10 minutes.


NUMBER OF REPLICATIONS:
- experiment 1: 4h exposure, 18h sampling time, with and without metabolic activation
- experiment 2: 18h exposure, 18h sampling time, without metabolic activation; 18 h exposure, 28 hours sampling time, without metabolic activation; 4 hours exposure, 28 hours sampling time, with metabolic activation

NUMBER OF CELLS EVALUATED: 100 consecutive well-spread metaphases of each culture were counted for all test groups


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (1 000 cells, incl. mitotic cells, were counted per culture)

Evaluation criteria:

As a rule, the first 100 consecutive well-spread metaphases of each culture were counted for all test groups, and if cells had 20 - 22 chromosomes, they were analyzed for structural chromosome aberrations. In the case of clearly increased aberration rates, the number of metaphases to be analyzed for this test group can be reduced to at least 50 metaphases per culture.
In addition, numerical chromosome aberrations were scored and recorded.

Statistics:

The statistical evaluation of the data was carried out using the MUCHAN program system (BASF SE). The proportion of metaphases with structural aberrations was calculated for each group. A comparison of each dose group with the negative control group was carried out using Fisher's exact test for the hypothesis of equal proportions. This test was Bonferroni-Holm corrected versus the dose groups separately for each time and was performed one-sided. If the results of this test were statistically significant compared with the respective negative control, labels are printed in the tables.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: was not influenced by test substance treatment
- Effects of osmolality: was not influenced by test substance treatment


RANGE-FINDING/SCREENING STUDIES:
In the pretests for toxicity 5 000 μg/mL test substance was used as top concentration. The cells were prepared at a sampling time of 18 hours (about 1.5-fold cell cycle time) after 4 and 18 hours exposure time without S9 mix and after 4 hours exposure time with S9 mix.
The pretests were performed following the method described for the main experiment. As indication of test substance toxicity mitotic index, cell count, cell attachment (morphology) and quality of the slides were determined for dose selection.
In the pretests the parameters pH value and osmolarity were not relevant influenced by the addition of the test substance preparation to the culture medium at the concentrations measured.
Regarding the toxicity of the test substance reduced cell numbers of below 50% of control were observed at 5 000 μg/mL after 4 hours treatment in the absence of S9 mix only. No cytotoxicity occurred after 4 hours treatment in the presence of S9 mix or after 18 hours treatment in the absence of S9 mix.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Mitotic index: In the main experiments, according to the results of the determination of the mitotic index, no suppression of the mitotic activity was observed under all experimental conditions up to the highest applied concentration in the absence and presence of S9 mix.
- Cell counts: According to the results of the determination of the cell count in the main experiments, no growth inhibition was observed under all experimental conditions up to the highest applied concentration in the absence and presence of S9 mix.
- Cell morphology: Cell attachment was not influenced at any dose evaluated for structural chromosomal aberrations in the absence and presence of S9 mix.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Results:

