Trizinc bis(orthophosphate)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Not reported

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Not applicable

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

50, 150, 500, 1500 and 5000 µg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Test material was soluble in DMSO

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide, 4-nitro-o-phenylenediamine, 2-aminofluorene, 2-aminoanthracene, 9-aminoacridine hydrochloride monohydrate, N-methyl-N'-nitro-N-nitrosoguanidine

Details on test system and experimental conditions:

METHOD OF APPLICATION: In agar (plate incorporation)

NUMBER OF REPLICATIONS: Triplicate

Evaluation criteria:

no information

Statistics:

Not available

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

none

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

None

Conclusions:

Interpretation of results (migrated information):
negative

The test material was considered to be non-mutagenic for all the used bacterial strains (Salmonella typhimurium as well as Escherichia coli) with as well as without metabolic activation

Executive summary:

A study was conducted to determine the potential mutagenicity of the test material using bacterial reverse mutation assay (e.g. Ames test). 

Four indicator Salmonella typhimurium strains TA98, TA100, TA1535 and TA 1537 and one indicator Escherichia coli WP2 uvrA strain were treated with the test material suspended in dimethylsulfoxide using the plate incorporation method at doses of 50 -5000 µg/plate).

 

No significant increases in the frequency of revertant colonies were recorded at any dose level.

 

The test material was considered to be non-mutagenic under the conditions of this test.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Used in EU risk assessment report for zinc metal. Study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: human

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Test concentrations with justification for top dose:

Concentration in (ug/ml)
without metabolic activation 5-20
with metabolic activation 10-40

Vehicle / solvent:

none

Details on test system and experimental conditions:

No data

Evaluation criteria:

No data

Statistics:

No data

Species / strain:

lymphocytes: human

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

at 30 and 40 μg/ml

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 40 μg/ml

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

No data

None

Conclusions:

The information summarised in this study record was taken from the EU Risk Assessment report for zinc (EU RAR 2004) where it has been thoroughly reviewed. Due to the unavailability of the primary reference, the study was rated with reliability 4 for formal reasons. However, the results of this study will be used in the hazard assessment of the zinc category substances in a weight of evidence approach.

positive: with metabolic activation at 30 and 40 ug/ml.

Executive summary:

In an OECD 473 guideline study, zinc monoglycerolate was tested at concentrations of 5 -20 ug/ml without metabolic acitivation and 10 -40 ug/ml with metabolic activation in human lymphocytes. Zinc monoglycerolate was positive with metabolic activation at 30 and 40 ug/ml.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Not applicable

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Used in EU risk assessment for zinc metal. Study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

(No data of negetive control)

Principles of method if other than guideline:

Not applicable

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

No data

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

No data

Additional strain / cell type characteristics:

not specified

Metabolic activation:

not specified

Test concentrations with justification for top dose:

50-5000 µg/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

not specified

Details on test system and experimental conditions:

refer to reference

Evaluation criteria:

Positive response: Induction of reproducible dose related increases in the number of his+ revertants in the treated group compared to the control.

Statistics:

No data

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

None

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

None

Conclusions:

The information summarised in this study record was taken from the EU Risk Assessment report for zinc (EU RAR 2004) where it has been thoroughly reviewed. Due to the unavailability of the primary reference, the study was rated with reliability 4 for formal reasons. However, the results of this study will be used in the hazard assessment of the zinc category substances in a weight of evidence approach.

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

No toxicity observed up to 5000 ug/plate. The test material was considered to be non-mutagenic under the condition of this test.

Executive summary:

The study by Jones et al. (1994) was conducted to determine the potential mutagenicity of zinc monoglycerolate using bacterial reverse mutation assay. Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 were treated with the test material using the plate incorporation method at dose levels ranging from 50 to 5,000 µg/plate in triplicate in presence and absence of a metabolic activation system. No toxicity observed up to 5000 ug/plate. No significant increases in the frequency of his+ revertant colonies were recorded at the dose range tested. The test material was considered to be non-mutagenic under the conditions of this test. The information summarised in this study record was taken from the EU Risk Assessment report for zinc (EU RAR 2004) where it has been thoroughly reviewed. Due to the unavailability of the primary reference, the study was rated with reliability 4 for formal reasons. However, the results of this study will be used in the hazard assessment of the zinc category substances in a weight of evidence approach.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Used in EU risk assessment report for zinc metal. Study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

not specified

Type of assay:

mammalian cell gene mutation assay

Species / strain / cell type:

mouse lymphoma L5178Y cells

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Test concentrations with justification for top dose:

Concentration in (ug/ml)
without metabolic activation 1 - 15
with metabolic activation 1-30

Vehicle / solvent:

none

Details on test system and experimental conditions:

No data

Evaluation criteria:

No data

Statistics:

No data

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

15 μg/ml

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 30 μg/ml

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

from 10 μg/ml

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 15 μg/ml

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

No data

None

Conclusions:

The information summarised in this study record was taken from the EU Risk Assessment report for zinc (EU RAR 2004) where it has been thoroughly reviewed. Due to the unavailability of the primary reference, the study was rated with reliability 4 for formal reasons. However, the results of this study will be used in the hazard assessment of the zinc category substances in a weight of evidence approach.

Interpretation of results (migrated information):
positive

positive: without metabolic activation from 10 μg/ml
with metabolic activation from 15 μg/ml

Executive summary:

In an OECD 476 guideline study by Adams et al. (1994), zinc monoglycerolate was tested at concentrations of 1-15 µg/ml without metabolic activation and 1 -30 µg/ml with metabolic activation in mouse lymphoma cells. The results showed the following positive results: without metabolic activation from 10 μg/ml with metabolic activation from 15 μg/ml. The information summarised in this study record was taken from the EU Risk Assessment report for zinc (EU RAR 2004) where it has been thoroughly reviewed. Due to the unavailability of the primary reference, the study was rated with reliability 4 for formal reasons. However, the results of this study will be used in the hazard assessment of the zinc category substances in a weight of evidence approach.

Reference 5

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

study was conducted before the implementation of the OECD guideline for in vitro mutation in the tk locus

Deviations:

yes

Remarks:

toxicity not reported, no continuous 24 hrs treatment, no colony sizing performed

Principles of method if other than guideline:

Cells deficient in thymidine kinase (TK) due to the mutation TK+/- to TK-/- are resistant to the cytotoxic effects of trifluorothymidine (TFT). Thymidine kinase proficient cells (TK+/-) are sensitive to TFT, which causes the inhibition of cellular metabolism and halts further cell division. Thus mutant cells are able to proliferate in the presence of TFT, whereas normal cells, which contain thymidine kinase, are not able to proliferate.

GLP compliance:

not specified

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

ZnCI2 obtained from Mallinckrodt

Target gene:

Thymidine kinase locus/TK +/-

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 medium
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically checked for karyotype stability: No data
- Periodically "cleansed" against high spontaneous background: Yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

without

Metabolic activation system:

No data

Test concentrations with justification for top dose:

1.21-12.13 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Normal saline (1 %)
- Justification for choice of solvent/vehicle: Not reported

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Remarks:

None

Details on test system and experimental conditions:

Zinc chloride was first dissolved in normal saline and filter-sterilized, then immediately added to individual cell cultures containing 6X1E06 cells each in 10ml. Final solvent concentration was 1% in all cases. Light exposure was minimal and duration of exposure to either solvent only (controls) or test agents dissolved in that solvent was 3 h at 37 degrees Celsius in all cases and terminated by repeated washing of cells with Rs media. All cells were then maintained 48 h at 37 degrees Celsius in log phase growth to permit recovery and mutant expression.

48 h after treatment, metal-treated cells and solvent controls were cloned in soft-agar media containing RPMI-1640 medium, 10% horse serum (Microbiological Associates), no antibiotics, 1 mM sodium pyruvate, 0.02% (w/v) Pluronic F-68, and 0.37% Difco Bacto agar which had been purified by washing with acetone and distilled water. Cells suspended in this medium were plated into Fisher 100-mm petri dishes as previously described (Amacher et al., 1979). Final cell densities were 30000/ml (1 X 106 /plate) for mutant selection and 15/ml (500/plate) for viability determinations. Trifluorothymidine resistance (TFTres) was determined by adding 4 µg/ml TFT to one set of plates. Stock TFT had been dissolved in saline as a 100X solution and stored frozen in the dark until 30 min before use. Plates were incubated at 37 degrees Celsius in a humid 5% CO2 95% air atmosphere. All colonies growing either in the presence of TFT (TFTres) or its absence (viable count colonies) were counted on day 7 with a Artek model 870 bacterial Colony Counter.

Evaluation criteria:

Colonies growing in the presence of triflurothymidine (TFT resistant) or its absence (viable count colonies) were counted. TFT Resistant colonies which were equivalent in size to colonies growing in the solvent control viable count plates ie., large, were scored as mutants.

Statistics:

Not reported

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

the highest concentration showed less than 20% cell survival

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Remarks:

reported in a previous publication

Additional information on results:

average background mutation frequency and absolute cloning efficiency for the solvent controls used in these experiments were: 0.46 ± 0.16 and 119 ± 13%, resp.
positive control results with methyl methanesulfonate (MMS) are reproted as given in a previous publication, a significant and dose-dependent increase of the mutation frequency in L5178Y was seen when treated with concentrations ranging from 0.016-0.12 mM (Clive, D. (1973) Recent developments with the L5178Y TK-heterozygote mutagen assay system, Environ. Health Perspect., 6, 119-125.)

Graph showing Average trifluorothymidine resistance (TFTRes) mutant counts versus test material has been attached as 'attached background material'.

Conclusions:

Interpretation of results (migrated information):
negative

The test material was found to be non-mutagenic under the test conditions.

Executive summary:

Amacher and Paillet (1980) tested zinc chloride for induction of tk mutations in mouse lymphoma cells. Treatment was only for 3 hrs in the absence of metabolic activation. Concentrations ranged from 1.21 -12.13 µg/mL, but there is no indication what levels of toxicity (if any) these treatments induced. There were no increases in mutant frequency as a result of these treatments. However, at the time this study was conducted there was no optimisation of the assay to detect small colony mutants (which would be expected to be representative of clastogenic or aneugenic modes of action), and no continuous 24 hr treatment in the absence of metabolic activation, as is now routine. Zinc dichloride was found to be non-mutagenic under the test conditions.

Reference 6

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Not reported

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Study well documented, meets generally accepted scientific principles, acceptable for assessment.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

no guideline followed

Principles of method if other than guideline:

Chromosome aberrations were studied in human lymphocyte cultures using three subtoxic doses of test material added to 48 and 72 h cultures at 0 and 24 h after initiation. Metaphases from each culture were evaluated for the presence of numerical and structural aberrations.

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

- Type and identity of media: Ham's F 10 media

Additional strain / cell type characteristics:

not specified

Metabolic activation:

not specified

Metabolic activation system:

No data

Test concentrations with justification for top dose:

3 X 10-3, 1.5 X 10-3, 3 X 10-5 and 3 X 10-4 M

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: no data

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Details on test system and experimental conditions:

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER: Test material was added to 48 and 72 h cultures at 0 and 24 h after initiation.

