Hafnium

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Mutagenicity testing in bacterial systems is not appropriate for metal. So, testing is not required. No relevant articles is available for Hf to assess in vitro cytogenicity/ mutagenicity in mammalian cells. Nevertheless, an in vitro cytogenicity studies in mammalian cells with ZrO2 and in vitro gene mutation study in mammalian cells with ZrO2 (according to OECD 473 and 476 guideline) have been performed. Their results were negative.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well documented GLP study performed according to OECD Guideline 476 but quoted with reliability 2 because of the read across

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Deviations of temperature and humidity caused by adjustment after opening of the incubator door. However the study integrity was not adversely affected by the deviations

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Deviations of temperature and humidity caused by adjustment after opening of the incubator door. However the study integrity was not adversely affected by the deviations

GLP compliance:

yes (incl. QA statement)

Remarks:

Food and Consumer Product Safety Authority (VWA), Prinses Beatrixlaan 2, 2595 AL Den Haag, Postbus 19508, 2500,CM Den Haag, The Netherlands

Type of assay:

mammalian cell gene mutation assay

Target gene:

thymidine-kinase (TK) locus L5178Y

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9-mix induced by a combination of phenobarbital and beta-naphtoflavone

Test concentrations with justification for top dose:

0.03, 0.1, 1, 3, 10, 33 and 100 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: no data

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without metabolic activation; MMS was dissolved in dimethyl sulfoxide. The stock solutions of MMS were prepared immediately before use.

Migrated to IUCLID6: at concentration of 15 and 5 µg/mL for a 3 and 24 hours treatment period, respectively

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation; CP was dissolved in Hanks' balanced salt solution (HBSS) without calcium and magnesium. The stock solutions of CP were stored in aliquots at < or = -15°C in the dark and one sample was thawed immediately before use

Migrated to

Details on test system and experimental conditions:

In a first experiment, cell cultures were exposed for 3 hours to zirconium dioxide in exposure medium in the absence and presence of S9-mix. In a second experiment, cell cultures were exposed to Zirconium dioxide in exposure medium for 24 hours in the absence of S9-mix and for 3 hours in the presence of S9-mix.

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: not applicable
- Exposure duration: 3 hours or 24 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 11 or 12 days (TFT selection)
- Fixation time (start of exposure up to fixation or harvest of cells): 2 hours (MTT staining)

SELECTION AGENT (mutation assays): TFT
SPINDLE INHIBITOR (cytogenetic assays): not applicable
STAIN (for cytogenetic assays): not applicable

NUMBER OF REPLICATIONS: 2 independent experiments

NUMBER OF CELLS EVALUATED: for the determination of mutation frequency a total number of 9.6 x 1E05 cells/concentration were plated in five 96-well microtiter plates, each well containing 2000 cells in selective medium, with the exception of the positive control groups (MMS and CP) where a total number of 9.6 x 1E05 cells/concentration were plated in ten 96-well microtiter plates, each well containing 1000 cells in selective medium (trifluorothymidine-selection).

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

OTHER:
- Determination of polyploidy: not applicable
- Determination of endoreplication: not applicable
- Type and identity of media: horse serum was inactivated by incubiation at 56°C for at least 30 minutes. Basic medium: RPMI 1640 Hepes buffered medium (Dutch modificiation) containing penicillin/streptomycin (50 U/mL and 50 µg/mL, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin. Growth medium: basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium). Exposure medium: for 3 hour exposure: cells were exposed to the test substance in basic medium supplemented with 5% (v/v) heat-inactivated horse serum (R5-medium). For 24 hour exposure: cells were exposed to the test substance in basic medium supplemented with 10% (v/v) heat-inactivated horse serum (R10-medium). Selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20) and 5 µg/mL trifluorothymidine (TFT) (Sigma). Non-selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20).
- State of the suspension/solution according to the concentration: at a concentration of 0.12 mg/mL and higher zirconium dioxide was suspended in dimethyl sulfoxide (DMSO, SeccoSolv, Merck Darmdstadt, Germany). At concentration of 0.04 mg/mL and lower the test substance was dissolved in dimethyl sulfoxide. The stock solution was treated with ultrasonic waves to obtain a homogeneous suspension. Zirconium dixoide concentrations were used within 1 hour after preparation. The final concentration of the solvent in the exposure medium was 0.8% (v/v).