			Genotoxicity				Cytotoxicity
Schedule	Test group	S-9 mix	aberrant cells (%)			polyploid cells (%)
Exposure/Preparation period			incl gaps	excl. Gaps	with exchanges		Cell number (%)	Mitotic index (%)
4/18 h	vehicle	-	3.5	1.5	1	2	100	100
	0.5 µg/ml	-	-	-	-	-	101.1	-
	1 µg/ml	-	9.5s	2	1	0.5	121.3	124.9
	2 µg/ml	-	9.5s	5	2	2	105.2	104
	4 µg/ml	-	6	2	1	1	98.3	109
	8 µg/ml	-	-	-	-	-	110.9	-
	EMS	-	22.0s	20.0s	11s	0		105.1
18/18 h	vehicle	-	5	2.5	0.5	0.5	100	100
	0.5 µg/ml	-	-	-	-	-	108.5	-
	1 µg/ml	-	6	2	1	0.5	110	110.3
	2 µg/ml	-	6	2	1.5	0	116.6	119.3
	4 µg/ml	-	6	3	1.5	0.5	117.5	104.1
	8 µg/ml	-	-	-	-	-	104.7	-
	EMS	-	29.0s	24.0s	15.0s	0		81.4
18/28 h	vehicle	-	4.5	3	0.5	0.5	100	100
	2 µg/ml	-	-	-	-	-	100.6	-
	4 µg/ml	-	5	4	2.5	0	83.1	101.4
	8 µg/ml	-	-	-	-	-	73.9	-
	EMS		23.0s	22.0s	19.0s	0		90.8
4/18 h	vehicle	+	7.5	1.5	0.5	1.9	100	100
	0.5 µg/ml	+	-	-	-	-	80.8	-
	1 µg/ml	+	8.5	2.5	1	2.9	81.2	90.4
	2 µg/ml	+	11.5	3.5	1.5	3.4	76.6	105.7
	4 µg/ml	+	9	3	2	1.9	79.5	79.7
	8 µg/ml	+	-	-	-	-	83.3	-
	CPP	+	22.0s	19.0s	14.0s	0		81.2
4/28 h	vehicle	+	9.5	4	1	0.5	100	100
	0.5 µg/ml	+	-	-	-	-	11.6	-
	1 µg/ml	+	3.5	1	0.5	1.5	114.9	115.9
	2 µg/ml	+	3	2.5	0	0.5	101.8	1117.7
	4 µg/ml	+	8.5	1	0	1	103.5	86.8
	8 µg/ml	+	-	-	-	-	111.4	-
	CPP	+	15	15.0s	9.0s	1		129.1

s: aberration frequency statistically significant higher than corresponding control values

Reference 2

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:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Experimental Toxicology and Ecology, BASF AG

Type of assay:

bacterial reverse mutation assay

Target gene:

his

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S-9 mix

Test concentrations with justification for top dose:

Standard plate test: 20, 100, 500, 2500 and 5000 μg/plate
Preincubation test: 312.5, 625, 1250, 2500 and 5000 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: due to the limited solubility of the test substance in water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2-AA); 2.5 μg/plate, dissolved in DMSO for strains TA 1535, TA 100, TA 1537, TA 98; 60 μg/plate, dissolved in DMSO for strain Escherichia coli WP2 uvrA

Remarks:

with metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG); 5 μg/plate, dissolved in DMSO for strains TA 1535, TA 100

Remarks:

without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylendiamine (NOPD); 10 μg/plate, dissolved in DMSO for strain TA 98

Remarks:

without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without metabolic activation Migrated to IUCLID6: 100 μg/plate, dissolved in DMSO for strain TA 1537

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without metabolic activation Migrated to IUCLID6: 5 μg/plate, dissolved in DMSO for strain E. coli WP2 uvrA

Details on test system and experimental conditions:

METHOD OF APPLICATION:in agar (plate incorporation); preincubation


DURATION
- Preincubation period: 20 min, 37°C
- Exposure duration: 48-72h, 37°C


NUMBER OF REPLICATIONS: triplates


DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertants, clearing or diminution of the background lawn (= reduced his- or trp- background growth), reduction in the titer

Evaluation criteria:

The test chemical is considered positive in this assay if the following criteria are met:
- A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.

A test substance is generally considered non-mutagenic in this test if:
- The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in at least two experiments carried out independently of each other.

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Weak bacteriotoxic effects were occasionally observed depending on the strain and the test conditions from about 2 500 μg/plate onward

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: precipitation was found from 2 500 μg/plate onward with and without S9 mix.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Results standard plate assay:

Dose µg/plate	metabolic activation	TA98			TA100			TA1535			TA1537			E.coli WP2 uvra
		Trial 1	Trial 2	Trial 3	Trial 1	Trial 2	Trial 3	Trial 1	Trial 2	Trial 3	Trial 1	Trial 2	Trial 3	Trial 1	Trial 2	Trial 3
0	+	46	45	45	101	124	118	15	14	20	7	10	10	50	53	55
20	+	52	52	44	134	128	124	18	14	14	15	14	13	50	52	22
100	+	45	55	37	122	127	110	16	18	20	13	12	15	60	71	52
500	+	44	53	51	137	128	127	19	15	21	13	12	14	47	44	53
2500	+	52P	42P	51P	121P	124P	135P	24P	17P	15P	14P	7P	10P	59P	48P	46P
5000	+	48P	34P	61P	168P	110P	122P	16P	14P	18P	12P	15P	11P	47P	50P	42P
60 µg 2 -Aminoanthracene	+													270	281	226
2.5 µg 2-Aminoanthracene	+	669	828	796	917	756	704	190	169	185	157	186	155
0	-	41	37	38	102	115	103	15	12	16	7	9	11	44	43	33
20	-	42	30	36	103	114	108	17	21	13	5	9	6	63	40	47
100	-	52	36	39	111	91	122	14	11	19	5	16	6	42	44	41
500	-	35	37	35	113	112	113	14	18	12	7	12	4	39	33	52
2500	-	31P	30P	30P	115P	116P	110P	12P	15P	13P	11P	7P	6P	46P	52P	39P
5000	-	32P	34P	29P	105P	116P	110P	14P	13P	15P	9P	8P	2P	46P	48P	51P
5 µg MNNG	-				626	600	660	514	559	604
10 µg 4-Nitro-o-phenylendiamin	-	700	725	675
5 µg 4-nitroquinoline-N-oxide	-													974	774	664
100 µg 9-amninoacridine	-										307	334	352

P: precipitation

Results preincubation test:

Dose µg/plate	metabolic activation	TA98			TA100			TA1535			TA1537			E.coli WP2 uvra
		Trial 1	Trial 2	Trial 3	Trial 1	Trial 2	Trial 3	Trial 1	Trial 2	Trial 3	Trial 1	Trial 2	Trial 3	Trial 1	Trial 2	Trial 3
0	+	36	28	27	119	130	102	17	18	14	11	9	11	45	46	38
312.5	+	27	30	24	104	102	95	12	17	15	8	8	12	29	39	44
625	+	29	22	26	111	100	114	13	16	17	9	5	13	51	35	34
1250	+	31	25	27	127	120	119	11	14	17	9	7	6	39	36	42
2500	+	20P	24P	22P	107P	114P	115P	16P	17P	16P	9P	7P	9P	45P	30P	34P
5000	+	17P	14P	14P	107P	120P	106P	8P	10P	11P	6P	6P	3P	38P	28P	21P
2.5 µg 2 -Aminoanthracene	+	594	600	544	888	832	709	111	104	129	109	115	120
60 µg 2-Aminoanthracene	+													214	228	253
0	-	22	28	29	124	119	137	15	16	17	7	7	10	33	42	34
312.5	-	34	32	26	117	111	97	17	17	17	11	8	6	24	39	39
625	-	24	27	24	93	112	120	18	21	18	8	5	7	44	43	30
1250	-	27	27	23	104	113	114	19	14	14	9	8	6	32	37	45
2500	-	25P	26P	21P	113P	124P	11P	10P	15P	14P	7P	8P	9P	34P	31P	36P
5000	-	16P	17P	11P	108P	93P	104P	8P	8P	9P	3P	3P	5P	22P	17P	20P
5 µg MNNG	-				774	734	708	559	652	571
10 µg 4-Nitro-o-phenylendiamin	-	582	468	447
5 µg 4-nitroquinoline-N-oxide														509	558	517
100 µg 9-amninoacridine	-										335	402	347

P: precipitation

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

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:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

adopted: 21 st July 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

1st Experiment:
- Without S9 mix 4-hour exposure: 2.5 μg/mL, 5.0 μg/mL, 10.0 μg/mL, 20.0 μg/mL, 400.0 μg/mL, 800.0 μg/mL, 1600.0 μg/mL, 3200.0 μg/mL
- With S9 mix 4-hour exposure: 1.3 μg/mL, 2.5 μg/mL, 5.0 μg/mL, 10.0 μg/mL, 20.0 μg/mL, 40.0 μg/mL