Evaluation criteria:

100 well-spread metaphases from each culture were evaluated for the presence of numerical and structural aberrations.

Statistics:

Chi-square test was used to compare the incidence of dicentrics in the cells treated with zinc and controls.

Species / strain:

lymphocytes: Human

Metabolic activation:

not applicable

Genotoxicity:

ambiguous

Remarks:

Chromosome aberration observed only at the lowest concentration tested, since higher concentrations were unsuitable for the analysis due to cytotoxicity. However, the findings were not significant statistically when compared to the controls only.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

None

Table 1: Chromosome analysis of leucocyte cultures contaminated by zinc chloride

Duration of the culture (h)	Dose of salt administered (M)	Interval of time between initiation of the culture and salt administration (h)	Number of cells analysed	Numerical aberrations		Structural aberrations	Type and number of structural aberrations
				Aneuploid cells	Cells in endoreduplication		Chromatid aberrations	Chromosome aberrations
							Gap	Gap	Fragment	Dicentric
48	0	-	100	3	0	1	1
	3 X 10^-4	0	100	1	0	2	2
		24	100	4	0	4	2		2
	3 X 10^-5	0	100	4	0	3	3			1
		24	100	4	0	4	2		2
72	0	-	100	2	0	3	3
	3 X 10^-4	0	100	3	0	0
		24	100	6	0	4	3		1
	3 X 10^-5	0	100	4	0	5	3			2
		24	100	2	1	3	2	1

Conclusions:

The information summarised in this study record was taken from the EU Risk Assessment report for zinc (EU RAR 2004) where it has been thoroughly reviewed. Due to the unavailability of the primary reference, the study was rated with reliability 4 for formal reasons. However, the results of this study will be used in the hazard assessment of the zinc category substances in a weight of evidence approach.

Interpretation of results (migrated information):
ambiguous

Chromosome aberrations (dicentric chromosomes) were observed at the lowest concentration of 3 X 10-5 M of the test material . However, the results were found to be insignificant when compared to only controls in chi-square analysis.

Executive summary:

A chromosome aberration test was conducted on human lymphocyte cultures to determine the mutagenic potential of Zinc chloride.

The concentration of zinc chloride inhibiting mitotic activity was found to be 3 X 10-3 M. Three subtoxic doses i.e., 1.5 X 10-3, 3 X 10 -5 and 3 X 10-4 M were taken for the study. Human lymphocytes were obtained from a healthy donor and cultured for 48 or 72 h in Ham's F 10 medium. The test material was added to 48 and 72 h cultures at 0 and 24 h after initiation. Chromosome preparations were prepared and 100 well-spread metaphases from each culture were evaluated for the presence of numerical and structural aberrations.

Chromosome aberrations (dicentric chromosomes) were observed at the lowest concentration of 3 X 10-5 M of the test material . However, the results were found to be insignificant when compared to only controls in chi-square analysis.

Reference 7

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

not specified

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

Based on the following shortcomings the reference is considered not reliable and therefore disregarded for the hazard assessment: Test item is insufficiently characterised; cytotoxicity not evaluated using the recommended parameters (OECD TG 487, 2016); cytotoxicity results not shown; micronucleus test performed in a cell line, which is not recommended by OECD TG 487 (2016); description of methodology lacks some details; concentrations specified in µg/mL; no information on potential effects on pH and osmolality; information on evaluation criteria are not specified; historical control data missing

Qualifier:

no guideline followed

Principles of method if other than guideline:

Kononenko, V. et al. (2017) analysed the chromosomal damage in the MDCK (Madin-Darby Canine Kidney) cell line after treatment with ZnO NP, ZnO macroparticles (MPs), and ZnCl2. The chromosomal damage was evaluated in a cytokinesis-block micronucleus using cytochalasin B. At least 500 randomly selected cells per experiment were scored for micronucleus occurrence. For each treatment, at least nine experimental repeats were performed. The cell viability was examined in by calculation of the nuclear division index (NDI). In addition, cytotoxicity of the test materials was evaluated in separate experiments utilising the MTT, NRU, and the trypan blue exclusion assay. Moreover, the ZnO NP dissolution in the cell culture medium was analysed. In addition, activity of catalase and glutathione-S-transferase were measured, and ROS generation induced by the test materials was analysed in a cell-free suspension.

GLP compliance:

not specified

Remarks:

publication

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

not applicable

Species / strain / cell type:

other: MDCK (Madin-Darby Canine Kidney cell line)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells:
epithelial cells (Institute of Cell Biology, University of Ljubljana; Slowenien)

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature: Cultured in 1:1
(v/v) mixture of Dulbecco's modified Eagle's medium (DMEM) and F-12K medium. Medium was supplemented with 4 mM L-glutamine and 5% (v/v) FBS. Cellswere grown at 37 °C in a humidified atmosphere with 5% CO2.

Cytokinesis block (if used):

Cytochalasin B (4 µg/mL)

Metabolic activation:

not specified

Test concentrations with justification for top dose:

12, 61, and 123 µM (corresponding to 1, 5, and 10 µg/mL)

Vehicle / solvent:

- Vehicle used: culture medium

Untreated negative controls:

yes

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Details on test system and experimental conditions:

MICRONUCLEUS TEST
MDCK cells were plated in 12-well plates (1.2x10^4 cells/cm²) with previously inserted sterile cover glasses. After 24 h incubation, when cells adhered to the cover glasses, the medium was replaced with sub-cytotoxic concentrations of ZnO NPs, ZnO MPs or ZnCl2 (12, 61, and 123 μM) prepared in cell culture medium. MMS solution (0.45 μM) was used as a positive control for genotoxicity. After the 24 h treatment, cells on coverslips were rinsed with DPBS and fresh cell culture medium with 4 μg/mL cytochalasin-B was added to cells in order to arrest cytokinesis. After 24 h incubation, cells on coverslips were fixed in the 25% v/v acetic acid in methanol and washed with DPBS. Cells were stained with 1.5 μg/mL Hoechst 33258 and 1 μg/mL propidium iodide. Coverslips with cells were placed on microscopic slides and examined by epifluorescent microscope. At least 500 randomly selected binucleated cells per sample were analysed for micronuclei presence on the basis of scoring criteria described previously*. In order to provide information on the proliferative status of viable cells and on the cytotoxicity, the frequency of non-viable cells (apoptotic or necrotic cells) and of mononucleated, binucleated, trinucleated and tetranucleated cells were evaluated. For each treatment condition at least 9 independent experimental repeats were performed.

CYTOTOXICITY
H2O2 (0.5 mM) was used as a positive control for all cytotoxicity assays. Cells were seeded in 96/12-well plates at a density of 2.2× 10^4 cells/cm² and incubated for 24 h to allow attachment.
- In the modified MTT assay, the cells were treated with ZnO NPs, ZnOMPs and equimolar concentrations of ZnCl2, suspended in cell culture medium with final concentrations of 12, 61, 123, 184, 369, and 737 μM (which correspond to 1, 5, 10, 15, 30, and 60 μg/mL ZnO and 1.67, 8.37, 16.7, 25.1, 50.2, 100 μg/mL ZnCl2, respectively). After 24 h incubation, MTT (final concentration 0.5 mg/mL) was added, and after 3 h incubation the formed formazan crystals were diluted with DMSO and measured spectrophotometrically. For each treatment condition at least 4 independent experimental repeats of 5 replicates were performed.
- In the modified NRU assay, cells were treated for 24 h with suspensions of ZnO NPs, ZnO MPs or ZnCl2 solutions prepared in cell culture medium (12, 61, 123, 184, 369, 737 μM). After incubation, neutral red dye was added (final concentration 0.04 mg/mL) and cells were incubated for 2 h, allowing dye to become trapped inside acid organelles, then rinsed with DPBS, followed by releasing the internalized dye by a prepared solvent (50% v/v ethanol, 1% v/v acetic acid and 49% v/v deionized water). Fluorescence of released neutral red dye was measured spectrophotometrically. For each treatment condition at least 4 independent experimental repeats, each with 5 replicates, were performed.
- In the trypan blue exclusion assay, the medium was then replaced with ZnO NPs, MPs or ZnCl2 prepared in cell culture medium (12, 61, 123, 184, 369, 737 μM) in at least 6 independent replicates. After 24 h incubation, cells were harvested (Trypsin/EDTA) and stained with 0.2% (w/v) trypan blue solution. Evaluation of cell viability was performed using microscopic observation (100–200 cells/sample). For each treatment condition at least 6 independent experimental repeats were performed.

DISSOLUTION ASSAY
ZnONP suspensions in the cell culture medium were prepared at different concentrations (0, 61, and 123 μM). After 24-h incubation, 8 mL of cell medium samples were ultra-centrifuged at 100000 rcf for 30 min at 20°C. The supernatant from each sample was divided into two aliquots. 750 μL from each of the first aliquots were acidified with 750 μL 65% HNO3 and samples were subjected to closed-vessel digestion. Digestion was conducted at 200°C and 700 W power, with step 1 (heating) lasting 15 min, step 2 (constant temperature) lasting 15 min, and 45 min cooling to 60°C. Total Zn concentrations in the digests were measured by flame atomic absorption spectroscopy. The second aliquots were left unacidified and were analysed by square-wave anodic stripping voltammetry. The measurements were performed using the modular voltammetric analyzer Autolab PGSTAT. The usual three-electrode configuration was employed with the bismuth film electrode prepared in situ on a substrate glassy carbon disk electrode (d=2 mm) as the working electrode, a platinum wire as the counter electrode, and a double-junction saturated Ag/AgCl/KCl (sadt.) reference electrode containing 0.1 M HNO3 as the outer electrolyte. A computer-controlled magnetic stirrer rotating at approximately 300 rpm was employed during the accumulation and cleaning step. All experiments were carried out at 23±1°C in a 20 mL one-compartment voltammetric cell. A mixture of 0.1 M piperazine-N,N′-bis(2-ethanesulfonic acid) and 0.1 M KNO3 was used as the supporting electrolyte. The pH was adjusted to 6.5 with 0.5 M KOH. Water used throughout the work was first deionized and then further purified using Elix 10/Milli-Q Gradient unit. 100–300 μL of the samples were added to the supporting electrolyte before the measurements. Following an electrochemical accumulation step of 120 s at −1.6 V, and subsequent equilibration period of 15 s, an anodic stripping voltammogram was recorded in a quiescent solution by scanning the potential towards more positive values using a square-wave potential scan from −1.6 V to +0.4 V vs. the reference electrode, with a frequency of 25Hz, a potential step of 4mV, and an amplitude of 50mV. Before each measurement, a cleaning step was carried out by applying the potential of +0.3 V for 30 s. The non-particulate Zn species were measured by the method of three successive standard additions and their concentrations were calculated by linear regression.