Evaluation criteria:

The global evaluation factor (GEF) has been defined as the mean of the negative/solvent mutation frequency distribution plus one standard deviation. For the micro well version of the assay the GEF is 126. A test substance is considered positive (mutagenic) in the mutation assay if it induces a mutation frequency of more then mutation frequency (controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range. A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study. A test substance is considered negative (not mutagenic) in the mutation assay if: a) non of the tested concentrations reaches a mutation frequency of mutation frequency (controls) + 126; b) the results are confirmed in an independent repeated test.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

first and second experiment

Cytotoxicity / choice of top concentrations:

no cytotoxicity

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:no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: Zirconium dioxide precipitated in the exposure medium at concentration of 100 µg/mL and above. Zirconium dioxide was tested beyond the limit of solubility to obtain adequate cytotoxicity data, the concentration used as the highest test substance concentration for the dose range finding test was 333 µg/mL
- Other confounding effects: no data

RANGE-FINDING/SCREENING STUDIES: L5178Y mouse lymphoma cells were treated with a test substance concentration range of 3 to 333 µg/mL in the absence of S9-mix with a 3 and 24 hour treatment period and in the presence of S9-mix with a 3 hour treatment period. After 3 hours of treatment: both in the absence and presence of S9-mix, no toxicity in the relative suspension growth was observed up to and including the highest test substance concentration of 333 µg/mL compared to the suspension growth of the solvent control. After 24 hours of treatment with various concentrations of Zirconium dioxide, no toxicity in the relative suspension growth was observed up to and including the highest test substance concentration of 333 µg/mL compared to the suspension growth of the solvent control.

COMPARISON WITH HISTORICAL CONTROL DATA: The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range.

ADDITIONAL INFORMATION ON CYTOTOXICITY: No toxicity was observed and all dose levels were evaluated in the absence and presence of S9-mix.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

The growth rate over the two-day expression period for cultured treated with DMSO was between 20 and 28 (3 hours treatment) and 40 and 50 (24 hours treatment).

Mutation frequencies in cultures treated with positive control chemicals were increased by 26- and 14 -fold for MMS in the absence of S9 -mix, and by 19 -fold for CP in the presence of S9 -mix, in the first and second experiment respectively. It was therefore concluded that the test conditions, both in the absence and presence of S9 -mix, were appropriate for the detection of a mutagenic response and that the metabolic activation system (S9 -mix) functioned properly. In addition the observed mutation frequencies of the positive control substances were within the acceptability criteria of this assay.

Experiment 1: Cytotoxic and mutagenic response of Zirconium dioxide in the mouse lymphoma L5178Y test system (3 hours treatment)

Without metabolic activation

dose (µg/mL)	RSG (%)	CE day2 (%)	RS day2 (%)	RTG (%)	Mutation frequency x 1E-06
					total	(small	large)
SC1	100	118	100	100	53	31	20
SC2	100	113	100	100	51	31	19
0.03	112	101	87	98	50	23	25
0.1	105	110	95	100	54	29	23
0.3	110	94	81	90	54	26	26
1	117	111	96	113	50	21	28
3	112	101	87	97	49	25	22
10	106	98	85	90	58	34	23
33	102	97	84	85	58	30	27
100 (1)	103	105	91	94	52	29	22
MMS	66	57	49	32	1334	804	318

With 8% (v/v) metabolic activation

dose (µg/mL)	RSG (%)	CE day2 (%)	RS day2 (%)	RTG (%)	Mutation frequency x 1E-06
					total	(small	large)
SC1	100	88	100	100	54	32	21
SC2	100	89	100	100	53	29	23
0.03	100	102	116	116	53	34	18
0.1	99	83	94	93	54	38	15
0.3	99	79	90	89	59	32	26
1	100	81	92	93	67	33	33
3	92	74	83	77	78	47	29
10	99	86	98	97	60	31	27
33	92	90	102	94	56	33	21
100 (1)	100	77	87	87	61	32	28
CP	53	72	82	44	1000	674	191

 