2nd Experiment:
- Without S9 mix 24-hour exposure: 2.5 μg/mL, 5.0 μg/mL, 10.0 μg/mL, 20.0 μg/mL, 400.0 μg/mL, 800.0 μg/mL
- With S9 mix 4-hour exposure: 1.9 μg/mL, 3.8 μg/mL, 7.5 μg/mL, 15.0 μg/mL, 30.0 μg/mL

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Without metabolic activation: 300 μg/mL ethyl methanesulfonate (EMS); With metabolic activation: 20 μg/mL methylcholanthrene (MCA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: Seeding of the cells pretreated with "HAT" medium for one day
- Exposure duration: 4-hour and 24-hour
- Selection time:
- Fixation time (start of exposure up to fixation or harvest of cells): Day 7 - 9: 2nd passage of the treated cells with seeding in the selection medium ("TG" medium); 2nd cytotoxicity determination (cloning efficiency 2: viability). From day 16: Drying, fixation, staining and counting of the selected colonies.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

Acceptance criteria:
The HPRT assay is considered valid if the following criteria are met:
• The absolute cloning efficiencies of the negative/vehicle controls should not be less than 50% (with and without S9 mix).
• The background mutant frequency in the negative/vehicle controls should fall within the historical negative control data range of 0 – 15.95 mutants per 10e6 clonable cells.
• The positive controls both with and without S9 mix must induce distinctly increased mutant frequencies.
• At least 4 dose levels ranging up to a toxic concentration or up to or beyond the limit of solubility under culture conditions should be tested. Freely soluble and apparently non-toxic substances are not tested at concentrations higher than 5 mg/mL or 10 mM.

Assessment criteria:
A finding is assessed as positive if the following criteria are met:
• Increase of the corrected mutation frequencies (MFcorr.) both above the concurrent negative control values and the historical negative control data range.
• Evidence of reproducibility of any increase in mutant frequencies.
• A statistically significant increase in mutant frequencies and the evidence of a doseresponse relationship.
Isolated increases of mutant frequencies above the historical negative control range (i.e. 15 mutants per 10e6 clonable cells) or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.
The test substance is considered non-mutagenic according to the following criteria:
• The corrected mutation frequency (MFcorr.) in the dose groups is not statistically significant increased above the concurrent negative control and is within the historical negative control data range.

Statistics:

Due to the clearly negative findings, a statistical evaluation was not carried out.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Without S9 mix, there was a decrease in the number of colonies from 800 μg/mL onward in the 1st and 2nd Experiment. Besides in both experiments after 4 hours exposure in the presence of S9 mix no change in colony numbers were obtained.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not influenced by test substance treatment
- Effects of osmolality: not influenced by test substance treatment
- Precipitation: In the absence of S9 mix test substance precipitation in culture medium at the end of treatment was observed macroscopically from 400 μg/mL onward in the 1st Experiment and from 20.0 μg/mL onward in the 2nd Experiment. Besides, in the presence of S9 mix test substance precipitation in culture medium occured 4 hours after start of treatment from 40 μg/mL onward in the 1st Experiment and from 30 μg/mL onward in the 2nd Experiment.


COMPARISON WITH HISTORICAL CONTROL DATA: The mutant frequencies at any concentration were within the range of the concurrent vehicle control values and within the range of the historical negative control data.
The mutation frequencies of the vehicle control groups were within the historical negative control data range including all vehicles used in the laboratory and, thus, fulfilled the acceptance criteria of this study.
The increase in the frequencies of mutant colonies induced by the positive control substances EMS and MCA clearly demonstrated the sensitivity of the test method and of the metabolic activity of the S9 mix employed. The values were within the range of the historical positive control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
-CELL MORPHOLOGY: Observations on adversely effected cell morphology (cell attachment) were obtained in the absence of S9 mix from 1600 μg/mL onward in the 1st Experiment after 4 hours exposure and from 400 μg/mL onward in the 2nd Experiment after 24 hours exposure. In the presence of S9 mix no changes in cell morphology (cell attachment) were observed up to the highest applied concentration in both experiments.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Bacteria gene mutation