CATALASE AND GLUTATHIONE-S-TRANSFERASE ACTIVITY ASSAYS
MDCK cells were seeded in 6-well plates (2.2x10^4 cells/cm²) and allowed to attach and grow for 24 h. The cell culture medium was then replaced with the test material at concentrations of 12, 61, and 123 μM, prepared in cell culture medium. H2O2 solution (0.5mM) was used as a positive control for the impairment of the enzymatic function. After 24 h incubation, cells were washed, detached (using a silicon-based cell scraper), centrifuged (300 g, 10 min), then the pellet was resuspended in 300 μL 0.1% Triton X-100 and homogenized by probe sonication for 1.5 min, with 15 s on-off cycles at a 40% amplitude (cooled on ice). The total protein concentration in the cell homogenates was measured by Pierce Bicinchoninic Acid (BCA) Protein Assay Kit (Life Technologies, Carlsbad, USA), according to the manufacturer's protocol.
- In the CAT activity assay, the assay mixture containing 950 μL of 0.0367 M H2O2 in DPBS and 50 μL of the cell homogenate was pipetted into the UV-light transparent cuvette. The rate of H2O2 decomposition was measured by monitoring the decrease in absorbance at 240nmfor 3 min. CAT activity per total cellular protein mass was calculated using an extinction coefficient for H2O2 (Ɛ240=43.6mM^−1 cm^−1). Results are expressed as a % activity of negative control. For every treatment condition at least 6 independent repeats of 3 replicates were performed.
- In the GST activity assay, the reaction mixture, consisted of 1 mM EDTA in DPBS (pH = 6.5), 1 mM glutathione (GSH) and 1 mM 1-chloro-2,4-dinitrobenzene (CDNB), was added to cell homogenate in each well of the transparent 96-well plate. Formation of the CDNBGSH conjugate, reaction catalyzed by GST, was monitored for 6 min by spectrophotometrically. GST activity per mass of total cellular protein was calculated using extinction coefficient for CDNB-GSH conjugate (Ɛ340 =0.0096 μM^−1 cm^−1). Results were expressed as a % activity of negative control. For each treatment condition at least 6 independent repeats of 3 replicates were performed.

ROS GENERATION
DCFH-DA was chemically hydrolysed to 2′,7′-dichlorofluorescein (DCFH) by mixing 1 mL 1 mM DCFH-DA with 9 mL 10 mM NaOH for 30 min. Reaction was stopped by neutralization with 20 mL of DPBS. Then, 100 μL of ZnO NPs, ZnO MPs or ZnCl2 prepared in cell culture medium and 50 μL of 33 μM DCFH was added to black 96-well plates. After 24 h incubation, the fluorescence of oxidised DCFH(DCF) was measured spectrophotometrically. For the negative control, only cell culture medium was added to the wells, and 0.025 mM H2O2 was used as a positive control. For every treatment condition at least 5 independent repeats of 3 replicates were performed.

*References
- Fenech, M., 2007. Cytokinesis-block micronucleus cytome assay. Nat. Protoc. 2, 1084–1104.

Evaluation criteria:

not specified

Statistics:

Results from the comet assay were statistically analysed by one-way analysis of variance (ANOVA), followed by Dunnett's multiple comparison test.

Species / strain:

other: MDCK (Madin-Darby Canine Kidney cell line)

Metabolic activation:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

MICRONUCLEUS TEST
- Compromised cell viability and cytostatic effects were not observed for ZnO NP, ZnO MP, and ZnCl2 at the concentrations tested.
- MDCK cells showed statistically significantly increased number of micronuclei per 1000 binucleated only after treatment with ZnO NPs at the two highest concentrations tested. Neither ZnO MPs nor ZnCl2 induced statistically significant micronuclei formation.

CYTOTOXICITY
In the MTT and NRU assays, all three zinc test materials induced no significant toxicity up to a concentration of 123 µM. The grade of cytotoxicity was comparable between the test materials. In the trypan blue exclusion assay, significant cytotoxicity was evident only at 369 and 737 µM. Notably, ZnCl2 treatment resulted in a complete loss of cell membrane stability already at a concentration of 369 µM.

DISSOLUTION ASSAY
Dissolution of the ZnO NPs was found to be high. About 50% of Zn was present in non-particulate form.

CATALASE AND GLUTATHIONE-S-TRANSFERASE ACTIVITY ASSAYS
- GST activity was significantly reduced only after treatment with ZnO NPs at the highest concentration tested, when compared to untreated control. Exposure to ZnO MPs and ZnCl2 did not result in a significant response.
- Catalase activity showed a concentration-related and significant reduction after ZnO NP exposure at 61 and 123 µM. Moreover, ZnCl2 resulted in a slight but significant decrease in catalase activity at the highest concentration tested, when compared to the untreated control.

ROS GENERATION
- ROS levels were not significantly changed by any of the test materials examined, as evidenced by DCF fluorescence after test material treatment in a cell-free environment.

Remarks on result:

not determinable because of methodological limitations

Conclusions:

Kononenko, V. et al. (2017) examined potential chromosomal damage induced by ZnO NPs, ZnO macroparticles (MPs), and ZnCl2 treatment in MDCK cells. The chromosomal damage was evaluated in a cytokinesis-block micronucleus test. Cell viability and cytostatic effects were examined concurrently by analysis of the nuclear division index and scoring of apoptotic or necrotic cells. Moreover, cytotoxicity was investigated utilising the MTT, NRU, and trypan blue exclusion assays. In further experiments, the authors examined ROS generation, ZnO NP dissolution, and the activity of catalase and glutathione-S-transferase (GST) after test material treatment.

According to the authors, cell viability and proliferation were not affected under the conditions tested in the micronucleus test. Chromosomal damage was only increased after treatment with ZnO NPs at concentrations of 61 and 123 µM, when compared to untreated controls. Moreover, the test materials induced statistically significant cytotoxicity at concentration of 184 µM in the MTT and NRU assay, whereas the trypan blue exclusion assay indicated considerable cytotoxicity at concentrations of 360 µM and above. GST activity was statistically significantly decreased only after ZnO NP treatment, whereas catalase activity was reduced in treatment with both ZnO NPs and ZnCl2. However, catalase activity was only slightly impaired after ZnCl2 treatment. Acellular measurement of ROS indicated no significant in ROS levels in cell culture media after the 24 h incubation with either ZnO NPs, ZnO MPs, or ZnCl2.

No conclusion can be drawn due to reporting and methodological deficiencies.

The test item is insufficiently characterised, since information on the purity, impurity elements and surface modifications are missing. Cytotoxicity was not evaluated using parameters recommend by the current test guideline (OECD TG 487, 2016) and the results of the NDI calculation and apoptosis/necrosis evaluation are not shown. The micronucleus test was performed in a cell line, which is not recommended by the current test guideline. Moreover, details on the cell lines, including cell doubling time, are missing. The description of the micronucleus test lacks some details. Furthermore, the authors did not state on precipitation and distribution of ZnO NPs during the treatment. Potential effects on pH or osmolality of the culture medium induced by ZnO NPs were not discussed. Information on evaluation criteria are unclear or missing. Exposure concentrations are stated in µg/mL; however, the cell line is composed of adherent cells, and thus, it would be appropriate to specify exposure concentrations in µg/cm². Cellular uptake of the test material was not investigated. Historical control data is not provided. The micronucleus frequency was only slightly increased and micronucleus frequencies show considerable variability within groups.

Based on the above-mentioned shortcomings the reference is considered not reliable.

Reference 8

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Not applicable

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

This assay detects agents that interact with cellular DNA to produce growth inhibition or killing. This interaction is recognized by specific cellular repair systems. The assays are based upon the use of paired bacterial strains that differ by the presence or absence of specific DNA repair genes. The response is expressed in the preferential inhibition of growth or the preferential killing of the DNA repair deficient strain since it is incapable of removing certain chemical lesions from its DNA.

GLP compliance:

not specified

Type of assay:

Bacillus subtilis recombination assay

Target gene:

Not applicable

Species / strain / cell type:

bacteria, other: Bacillus subtilis

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

other: H17 Rec+ (rec+ arg try) and M45 Rec- (rec45 arg try)

Metabolic activation:

not specified

Test concentrations with justification for top dose:

Not reported

Vehicle / solvent:

Not reported

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- Culture medium: Each thawed culture cells previously kept at -80 °C in broth culture (3 mL) with 50 % glycerol supplementation (1 mL), were streaked on the dry surface of broth agar (15 g/L of agar added to the B-2 broth)
- Test material: As impregnation on paper disks (16 mm diameter), over the starting points of the streaks


DURATION AND TEMPERATURE:
- Preincubation period: 24 h at 4-5 °C
- Incubation period: 20 h at 37 °C
- Exposure duration: 44 h


DETERMINATION OF GENOTOXICITY
- Method: Length of zone of inhibition

Evaluation criteria:

Not reported

Statistics:

Not reported

Species / strain:

bacteria, other: Bacillus subtilis

Metabolic activation:

not specified

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

None

None

Conclusions:

The information summarised in this study record was taken from the EU Risk Assessment report for zinc (EU RAR 2004) where it has been thoroughly reviewed. Due to the unavailability of the primary reference, the study was rated with reliability 4 for formal reasons. However, the results of this study will be used in the hazard assessment of the zinc category substances in a weight of evidence approach.

Interpretation of results (migrated information):
negative

Under the given test conditions, zinc chloride was found to be negative in B.subtilis recombination assay

Executive summary:

A Bacillus subtilis recombination assay (rec-assay) was conducted to evaluate the potential genotoxicity of the test material using H17 Rec+ (rec+ arg try) and M45 Rec- (rec45 arg try) strains. No guideline or GLP compliance was documented in the study report.

Thawed culture cells, previously kept at -80 °C in broth culture with 50 % glycerol supplementation, were streaked on the dry surface of broth agar. Impregnated paper disks (16 mm diameter) with test material solution were placed over the starting points of the streaks. The plates were first kept at 4-5 °C for 24 h, and then incubated at 30 °C for about 20 h. Then the length of the inhibition zone was measured. 

The test material did not show an increased lethal activity on Rec- in comparision with Rec+ cells and therefore did not cause cellular damage.

Under the given test conditions, the test material was found to be negative in B.subtilis recombination assay.

Reference 9

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study well documented, meets generally accepted scientific principles, acceptable for assessment.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

no guideline followed

Principles of method if other than guideline:

The clastogenic activity of zinc chloride was determined by studying chromosome aberrations in human dental pulp cells in vitro, both in the presence and absence of exogenous metabolic activation.

GLP compliance:

no

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

other: Human dental pulp cells (D824 cells)

Details on mammalian cell type (if applicable):

- Dental pulp tissue obtained from a lower third molar extracted from a 22 years old woman
- Type and identity of media: α-minimum essential medium supplemented with 20 % fetal bovine serum (FBS), 100 µM L-ascorbic acid phosphate magnesium salt n-hydrate, 2 mM L-glutamine, 0.22% NaHCO3 and 100 µg/mL streptomycin

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

colcemid, conc. not reported

Metabolic activation:

with and without

Metabolic activation system:

5% rat liver postmitochondrial supernatant (PMS) mixture

Test concentrations with justification for top dose:

30, 100 and 300 µM

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (60mM)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

None Migrated to IUCLID6: 50 µM

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: Overnight
- Exposure duration: 3 h

NUMBER OF REPLICATIONS: Triplicate

NUMBER OF CELLS EVALUATED: 1.6×10 (5) cells/dish

DETERMINATION OF CYTOTOXICITY: Yes
- Method: The cytotoxicity of the test material was determined as the number of cells treated with the test material relative to the number of cells in the control cultures×100

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes

OTHER: The pH range of the culture media containing the highest concentrations of test agents was approximately 7.2–7.5.