Note: all calculations were made without rounding off

RSG = Relative Suspension Growth; CE = Cloning efficiency; RS = Relative Survival; RTG = Relative Total Growth; SC = Solvent Control = DMSO; MMS = Methylmethanesulfonate; CP = cyclophosphamide

(1) zirconium dioxide precipitated in the exposure medium

Experiment 2: Cytotoxic and mutagenic response of Zirconium dioxide in the mouse lymphoma L5178Y test system (24 hours)

Without metabolic activation

dose (µg/mL)	RSG (%)	CE day2 (%)	RS day2 (%)	RTG (%)	Mutation frequency x 1E-06
					total	(small	large)
SC1	100	118	100	100	57	32	23
SC2	100	104	100	100	63	36	25
0.03	120	88	79	95	72	43	27
0.1	127	107	96	122	66	34	29
0.3	137	120	108	148	50	29	20
1	127	111	100	128	54	34	18
3	139	110	99	138	55	37	17
10	140	91	82	115	80	48	29
33	138	115	103	143	69	41	25
100 (1)	153	97	87	133	54	38	15
MMS	119	77	69	83	815	564	157

With 12% (v/v) metabolic activation:

dose (µg/mL)	RSG (%)	CE day2 (%)	RS day2 (%)	RTG (%)	Mutation frequency x 1E-06
					total	(small	large)
SC1	100	111	100	100	67	40	25
SC2	100	80	100	100	85	44	37
0.03	107	77	80	86	85	57	26
0.1	97	86	90	87	86	45	37
0.3	99	102	107	105	64	34	28
1	97	107	111	108	69	43	24
3	99	97	101	100	75	53	20
10	90	99	104	93	77	54	20
33	89	107	111	99	94	49	40
100 (1)	91	102	107	97	71	45	24
CP	42	54	56	24	1422	832	355

(1) = Zirconium dioxide precipitated in the exposure medium

Note: all calculations were made without rounding off

RSG = Relative Suspension Growth; CE = Cloning efficiency; RS = Relative Survival; RTG = Relative Total Growth; SC = Solvent control = DMSO; MMS = Methylmethanesulfonate; CP = Cyclophosphamid (1) = Zirconium dioxide precipitated in the exposure medium

Conclusions:

Interpretation of results (migrated information):
negative

In conclusion, zirconium dioxide is not mutagenic in the TK mutation test system under the specified experimental conditions.

Executive summary:

A mouse lymphoma test was performed according to OECD guideline 476. Mouse lymphoma L5178Y cells were exposed to 0.03, 0.1, 1, 3, 10, 33 and 100 µg/mL zirconium dioxide with and without metabolic activation. In a first experiment, cell cultures were exposed for 3 hours to zirconium dioxide in exposure medium in the absence and presence of S9-mix. In a second experiment, cell cultures were exposed to Zirconium dioxide in exposure medium for 24 hours in the absence of S9-mix and for 3 hours in the presence of S9-mix.

Zirconium dioxide tested negative in both experiments with and without metabolic activation. No cytotoxicity was observed and positive and vehicle controls were considered valid.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

• Concerning the Ames test

Tests in bacterial systems are not appropriate for metal due to a lack of sensitivity related to either probable mechanism of action or lack of metal uptake.The high prevalence of false negatives for metal compounds might suggest that mutagenesis essay with mammalian cells, as opposed to bacterial cells, would be preferred starting point.

 

• Concerning the in vitro mammalian cells tests

Although no data is available on the genotoxic potential of metallic hafnium, one may safely assume that metallic hafnium as such cannot cross cellular membranes, but instead only the hafnium cation, the active form. Therefore, read across with reference to information on the mutagenicity of other similar substances (like ZrO2) is clearly warranted.

 

An in vitro cytogenicity study in mammalian cells with ZrO2 (according to OECD 473 guideline) and vitro gene mutation study in mammalian cells with ZrO2 (OECD 476 guideline) were realised. The soluble Zr cation, the active form, was tested, so the results could be used to read across. The result of the two tests performed with ZrO2 was negative: under the experimental conditions, no chromosome aberration or gene mutation was induced in mammalian cells.

 

Justification for classification or non-classification

Based on the available data, the substance was not classified under the CLP Regulation 1272/2008 and the directive Classification and Labelling 67/548.