The test substance was evaluated for its ability to cause bacteria mutation in a test protocol conucted under GLP according to OECD guideline 471. Bacteria strains S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A were treated with 20 – 5000 µg/mL test substance with and without metabolic activation by Aroclor 1254-induced rat liver S-9 mix (BASF, 2007). In both, the standard plate assay and the preincubation test, weak bacteriotoxic effects and precipitation were occasionally observed depending on the strain from about 2500 μg/plate. No increase reverse mutation. Therefore, according to the results of the study, the test substance is not mutagenic in bacteria cells under the test conditions chosen.

A supporting study according OECD TG 471 was performed (Johnson Matthey Speciality Coatings, 2004). Salmonella typhimurium strains TA1535, TA1537, TA102, TA98 and TA100 were treated with suspensions of the test material using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate in the first and second experiment. The vehicle (sterile distilled water) control plates gave counts of revertant colonies generally within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A slight yellow film was observed at 5000 µg/plate, this did not prevent the scoring of revertant colonies. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. The test material was considered to be non-mutagenic under the conditions of this test.

DNA damage/clastogenicity

The test substance was evaluated for its ability to induce chromosome aberration in mammalian cells in a test protocol conducted under GLP according to OECD guideline 473, where Chinese hamster lung fibroblasts (V79) were treated with concentrations of 0.5, 1, 2, 4 and 8 µg/mL 4 or 18 hours. After the additional sampling time of 18 or 28 hours, the cells were harvested, fixed and analyzed for chromosomal aberrations. No increase in chromosomal damages was found. No increase in the number of structural chromosomal aberrations was observed after treatment with the test substance. Therefore, the test substance is not a chromosome damaging agent in V79 celss under the test conditions chosen.

A supporting study according OECD TG 473 was conducted (Johnson Matthey Speciality Coatings, 2004). Duplicate cultures of human lymphocytes, treated with the test material, were evaluated for chromosome aberrations at three dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study, ie. In Experiment 1, 4 hours in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2, the 4 hours exposure with addition of S9 was repeated (using a 1% final S9 concentration), whilst in the absence of metabolic activation the exposure time was increased to 24 hours.

All vehicle (solvent) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.

All the positive control materials induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test material was slightly toxic but did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range that included the maximum recommended dose level of 5000 µg/mL. The test material was considered to be non-clastogenic to human lymphocytes in vitro.

Mammalian cell gene mutation

The test substance was evaluated for its ability to induce gene mutation in mammalian cells in a test protocol under GLP according to the OECD guideline 476 using Chinese hamster ovary (CHO) cells which were treated with 1.3 - 800 μg/mL test substance Tin titanium zinc oxide for 4 hours both with and without metabolic activation and 24 hours without metabolic activation (BASF, 2011). In the 1st and 2nd Experiment in the absence of metabolic activation the highest concentrations tested for gene mutations were clearly cytotoxic. Besides, in both experiments in the presence of metabolic activation no cytotoxicity was observed up to the highest tested concentration. In the absence of S9 mix test substance precipitation in culture medium at the end of treatment was observed macroscopically from 400 μg/mL onward in the 1st Experiment and from 20.0 μg/mL onward in the 2nd Experiment. Besides, in the presence of S9 mix test substance precipitation in culture medium occured 4 hours after start of treatment from 40 μg/mL onward in the 1st Experiment and from 30 μg/mL onward in the 2nd Experiment. The test substance did not cause any relevant increase in the mutant frequencies both without S9 mix and after adding a metabolizing system in two experiments performed independently of each other. Thus, under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The available test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Thus, the test item is considered not to be classified for genotoxicity under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EU) 2019/521.