Evaluation criteria:

No data

Statistics:

χ2-test was used to assess the significance of the difference in the incidences of chromosome aberrations between control cultures and cultures treated with test agents. The level of significance in the statistical analysis was determined at p<0.05.

Species / strain:

other: Human dental pulp cells (D824 cells)

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:

No data

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1: Chromosome aberrations in D824 cells induced by treatment with ZnCl2 for 3 h and 30 h:

Test material	Time	Concentration	Relative cell number (%)	Number of metaphases scored	Type of structural aberrations(a) (%)								Aberrant metaphases (%)	Polyploidy and endoreduplication (%)
					ctg	csg	ctb	csb	cte	D	O	F
Control	3 h	0	100	500	0.8	0	0	0	0	0	0	0	0.8	2
Zinc chloride (µM)		30	91	100	0	0	0	0	0	0	0	0	0	0
		100	74	100	1	0	0	0	0	0	0	0	1	1
		300	77	100	0	0	0	0	0	0	0	0	0	1
Control	30 h	0	100	500	1	0	0	0	0	0	0	0	1	2
Zinc chloride (µM)		30	85	100	0	0	0	0	0	0	0	0	0	2
		100	77	100	1	0	0	0	0	0	0	0	1	2
		300	74	200	3.5	0	0.5	0	0	0	0	0	4	0

a = ctg, chromatid gaps; csg, chromosome gaps; ctb, chromatid breaks; csb, chromosome breaks; cte, chromatid exchanges; D, dicentric chromosomes; O, ring chromosomes; F, fragmentations.

 

Conclusions:

Interpretation of results (migrated information):
negative

Under the test conditions, the test material was considered to be non-clastogenic to human dental pulp cells in vitro.

Executive summary:

A study was conducted to investigate the ability of Zinc chloride to induce chromosome aberrations in human dental pulp cells.

Human dental pulp cells (D824 cells) treated with the test material, were evaluated for chromosome aberrations at up to 3 dose levels, together with vehicle and positive controls. Rat liver (5%) post mitochondrial supernatant mixture was used as the exogenous metabolic activator. Ability to induce chromosome aberrations was examined in cells treated with test material for 3 and 30 h.

The test material failed to induce chromosome aberrations in the presence or absence of exogenous metabolic activation. The percentages of cells with polyploid or endoreduplication were not enhanced by test material.

 

Under the test conditions, the test material was considered to be non-clastogenic to human dental pulp cells in vitro.

Reference 10

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Not reported

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Not applicable

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

50, 150, 500, 1500 and 5000 µg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Test material was soluble in DMSO

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide, 4-nitro-o-phenylenediamine, 2-aminofluorene, 2-aminoanthracene, 9-aminoacridine hydrochloride monohydrate, N-methyl-N'-nitro-N-nitrosoguanidine

Details on test system and experimental conditions:

METHOD OF APPLICATION: In agar (plate incorporation)

NUMBER OF REPLICATIONS: Triplicate

Evaluation criteria:

no information

Statistics:

Not available

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

none

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

None

Conclusions:

Interpretation of results (migrated information):
negative

The test material was considered to be non-mutagenic for all the used bacterial strains (Salmonella typhimurium as well as Escherichia coli) with as well as without metabolic activation

Executive summary:

A study was conducted to determine the potential mutagenicity of the test material using bacterial reverse mutation assay (e.g. Ames test). 

Four indicator Salmonella typhimurium strains TA98, TA100, TA1535 and TA 1537 and one indicator Escherichia coli WP2 uvrA strain were treated with the test material suspended in dimethylsulfoxide using the plate incorporation method at doses of 50 -5000 µg/plate).

 

No significant increases in the frequency of revertant colonies were recorded at any dose level.

 

The test material was considered to be non-mutagenic under the conditions of this test.

Reference 11

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

zinc oxide, >99% pure, Kanto Chemical

Species / strain / cell type:

other: Syrian hamster embryo cells

Details on mammalian cell type (if applicable):

SHE cell cultures were grown as described in references:
Tsutsui T, Barrett JC. Detection of non-mutagenic carcinogens using cultured Syrian hamster embryo cells. AATEX (Alternatives to Animal Testing and Experimentation). 1991;1:65–73.

Tsutsui T, Hayashi N, Maizumi H, Huff J, Barrett JC. Benzene-, catechol-, hydroquinone- and phenol-induced cell transformation,
gene mutations, chromosome aberrations, aneuploidy, sister chromatid exchanges and unscheduled DNA synthesis in Syrian hamster embryo cells. Mutat Res. 1997;373:113–123.

Cytokinesis block (if used):

Colcemid

Metabolic activation:

without

Test concentrations with justification for top dose:

Concentration in (uM)
0, 60, 120, 180

Vehicle / solvent:

none

Details on test system and experimental conditions:

Zinc oxide was dissolved in 0.1 N HCl at 50 mM and filter-sterilized.
Cytotoxicity of the chemical agents tested was determined by the colony-forming efficiencies of SHE cells treated with these agents. SHE cells (5 x 105) in tertiary culture were plated into 75-cm2 flasks (Costar, Cambridge, MA, USA), incubated overnight, and treated with each of 14 chemical agents at varying concentrations for 24 h. After harvesting with 0.1% trypsin, the cells were replated in triplicate onto 100-mm dishes (Costar) at 2000 cells/dish and incubated for 7 days for colony formation. The relative colony-forming efficiency was expressed as the number of colonies in the treated dishes divided by the number in the control dishes x 100. Actual colony-forming efficiency of control cells was 13.0 +/- 0.7% (S.D.).

Evaluation criteria:

Cytotoxicities determined by the colony-forming efficiencies of SHE cells

Statistics:

Chi Square test

Species / strain:

other: Syrian hamster embryo cells

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

No data

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

None

Conclusions:

Hikiba et al. (2005) tested zinc oxide in an in vitro chromosomal aberration test in SHE cells. Zinc oxide was dissolved in 0.1N hydrochloric acid and incubated at concentration of 0, 60, 120 and 180 mM (corresponding to concentrations of 4.8, 9.6 and 19.2mg/L) for 24 hrs. The highest concentration caused 91% cytotoxicity, thus only 2 concentration could be evaluated. A statistical significant increase in chromosome breaks was observed in the mid concentration (P<0.05) at acceptable cytotoxicity. No positive control was used. No explanation was provided why zinc oxide was dissolved in HCl prior testing. No confirmatory experiment was performed and the results were obtained from a single culture. Experimental conditions and chromosome preparation is insufficiently described.

Executive summary:

To assess the genotoxicity of 14 chemical agents used in dental practice, the ability of these agents to induce chromosome aberrations was examined using Syrian hamster embryo (SHE) cells. Statistically significant increases in the frequencies of chromosome aberrations were induced in SHE cells treated with 7 of 10 chemical agents used as endodontic medicaments, that is, carbol camphor, m-cresol, eugenol, guaiacol, zinc oxide, hydrogen peroxide, and formaldehyde. The other 3 chemical agents, that is, thymol, glutaraldehyde, and iodoform, did not increase the levels of chromosome aberrations. Of the 4 chemical agents that are used as an antiseptic on the oral mucosa, chromosome aberrations were induced by iodine, but not by the other 3 antiseptics, benzalkonium chloride, benzethonium chloride, and chlorhexidine. Among the 6 chemical agents exhibiting a negative response in the assay, only thymol induced chromosome aberrations in the presence of exogenous metabolic activation. The authors conclude that chemical agents having a positive response in the present study are potentially genotoxic to mammalian cells and need to be studied further in detail.

Reference 12

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

not specified

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

Li, C.-H. et al. (2012) investigated on the mutagenic potential of ZnO using the bacterial reverse mutation assay. ZnO was tested, up to the limit dose, in Salmonella tester strains TA 98, TA 100, TA 102, TA 1535, and TA 1537 both in absence and presence of a metabolic activation system. The assay was performed according to the plate incorporation assay. Three independent experiments were performed.

GLP compliance:

not specified

Remarks:

publication

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine operon genes

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction*

*References:
- Cheng YW, Lee WW, Li CH, Lee CC, Kang JJ. 2004. Genotoxicity of motorcycle exhaust particles in vivo and in vitro. Toxicol Sci 81:103–111.
- Maron DM, Ames BN. 1983. Revised methods for the Salmonella mutagenicity test. Mutat Res 113:173–215.

Test concentrations with justification for top dose:

0.312, 0.625, 1.25, 2.5, and 5.0 mg/plate

Vehicle / solvent:

- Vehicle used: sterile water

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 4-Nitro-o-phenylenediamine

Remarks:

Positive control in w/o S9 plate: TA98, 2.5 µg/plate

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Positive control in w/o S9 plate: TA100 and TA1535, 5 µg/plate

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Positive control in w/o S9 plate: TA102, 0.5 µg/plate

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Positive control in w/o S9 plate: TA1537, 50 µg/plate.

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-aminoanthrene

Remarks:

Positive control in w/w S9 plate: TA100/TA102/TA1535/TA1537, 5 µg/plate.

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

Positive control in w/w S9 plate: TA98, 5 µg/plate

Details on test system and experimental conditions:

AMES TEST
Salmonella typhimurium histidine auxotrophs, TA98, TA100, TA1537, TA1535, and TA102 were purchased from Molecular Toxicology Inc. (Moltox, USA). Mutagenicity was determined by incorporation method with the presence and absence of S9 metabolic activation. A 100 mL of each test bacterial culture (10^9 cells/mL), 2 mL of soft agar (0.6% agar, 0.5% NaCl, 5 mM histidine, and 50 mM biotin, pH 7.4, 40~50°C), 0.5 mL S9 mixture (if necessary), and ZnO-MPs were mixed well in a test tube. Subsequently, the sample was immediately poured onto a minimal agar plate (1.5% agar, Vogel–Bonner E medium containing 2% glucose). Plates were incubated in an incubator for 48 h at 37°C in the dark. The revertant colonies were counted macroscopically.

Evaluation criteria:

A compound was considered positive for mutagenicity only when i) the number of revertants was at least double the spontaneous yield, ii) a statistical significance (p ≤ 0.05) was found, and iii) a reproducible positive dose-response was present.

Statistics:

An one-way ANOVA was perfomred to test for statistical difference in revertant colony number between treatment and solvent control groups.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

AMES TEST
- No significant changes in the number of revertants were observed at any concentrations of ZnO-MPs treatments in the five Salmonella tester strains, when compared to solvent control (please refer to 'any other information on results incl. tables').
- The positive control substances induced statistically significantly and markedly increased revertant colony numbers, when compared to the solvent control values.

Remarks on result:

not determinable because of methodological limitations

Table 1.Mutagenicity assay of ZnO NPs and ZnO MPs.

	Without S9 metabolic activation					With S9 metabolic activation
	TA98	TA100	TA102	TA1535	TA1537	TA98	TA100	TA102	TA1535	TA1537
Negative	27±3	113±9	124±10	17±1	6±1	35±2	113±6	256±4	20±2	7±1
Positive	419±87*	901±105*	1825±139*	904±57*	685±142*	240±8*	549±50*	1021±81*	656±38*	462±44*
ZnO-MPs (mg/plate)
0.312	25±3	160±9	145±9	26±1	6±1	43±4	134±33	246±15	21±3	9±4
0.625	27±3	150±13	166±10	19±1	6±1	53±4	134±80	242±14	21±3	14±2
1.25	28±2	127±11	119±11	19±3	9±1	48±1	154±75	292±18	20±4	12±1
2.5	34±2	154±3	131±6	23±2	9±1	31±3	156±83	250±14	22±4	14±2
5.0	33±2	168±5	141±7	26±1	8±0	38±2	159±82	269±10	26±3	17±1

Values were presented as Mean±S.D.(N³3). Solvent (Sterile water) was used as negative control. Positive control in w/o S9 plate: TA98, 4-Nitro-o-phenylenediamine 2.5 µg/plate; TA100 and TA1535, Sodium azide 5 µg/plate; TA102, Mitomycin C 0.5 µg/plate; TA1537, 9 -Aminoacridine 50 µg/plate. Positive control in w/w S9 plate: TA98, benzo(a)pyrene 5 µg/plate; TA100/TA102/TA1535/TA1537, 2-aminoanthrene 5 µg/plate.Statistical analysis: *p< 0.05, **p< 0.01, and ***p< 0.001, indicate a statistical difference with the control groupby One-way ANOVA.

Conclusions:

Li, C.-H. et al. (2012) performed a Salmonella reverse mutation assay in order to evaluate the mutagenic potential ZnO MPs. The assay was performed according to the plate incorporation method both in presence and absence of a metabolic activation system. Three independent experiments were performed.
According to the authors, ZnO MPs did not induce a significant increase in the revertant colony number in any of the strains tested, when compared to the solvent control. The positive controls elicit a significant response as compared to the solvent controls.
The test material is insufficiently characterised, since information on purities and impurities is missing. Information on confounding factors, i.e. precipitation and cytotoxicity, in the main experiments is not provided. No confirmatory experiment was performed. Historical control data is missing.
Based on the above-mentioned shortcomings the reference is suitable for the weight of evidence analysis of the genetic toxicity of ZnO.

Reference 13

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Not applicable

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Study or study documentation showed limitations however considered useful for the evaluation of the genotoxicity of zinc. Used in EU risk assessment for zinc oxide 2004.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

(No data of negetive control)

Principles of method if other than guideline:

Not applicable

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

No data

Species / strain / cell type:

S. typhimurium, other: 3 strains not specified

Details on mammalian cell type (if applicable):

No data

Additional strain / cell type characteristics:

not specified

Metabolic activation:

not specified

Metabolic activation system:

no data

Test concentrations with justification for top dose:

refer to reference

Vehicle / solvent:

no data

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Details on test system and experimental conditions:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Evaluation criteria:

Positive response: Induction of reproducible dose related increases in the number of his+ revertants in the treated group compared to the control.

Statistics:

No data

Species / strain:

S. typhimurium, other:

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

None

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

None

Conclusions:

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

The test material was considered to be non-mutagenic under the condition of this test.

Executive summary:

In a bacterial reverse mutation assay by Litton Bionetics (1976), three strains of Salmonella typhimurium were treated with zinc oxide at unknown concentrations. No significant increases in the frequency of his+ revertant colonies were recorded therefore the test material was considered to be non-mutagenic under the conditions of this test. The information summarised in this study record was taken from the EU Risk Assessment report for zinc (EU RAR 2004) where it has been thoroughly reviewed. Due to the unavailability of the primary reference, the study was rated with reliability 4 for formal reasons. However, the results of this study will be used in the hazard assessment of the zinc category substances in a weight of evidence approach.

Reference 14

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Not applicable

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Study or study documentation showed limitations however considered useful for the evaluation of the genotoxicity of zinc. Used in EU risk assessment for zinc oxide 2004.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

no guideline available

Principles of method if other than guideline:

no information

GLP compliance:

no

Type of assay:

sister chromatid exchange assay in mammalian cells

Target gene:

No data

Species / strain / cell type:

other: Syrian hamster embryo cells

Details on mammalian cell type (if applicable):

No data

Additional strain / cell type characteristics:

not specified

Metabolic activation:

not specified

Metabolic activation system:

no data

Test concentrations with justification for top dose:

refer to reference

Vehicle / solvent:

no data

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Details on test system and experimental conditions:

refer to reference

Evaluation criteria:

refer to reference

Statistics:

No data

Species / strain:

other: Syrian hamster embryo cells

Metabolic activation:

not specified

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

None

None

Conclusions:

Interpretation of results (migrated information):
ambiguous

The results were considered to be ambiguous under the condition of this test.

Executive summary:

In a cytogenetic assay conducted using Syrian hamster embryo cells, zinc oxide gave ambiguous results.

Reference 15

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

The clastogenic activity of zinc chloride was determined by studying chromosome aberrations in human dental pulp cells in vitro, both in the presence and absence of exogenous metabolic activation.

GLP compliance:

no

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Zinc oxide, 99.9% pure, Kanto Chemical (Tokyo, Japan)

Target gene:

Not applicable

Species / strain / cell type:

other: Human dental pulp cells (D824 cells)

Details on mammalian cell type (if applicable):

- Dental pulp tissue obtained from a lower third molar extracted from a 22 years old woman
- Type and identity of media: α-minimum essential medium supplemented with 20 % fetal bovine serum (FBS), 100 µM L-ascorbic acid phosphate magnesium salt n-hydrate, 2 mM L-glutamine, 0.22% NaHCO3 and 100 µg/mL streptomycin

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

colcemid, conc. not reported

Metabolic activation:

with and without

Metabolic activation system:

5% rat liver postmitochondrial supernatant (PMS) mixture

Test concentrations with justification for top dose:

30, 100 and 300 µM

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (60mM)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

None Migrated to IUCLID6: 50 µM

Details on test system and experimental conditions:

D824 cells at 6–8 passages were plated into 100-mm dishes (Costar) at 8×10E3 cells/cm2, equivalent to 4.5×10E5 cells/dish, and incubated overnight. The cells were treated with test agents for 3 h.

DURATION
- Preincubation period: Overnight
- Exposure duration: 3 h

NUMBER OF REPLICATIONS: Triplicate

NUMBER OF CELLS EVALUATED: 1.6×10 (5) cells/dish

DETERMINATION OF CYTOTOXICITY: Yes
- Method: The cytotoxicity of the test material was determined as the number of cells treated with the test material relative to the number of cells in the control cultures×100

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes

OTHER: The pH range of the culture media containing the highest concentrations of test agents was approximately 7.2–7.5.

Evaluation criteria:

No data

Statistics:

χ2-test was used to assess the significance of the difference in the incidences of chromosome aberrations between control cultures and cultures treated with test agents. The level of significance in the statistical analysis was determined at p<0.05.

Species / strain:

other: Human dental pulp cells (D824 cells)

Metabolic activation:

with and without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

No data

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1: Chromosome aberrations in D824 cells induced by treatment with ZnCl2 for 3 h and 30 h:

Test material	Time	Concentration	Relative cell number (%)	Number of metaphases scored	Type of structural aberrations(a) (%)								Aberrant metaphases (%)	Polyploidy and endoreduplication (%)
					ctg	csg	ctb	csb	cte	D	O	F
Control	3 h	0	100	500	0.8	0	0	0	0	0	0	0	0.8	2
Zinc oxide (µM)		30	88	100	0	0	0	0	0	0	0	0	0	4
		100	75	100	2	0	0	0	0	0	0	0	2	2
		300	59	100	1	0	0	0	0	0	0	0	1	6
Control	30 h	0	100	500	1	0	0	0	0	0	0	0	1	2
Zinc oxide (µM)		30	79	100	3	0	0	0	0	0	0	0	3	1
		100	58	100	5	0	0	0	0	0	0	0	5	3
		300	58	200	5	0	2	0	0	0	0	0	6,5	4

a = ctg, chromatid gaps; csg, chromosome gaps; ctb, chromatid breaks; csb, chromosome breaks; cte, chromatid exchanges; D, dicentric chromosomes; O, ring chromosomes; F, fragmentations.

 

Conclusions:

Interpretation of results (migrated information):
negative

Under the test conditions, the test material was considered to be non-clastogenic to human dental pulp cells in vitro.

Executive summary:

A study was conducted to investigate the ability of zinc oxide to induce chromosome aberrations in human dental pulp cells. Human dental pulp cells (D824 cells) treated with the test material, were evaluated for chromosome aberrations at up to 3 dose levels, together with vehicle and positive controls. Rat liver (5%) post mitochondrial supernatant mixture was used as the exogenous metabolic activator. Ability to induce chromosome aberrations was examined in cells treated with test material for 3 and 30 h. The test material induced weak chromosome aberrations in the presence or absence of exogenous metabolic activation. The percentages of cells with polyploid or endoreduplication were not enhanced by test material. The authors concluded that the findings suggest that the results of genetic-toxicity tests for zinc are equivocal, probably depending on the zinc compounds tested and the type of cells used.

Reference 16

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Not applicable

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Study or study documentation showed limitations however considered useful for the evaluation of the genotoxicity of zinc. Used in EU risk assessment for zinc oxide 2004.

Qualifier:

no guideline available

Principles of method if other than guideline:

no information

GLP compliance:

no

Type of assay:

DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro

Target gene:

No data

Species / strain / cell type:

other: Syrian hamster embryo cells

Details on mammalian cell type (if applicable):

No data

Additional strain / cell type characteristics:

not specified

Metabolic activation:

not specified

Metabolic activation system:

no data

Test concentrations with justification for top dose:

0.3, 1.0, 3.0, 10 and 30 ug/ml

Vehicle / solvent:

no data

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Details on test system and experimental conditions:

refer to reference

Evaluation criteria:

refer ro reference

Statistics:

No data

Species / strain:

other: Syrian hamster embryo cells

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

>1 µg/ml

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

None

None

Conclusions:

Interpretation of results (migrated information):
positive without metabolic activation

Positive without metabolic activation.

Executive summary:

Zinc oxide tested at concentrations of 0.3, 1.0, 3, 10 and 30 ug/ml produced unscheduled DNA synthesis at >1ug/ml without metabolic activation in Syrian hamster embryo cells.

Reference 17

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From January 12, 2010 to June 30, 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Additional strain / cell type characteristics:

other: Doubling time 16 h

Cytokinesis block (if used):

Colcemid

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Test substance: 0, 5, 10, and 20 µg/mL (4 h without S9-mix); 0, 1, 3, 10, and 15 µg/mL (24 h without S9-mix); 0, 12.5, 25, and 50 µg/mL (4 h with S9-mix)
Reference substance (Z-COTE® and microscaled ZnO): 1µg/mL (4 h without S9-mix), 12.5 µg/mL (4 h with S9-mix), and 3 µg/mL (24 h without 59-mix)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Phosphate buffer pH 7.4 with 100 µg/mL soy lecithin

Untreated negative controls:

yes

Remarks:

Growth medium

Negative solvent / vehicle controls:

yes

Remarks:

Phosphate buffer pH 7.4 with 100 µg/mL soy lecithin

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 10E6 per 25cm² flask
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 24 hres
- Exposure duration/duration of treatment: 4 hrs (+ and - S9) and 24 hrs (- S9)
- Harvest time after the end of treatment (sampling/recovery times): for the 4 hrs treatmentapprox. 1.5 cycle lengths recovery

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): 2-3 hrs prior cell harvest 0.2mL colcemid stock solution (10 µg/mL) was added
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): 4slides per culture, faining with Giemsa (10% Giemsa freshly prepared)
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): 100 well spread metaphases with 22 +/- 2 centromeres/chromosomes were analysed per culture
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): according to Buckton and Evans (1973), ISCN (1985), Savage (1976 and 1983)

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: mitotic index (MI)
- Any supplementary information relevant to cytotoxicity: 1000 cells were counted per concentration and number of metaphases recorded

METHODS FOR MEASUREMENTS OF GENOTOXICIY

- OTHER:

Evaluation criteria:

- Gaps: lowest relevant aberrations
- Exchanges: Highest biologically relevant aberrations
- Pulverization or shattering: Toxic effect rather than clastogenic effect
- Positive result: Aberration (including gaps) in > 5% of the analyzed cells in each of the two technical replicates at one dose level
- Dose dependency may provide further evidence to clastogenicity
- Negative result: Only gaps in single test group without dose relation

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Dose dependent without S9-mix and only at highest dose with S9-mix

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No changes
- Effects of osmolality: No changes
- Evaporation from medium: Test substance is solid
- Water solubility: Insoluble

RANGE-FINDING/SCREENING STUDIES: Dose finding as pre-test measuring cytotoxicity

COMPARISON WITH HISTORICAL CONTROL DATA: Concurrent controls were in line with historical controls

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Detail on results:

Estimation of cytotoxicity (pre-experiments): Cytotoxicity of the test substance and the particulate reference substances was evaluated in pre-experiments without S9-mix, using four concentrations for the test substance (4 h: 10, 30, 40, 50 µg/mL; 24 h: 5, 10, 20, 40 µg/mL) and also four concentrations for the particulate reference substances (4 h: 10, 30, 40, 50 µg/mL; 24 h: 1, 5, 10, 20 µg/mL). Using the M.I. as a measure for cell viability/cytotoxicity, there was a more or less concentration-dependent decrease in cell viability after 4 h of incubation with the test substance in the absence of S9-mix. At the highest concentration (50 µg/mL) M.I. was reduced to 50.4 % of the negative control and 52.0 % of the vehicle control, respectively. In contrast, the vehicle was nearly nontoxic, as compared to the negative control. For the main experiments the maximum concentration of test substance was reduced to 25 µg/mL to ensure both an appropriate high cytotoxicity of the maximum concentration (at least 50 % reduction in viability), but also enough metaphases for analysis of chromosome aberrations. In contrast to test substance, there was profound concentration-dependent cytotoxicity after 4 h of incubation for the particulate reference substances. At 40 and 50 µg/mL, M.I. was markedly reduced to 0.8 and 0.0 % of the negative control for uncoated nanoscaled ZnO and to 7.7 and 4.7% of the negative control for micron-scaled ZnO, respectively. Thus, it was decided to use no more than 10 µg/mL of the particulate reference substance as maximum concentration in the short term main experiment without S9-mix. As expected, cytotoxicity of the test substance was more intense after 24 h of incubation without S9-mix than after 4 h of incubation, perhaps due to potentially increased particle-like effects or test substance dissolution. M.I., at the highest concentration used (40 µg/mL), amounted to only 5.8 %, as compared to the negative control. The vehicle was, again, only marginally cytotoxic. To ensure a sufficient number of metaphases for chromosome analysis, but also to reach an appropriate high level of cytotoxicity for the highest concentration in the main experiment, 15 µg/mL of the test substance were chosen as the maximum concentration. In the long term pre-experiment, uncoated nanoscaled ZnO was highly cytotoxic. Already at a concentration as low as 1 µg/mL, M.I. was reduced to 5.8 % of the negative control. In this preexperiment, micron-scaled ZnO was less cytotoxicity up to a concentration of 10 µg/mL, but there was complete loss of metaphases at 20 µg/mL. As cytotoxicity of the uncoated ZnOs after 24 h of incubation was more pronounced than that of the test substance, it was decided to use 3 µg/mL as exposure concentration for the particulate reference substances, and not the highest concentration chosen for test substance (15 µg/mL), to enable comparison of the different particles.

Main experiments:

-Osmolality and pH: To estimate unphysiological changes of the cell culture environment due to addition of the test and particulate reference substances, osmolality and pH of the test substance and particulate reference substance-containing incubation media without S9-mix were evaluated at the highest concentrations, used for the main experiments. Concerning osmolality, there was a slight decrease from 342 mOSMOL/kg for the negative control medium to 309 mOSMOL/kg for the vehicle control medium (negative control medium + phosphate buffer with soy lecithin) after 4 h of incubation, and from 345 mOSMOL/kg to 311 mOSMOL/kg after 24 h of incubation, respectively. Osmolality of test substance containing incubation medium (50 µg/mL) was in the same range as the vehicle control medium with 307 mOSMOL/kg after 4 h of incubation. Osmolality of the particulate reference substance (12.5 µg/mL) both amounted to 309 mOSMOL/kg, indicating no test- or reference substance-mediated changes in osmolality. Only after 24 h of incubation, osmolality of the test substance (15 µg/mL) with 336 mOSMOL/kg exceeded that of the vehicle control value, but, as compared to the 4 h values, seemed to represent a false positive result. For the particulate reference substance (3 µg/mL) osmolality resembled that of the vehicle control. All osmolalities were still in the physiological range. Concerning pH, the vehicle, the test substance, and the particulate reference substance uncoated nanoscaled ZnO and micron-scaled ZnO did not significantly change the pH of the incubation media, both after 4 and 24 h of incubation. There was only a very slight tendency towards an increase in pH in the particle-containing incubation media after 4 h. However, after 24 h of incubation no such increase was observed.

-Mitotic index (M.I.):

Short term treatment without S9-mix: Cells were treated for 4 h with different concentrations of the test substance (5, 10, 20, and 25 µg/mL) and one concentration (10 µg/mL) of the reference substance without S9-mix. M.I. was subsequently evaluated for both of the duplicate cultures to estimate cytotoxicity. As compared to the negative controls, the vehicle controls exhibited slight reduction in the number of metaphases, with a reduction of the mean M.1. to 84.5 % of the negative controls. Concerning test substance, there was a clear, concentration-dependent cytotoxic effect of the test substance. At the highest concentration used (25 µg/mL) mean M.I. amounted to 2.20 (=24.0 % of the negative control, M.1. = 9.25), as compared to 7.8 for the vehicle control. The reference substance uncoated nanoscaled and micron-scaled ZnO at 10 µg/mL exhibited mean MI of 4.1 (=44.5% of the negative control) and 5.05 (=54.5% of the negative control), respectively, with a higher mean MI of 5.95 (= 64.5% of the negative control) for test substance at the same concentration.

Short term treatment with S9-mix: Cells were treated for 4 h with different concentrations of the test substance (12.5, 25, 40, and 50 µg/mL) and one concentration (12.5 µg/mL) of the reference substance in the presence of S9-mix. The M.I. was evaluated for both of the duplicate cultures to estimate cytotoxicity. Interestingly, in contrast to the short treatment without S9-mix and the respective pre-experiment on cytotoxicity, there was no reduction in viability due to the vehicle exposure and less cytotoxic action of the test substance. Only at the highest Z-COTE® HP1 concentration (50 µg/mL) mean M.I. was significantly reduced to 4.95 (= 44.5 % of the negative control), as compared to 11 .45 102.5 % of the negative control) for the vehicle control. The reference substances at 12.5 µg/mL each mediated only slight reduction in mean M.I., with Z-COTE® treated cultures exhibiting a mean M.I. of 9.8 (= 88.0% of the negative control) and micron-scaled ZnO treated cultures exhibiting a mean M.I. of 10.5 94.5% of the negative control). Interestingly, at 12.5 µg/mL Z-COTE® HP1 rather increased than decreased viability of the V79 cells.

Long term treatment without S9-mix: Cells were treated for 24 h with five different concentrations of the test substance (1, 3, 10, 15 and 30 µg/mL) and one concentration (3 µg/mL) of the reference substances Z-COTE® and micron-scaled ZnO in the absence of S9-mix. The M.I. was evaluated for both of the duplicate cultures to estimate influence of the test and reference substance on cell viability. After 24 h of incubation, Z-COTE® HP1 concentration-dependently decreased cell viability with a clear reduction in mean M.I. to 3.35 (= 46 % of the negative control) at 15 µg/mL, as compared to 6.9 (= 78.5 % of the negative control) for the vehicle control. At 30 µg/mL of the test substance no metaphases were present. After 24 h of incubation, the vehicle (phosphate buffer and soy lecithin) mediated a slight decrease in cell viability with a mean M.I. of 6.9, as compared to 8.75 for the negative control. This was in line with the marginal cytotoxicity of the vehicle in the pre-experiment. The reference substances Z-COTE® and micron-scaled ZnO (both 3 µg/mL) mediated slightly more reduction in viability than the test substance. Mean M.I. amounted to 5.9 (= 68.0 % of the negative control) for Z-COTE® and 6.0 (= 69% of the negative control) for micron-scaled ZnO, as compared to 7.45 (= 86% of the negative control) for the test substance Z-COTE® HP1 at the same concentration.

Chromosome analysis: Negative and vehicle controls exhibited spontaneous aberration frequencies within the normal range for V79 cells. As positive controls, cells treated with EMS (without S9-mix) or CP (with S9-mix), were analyzed for structural (and numerical) chromosome aberrations. The positive control cultures demonstrated that the system was operating satisfactorily. The marked increase in aberrations indicated that the target cells were very sensitive to the effect of known clastogens and that the used metabolizing system (S9-mix) was quite efficient. The chromosome aberrations observed in the positive control cultures consisted of: g, G, b (ctb, ccmin), B (f, nud, dmin, su), ex (qr, tr, ctr, cte, cUnv), EX (ex, csr, die), and mao For historical positive control data. 

Short treatment without S9-mix: V79 cells, treated for 4 h in the absence of S9-mix with 5, 10, or 20 µg/mL of the test substance Z-COTE® HP1, or with 10 µg/mL of the reference substances Z-COTE® or micron-scaled ZnO, were analyzed for occurrence of chromosome aberrations. There was no evidence of a concentration-dependent induction of structural chromosome aberrations by treatment with Z-COTE® HP1, with the exception of chromatid gaps (g = ctg) at all concentrations analyzed, one chromosome gap (G = csg, at 20 µg/mL), and three B (f, 1 in each concentration). Chromatid gaps (g =ctg) were also observed in the negative and vehicle control cultures and one isochromatid gap (G =csg) was found in one of the negative control cultures. Aberration frequencies (gaps excluded) in the negative and vehicle control cultures amounted to 0.0 %, whereas the Z-COTE® HP1-treated cultures exhibited aberration frequencies (gaps excluded) of 0.5 % for all concentrations tested. The frequency of polyploid cells (py) was not significantly increased by Z-COTE® HP1, as compared to the vehicle control. Only chromatid (g = ctg) and isochromatid gaps (G = csg) were noted in the cultures treated with 10 µg/mL of the particulate reference substance. The particulate reference substances only demonstrated some gaps, which were also observed in the negative and vehicle control cultures.

Short treatment with S9-mix: V79 cells, treated for 4 h in the presence of S9-mix with 12.5, 25, and 50 µg/mL of the test substance Z-COTE® HP1 or 12.5 µg/mL of the particulate reference substances Z-COTE® or micron-scaled ZnO, were analyzed for the occurrence of chromosome aberrations. The respective negative and vehicle control cultures exhibited three (negative control) and four chromatid gaps (g =ctg, vehicle control), respectively. Concerning the Z-COTE® HP1treated cultures, there was evidence of six chromatid gaps (G =ctg) and one isochromatid gap (G =csg) (12.5 µg/mL), three chromatid gaps (g = ctg) and one isochromatid gap (G = csg) (25 µg/mL), and six chromatid gaps (g = ctg) and one isochromatid gap (G =csg) (50 [µg/mL). The aberration frequencies (gaps excluded) for all Z-COTE® HP1 concentrations tested as well as for the negative and vehicle control cultures both amounted to 0.0%. There was also no significant increase in the frequencies of polyploid cells (py) in Z-COTE® HP1-treated cultures, as compared to the vehicle controls. The particulate reference substances only demonstrated chromatid (g = ctg) and isochromatid gaps (G == csg), which were also observed in the negative and vehicle control cultures, and very slight elevation in polyploidy metaphases.

Long term treatment without S9-mix: V79 cell cultures, treated for 24 h without S9-mix with 1, 3, 10, and 15 µg/mL Z-COTE® HP1 or 3 µg/mL of the particulate reference substances Z-COTE® and micron-scaled ZnO, were analyzed for the occurrence of chromosome aberrations. The negative and vehicle control cultures only demonstrated occurrence of chromatid gaps (g = ctg) and one chromosome gap (G = csg, vehicle control). In the Z-COTE® HP1-treated cultures, only one chromatid gap (g = ctg) and one isochromatid gap (G = csg) (1 µg/mL), two chromatid gaps (g = ctg) (3 µg/mL, two chromatid gaps (g = ctg) (10 µg/mL), and six chromatid gaps (g = ctg), two isochromatid gaps (G = csg), and one chromosome break (B = 1 dmin) (15 µg/mL) were noted. The aberration frequencies (% cells with aberrations, gaps excluded) of the test substance-treated cells thus amounted to 0.0 % (1, 3, and 10 µg/mL) and 0.5 % (15 µg/mL), respectively. The aberration frequencies of the negative and vehicle control cultures both amounted to 0.0 %. The frequency of polyploid cells (py) in the test substance-treated cultures resembled that of the negative and vehicle control cultures for 1 - 10 µg/mL and was slightly elevated to six polyploidy metaphases, as compared to the negative and vehicle control cultures, which exhibited both one py. Like for the test substance, the particulate reference substances only demonstrated chromatid (g =ctg) and isochromatid gaps (G = csg), which were also observed in the negative and vehicle control cultures.

Reference items	4 h / -S9-mix			4 h / +S9-mix			24 h / -S9-mix
	%AC			%AC			%AC
	-g	+g	%M	-g	+g	%M	-g	+g	%M
Negative control	2.0	0.0	100	1.5	0.0	100	1.0	0.0	100
Vehicle control	2.0	0.0	84.3	2.0	0.0	102.7	2.0	0.0	78.9
EMS         150 µg/ml	55.5	36.5	45.9	-	-	-	-	-	-
EMS           50 µg/ml	-	-	-	-	-	-	79.5	70.5	25.7
CP             2.5 µg/ml	-	-	-	45.0	31.5	98.7	-	-	-
Z-COTE        3 µg/ml	-	-	-	-	-	-	3.5	0.0	67.4
Z-COTE     10 µg/ml	1.0	0.0	44.3	-	-	-	-	-	-
Z-COTE  12.5 µg/ml	-	-	-	2.0	0.0	87.9	-	-	-
ZnO             3 µg/ml	-	-	-	-	-	-	1.5	0.0	68.6
ZnO           10 µg/ml	3.0	0.0	54.6	-	-	-	-	-	-
ZnO       12.5 µg/ml	-	-	-	3.0	0.0	94.6	-	-	-
Z-COTE HP1 (µg/ml)
1	-	-	-	-	-	-	1.0	0.0	102.3
3	-	-	-	-	-	-	1.0	0.0	85.1
5	3.5	0.5	83.8	-	-	-	-	-	-
10	4.0	0.5	64.3	-	-	-	1.0	0.0	72.6
12.5	-	-	-	3.5	0.0	104.5	-	-	-
15	-	-	-	-	-	-	4.5	0.5	38.3
20	4.0	0.5	47.0	-	-	-	-	-	-
25	-	-	-	2.0	0.0	97.8	-	-	-
50	-	-	-	3.5	0.0	44.4	-	-	-

%AC: % aberrationt cells; %M: M.I. in percent of the negative control (mean of two technical replicates); +g: with gaps; -g: without gaps; -: not tested

Conclusions:

An in vitro mammalian chromosome aberration test was performed with V79 cells to assess the potential of microscaled ZnO to induce structural (and numerical) chromosome aberrations in somatic mammalian cells according to the OECD guideline 473 in compliance with GLP.

V79 Chinese hamster lung fibroblast cells were exposed to the test substance for 4 h both in the absence and presence ofmetabolic activation system.An initial experiment on cytotoxicity was conducted to determine the concentrations of the test substance to be used in main experiment. For the particulate microscaled ZnO one concentration per main experiment was used, with 10 µg/mL for the short term experiment without S9-mix, 12.5 µg/mL for the short term experiment with S9-mix, and 3.0 µg/mL for the long term experiment without S9-mix, respectively.

There was a decrease in M.I. for micron-scaled ZnO, primarily without S9-mix. Test performance and activity of the metabolizing system were satisfactory. Irrespective of its cytotoxic potential in the absence of S9-mix,coated the microscaled ZnO, under all treatment modalities, did not increase significantly the aberration frequency, as compared to the respective vehicle controls. Besides some gaps, which were also evident in the negative and vehicle controls, a sum of only 4 chromosome breaks was observed in all 3 main experiments. The aberration frequencies (without gaps) in no case exceeded 5 % and they all fell within the range of the historical negative controls.

Under the test conditions, microscaled ZnO is considered not to induce structural chromosome aberrations in cultured mammalian somatic cells.

Reference 18

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

thymidine kinase (TK)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Periodically checked for Mycoplasma contamination: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

1, 2, 4, 5, and 6 µg/ml (without S9-mix)
2.5, 5, 7.5, and 10 µg/ml (with S9-mix)

Vehicle / solvent:

phosphate buffer + 100 µg/ml soy lecithin (1 part) and RPMI-1640 medium + 5% horse serum (9 parts)

Untreated negative controls:

yes

Remarks:

RPMI-1640 medium + 5% horse serum

Negative solvent / vehicle controls:

yes

Remarks:

phosphate buffer + 100 µg/ml soy lecithin (1 part) and RPMI-1640 medium + 5% horse serum (9 parts)

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

cyclophosphamide as positive control with S9-mix Migrated to IUCLID6: positive control without S9-mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: no preinubation performed
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 14 -16 days

SELECTION AGENT (mutation assays): 5-fluorothymidine

NUMBER OF CELLS EVALUATED: 2,000 cells/well

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

criteria for positive results:
- concentration-related or reproducible increase in mutant frequency (MF)
- biological relevance of the results is considered firs
- increase in MF occuring only at highly toxic concentrations of the test item (i. e. less than 10% of total growth) is not considered biologically relevant
- relevant increase in present study is stated if MF of test item amounted to more than [(MF of positive/vehicle control) + 125], 125 represents the historical control data for this type of method
- a test item is considered to be mutagenic if both criteria (relevant increase and dose-dependency) are met
- if only one criteria is met the test item is reported as equivocal
- in case of no relevant increase and no dose-dependency the test item is considered to be non-mutagenic in the present test system

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

ambiguous

Remarks:

relevant increase in mutant frequency always linked to cytotoxicity

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: slight reduction of medium's pH directly after preparation but not after 4 hours of incubation
- Effects of osmolality: physiological range directly after preparation, lower physiologigal range after 4 hours of incubation
- Evaporation from medium: test item is solid
- Water solubility: insoluble
- Precipitation: turbidity did not change during 4 hours of incubation indicating stable treatment suspensions, slightly increased turbidity noted for Z-COTE HP1 at 10µg/ml (7.81 FAU), for Z-COTE at 7.5 µg/ml (8.31 FAU = formazine attenuation units) as well as microscaled ZnO (11.84 FAU) compared to negative and vehicle control (5.29 and 6.3 FAU). 50 µg/ml Z-COTE HP1 markedly increased turbidity (70.72 FAU).

RANGE-FINDING/SCREENING STUDIES: dose range study performed as pre-test, concentration of main test based on results of pre-test



ADDITIONAL INFORMATION ON CYTOTOXICITY:

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

After 4 hours of incubation the highest Z-COTE HP1 concentration (10 µg/ml) did not alter pH and osmolarity of the treatment media but induced concentration-dependent cytotoxicity with and without S9 -mix. Vehicle, negative, and positive control exhibited mutant frequencies (MF) within the normal range. Based on the evaluation criteria of the present study Z-COTE HP1 induced relevant increases (marked with * in the following tables) of the MF in both replicates at 6 µg/ml without S9 -mix and 7.5 and 10 µg/ml with S9 -mix, and in one replicate at 5 µg/ml without S9 -mix. However, significantly increased MF was always linked to acute cytotoxicity. In the presence of S9 -mix relevant increases in MF were also obvious for the particulate reference items at 7.5 µg/ml. All the other particle-treated cultures exhibited MFs which were within the normal range for negative control.

4 h without S9-mix
Treatment	Relative total growth	Mutant frequency
Vehicle control	1.00	90.3
Negative control	0.93	105.7
Z-COTE®HP1: 1 µg/ml	0.81	124.8
Z-COTE® HP1: 2 µg/ml	0.81	108.3
Z-COTE® HP1: 4 µg/ml	0.77	115.7
Z-COTE® HP1: 5 µg/ml	0.62	181.1*
Z-COTE® HP1: 6 µg/ml	0.21	982.1*
Z-COTE® : 4 µg/ml	0.79	128.3
ZnO microscaled: 4 µg/ml	0.82	149.4
Positive control (methyl methansulfonate 10 µg/ml)	0.43	697.9*

4 h with S9-mix
Treatment	Relative total growth	Mutant frequency
Vehicle control	1.00	110.3
Negative control	1.85	117.2
Z-COTE®HP1: 2.5 µg/ml	1.31	123.1
Z-COTE® HP1: 5.0 µg/ml	1.24	139.6
Z-COTE® HP1: 7.5 µg/ml	0.50	463.1*
Z-COTE® HP1: 10.0 µg/ml	0.36	574.3*
Z-COTE® : 7.5 µg/ml	0.43	537.9*
ZnO microscaled: 7.5 µg/ml	0.52	433.3*
Positive control (cyclophosphamide 2.5 µg/ml)	1.49	468.7*

*: relevant increase regarding evaluation criteria of the present study method

Conclusions:

Mouse lymphoma L5178Y/TK± cells in suspension culture were treated for 4 hours with different concentrations of zinc oxide with and without S9 -mix. Medium, medium with soy lecithine, methyl methanesulfonate and cyclophosphamide were used as negative control, vehicle control, positive control without S9 -mix and positive control with S9 -mix, respectively. Zinc oxide induced increases of MF with and without S9 -mix in the presence of S9 -mix. The increase mutant colonies were primarily characterised as small colonies, indicating a clastogenic event. The significantly increased MF was always linked to pronounced cytotoxicity. For this reason and the limited significance of the test system for particles, the test results should more likely be judged as questionable in L5178Y/TK cells under the conditions and restrictions of this assay, implying a possible false positive result. Consequently, the results are interpreted as equivocal, as the clastogenic findings were confounded with cytotoxicity.

Reference 19

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

not specified

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

No conclusion can be drawn on the results presented due to major deficiencies with regards to reporting and the methodology applied. The test material is insufficiently characterised, since information on purity, source, manufacturer, and physical appearance are missing. The number of cells scored for chromosomal damage was very low (n=100). The vehicle control and lowest dose did not show any cells with chromosomal damage. The test material was not tested up to toxic concentrations. Evaluation and scoring criteria are not specified. Historical control data was not included. The authors did not discuss confounding factors, such as test material precipitation, or pH and osmolality effects of the test material on the culture medium. Based on the above-mentioned shortcomings the reference is considered not reliable.

Qualifier:

no guideline followed

Principles of method if other than guideline:

An in vitro cytogenetic assay was conducted in human embryonic lung cells to evaluate the genotoxic potential of zinc sulfate.

GLP compliance:

no

Remarks:

pre-GLP study

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

mammalian cell line, other: human embryonic lung cells: WI-38

Details on mammalian cell type (if applicable):

No data

Metabolic activation:

without

Test concentrations with justification for top dose:

0.1, 1.0 and 10 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: saline

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

triethylenemelamine

Details on test system and experimental conditions:

CYTOGENETIC ASSAY
Human embryonic lung cultures (WI-38) which were negative for adventitious agents which may interfere were used. These cells were employed at passage level 19. The cells had been transferred using 0.025% trypsin and transferred into 40 mL culture medium. When growth was approximately 95% confluent, the cells were trypsinised, centrifuged, and frozen in tissue culture medium containing DMS. Cells were frozen in vials at a concentration of 2x10^6 cells/mL. When needed, the cells were thawed, washed, suspended in MEM tissue culture medium and transferred in milk dilution bottles at a concentration of 5x10^5 cells/mL. The test compound was added at three dose levels using three bottles for each level, 24 hours after planting. The dose levels required a preliminary determination of cytotoxicity. This was accomplished by adding logarithmic doses of the compound in saline to a series of tubes containing 5x10^5 cells/mL which were almost confluent. The cells were examined at 24, 48, and 72 hours. Any cytopathic effect (CPE) or inhibition of mitosis was scored as toxicity. Five more closely spaced dose levels were employed within the two logarithmic dosages, the higher of which showed toxicity and the lower no effect. The dose level below the lowest toxic level was employed as high level. Logarithmic dose levels were employed for the medium and low levels. Cells were incubated at 37°C and examined twice daily to determine when an adequate number of mitosis were present. Cells were harvested by shaking when sufficient mitoses were observed, usually 24-48 hours after planting, centrifuged, and fixed in absolute methanol: glacial acetic acid (3: 1) for 30 minutes. The specimens were centrifuged, decanted, and suspended in acetic acid-orcein stain (2%) and a drop of suspension placed on a slide and covered with a cover slip. A total of 100 cells were scored per concentration level.

Evaluation criteria:

not specified

Statistics:

not specified

Species / strain:

mammalian cell line, other: human embryonic lung cells: WI-38

Metabolic activation:

without

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

CYTOGENETIC ASSAY
- Zinc sulfate produced no significant aberration in the anaphase chromosomes of human tissue culture cells when tested at the dosage levels employed in this study.
- The mitotic index was not changed in cultures exposed to zinc sulfate, when compared to vehicle control cultures.

Remarks on result:

not determinable because of methodological limitations

Conclusions:

In a cytogenetic study (Litton Bionetics Inc., 1974), human embryonic lung cells (Wl-38) were exposed to zinc sulfate. The cells were treated at zinc sulfate concentration levels of 0.1, 1.0, and 10 µg/plate. Vehicle (saline) and positive (triethylenemelamine) control cultures were run concurrently. A total of 100 cells were scored per concentration level. In addition, the mitotic index was determined for cytotoxicity evaluations.

Zinc sulfate did not induce an increase of the proportion of cells with chromosomal damage in human embryonic lung cells (Wl-38), when compared to vehicle control cultures. The mitotic index was the same for zinc sulfate-exposed cells and vehicle control cultures. The positive control cultures showed marked increased in the proportion of cells with chromosomal damage.

No conclusion can be drawn on the results presented due to major deficiencies with regards to reporting and the methodology applied.
The test material is insufficiently characterised, since information on purity, source, manufacturer, and physical appearance are missing. The number of cells scored for chromosomal damage was very low (n=100). The vehicle control and lowest dose did not show any cells with chromosomal damage. The test material was not tested up to toxic concentrations. Evaluation and scoring criteria are not specified. Historical control data was not included. The authors did not discuss confounding factors, such as test material precipitation, or pH and osmolality effects of the test material on the culture medium.
Based on the above-mentioned shortcomings the reference is considered not reliable.

Reference 20

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Not reported

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Principles of method if other than guideline:

Method: According to "Maron DM & Ames BN (1983). Revised methods for the Salmonella mutagenicity test. Mutat. Res. 113: 173-215".

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Not applicable

Species / strain / cell type:

S. typhimurium TA 102

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

not specified

Metabolic activation system:

No data

Test concentrations with justification for top dose:

10, 30, 100, 300, 1,000 and 3,000 nM/plate. The selected doses were in logarithmic progression up to insolubility, or up to a threshold toxic dose that decreases the bacterial background lawn.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Water
- Justification for choice of solvent/vehicle: Test material was soluble in the water

Untreated negative controls:

yes

Remarks:

water

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: In agar (plate incorporation)

NUMBER OF REPLICATIONS: Triplicate

Evaluation criteria:

A test material was considered positive under the following conditions:
1. if there was a dose/effect relation,
2. if there was reproducibility of the effect and
3. if the number of revertants was twice the number of spontaneous revertants in the control

Statistics:

Not available

Species / strain:

S. typhimurium TA 102

Metabolic activation:

not specified

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 3,000 nM only

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

ADDITIONAL INFORMATION ON CYTOTOXICITY: 3000 nM was considered as the threshold toxic dose.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

None

Conclusions:

Interpretation of results (migrated information):
negative

The test material was considered to be non-mutagenic under the conditions of this test.

Executive summary:

 A study was conducted to determine the potential mutagenicity of the test material using bacterial reverse mutation assay (e.g. Ames test).  Salmonella typhimurium strain TA102 was treated with the test material using the plate incorporation method at six dose levels (10, 30, 100, 300, 1,000 and 3,000 nM/plate) in triplicate. Cytotoxicity was observed at 3,000 nM. No significant increases in the frequency of revertant colonies were recorded at any dose level. The test material was considered to be non-mutagenic under the conditions of this test. The test used only one tester strain instead of 4, as foreseen by OECD 471 (1983).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 December 2021 to June 2022

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study presented herein is a guideline study with a major deficiency under GLP conditions. Only one concentration level was tested, which precludes an evaluation of dose-response relationships.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

adopted 29 July 2016

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Specific details on test material used for the study:

Expiry date: 01 Mar 2022

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Wistar rats, Crl:WI(Han)
Rats were selected since this rodent species is recommended in the respective test guidelines. Wistar rats were selected since there is extensive experience available in the laboratory with this strain of rats.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland (Sulzfeld/Germany)
- Age at study initiation: approx. 7 weeks
- Weight at study initiation: The weight variation of the animals used did not exceed +/- 20 percent of the mean weight of each sex.
- Assigned to test groups randomly: yes: All animals were randomized before the start of the pre-exposure period (according to weight).
- Fasting period before study: No
- Housing: 5 rats per cage, Typ 2000P ca. 2065 cm2 (polysulfone cages) supplied by TECNIPLAST, Germany. Dust-free wooden bedding
- Diet: milled/ mouse and rat maintenance diet, GLP, 12 mm pellets, Granovit AG, Kaiseraugst, Switzerland; ad libitum
- Water: tap water; ad libitum
- Acclimation period: +/- 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 22-24°C
- Humidity: 45-65%
- Air changes: 15 air changes per hour
- Photoperiod: 12 hrs dark / 12 hrs light

Route of administration:

inhalation: aerosol

Vehicle:

- Vehicle(s)/solvent(s) used: clean air

Details on exposure:

TYPE OF INHALATION EXPOSURE: whole-body

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Generation of the inhalation atmospheres via a solid particle generators (brush-generator; BASF SE, Ludwigshafen, Germany) & Aerosol mixing tube (stainless steel; BASF SE, Ludwigshafen, Germany). Whole body exposure systems were used. The animals were kept singly in wire cages located in a glass steel inhalation chamber, volume of 1.1 m³ or 1.4 m³(BASF SE).
- Method of holding animals in test chamber: Whole body exposure systems. The animals were kept singly in wire cages located in a glass steel inhalation chamber, volume of 1.1 m³ or 1.4 m³(BASF SE). The chambers were located in exhaust hoods in an air conditioned room.
- Source and rate of air: Conditioned air from the central air conditioning system, compressed and exhaust air. Compressed air was produced by an oil-free compressor (HT 6, Josef Mehrer GmbH & Co KG, Germany). For this purpose, air is filtered by an inlet air strainer and introduced into the compressor. After passing through an second ultra filter (SMF 5/3, 108 mm, Donalson), the compressed air (15 bar) is stored in a storage of 1500 or 5000 L. The compressed air is conducted to the laboratories via pipes, where the pressure is reduced to 5 - 6 bar. In the laboratory, the compressed air can be taken as required.
- Method of conditioning air: Conditioned air from the central air conditioning system provides cold air of about 15°C. This cold air passes through an activated charcoal filter, is adjusted to room temperature of 20 to 24°C and passes through a second particle filter (H13 (HEPA) Camfil Farr, Germany). The so generated conditioned air was used to generate inhalation atmospheres.
- System of generating particulates/aerosols: The particles/aerosol was generated via a solid particle generator (brush-generator; BASF SE, Ludwigshafen, Germany) and an aerosol mixing tube (stainless steel; BASF SE, Ludwigshafen, Germany), according to the following method: For each concentration the dust aerosol was generated with the dust generator and compressed air inside a mixing stage; mixed with conditioned dilution air and passed into the inhalation system.
- Temperature, humidity, pressure in air chamber: Daily mean relative humidities in the inhalation systems ranged between 40.7 and 50.8 %. Daily mean temperatures in the inhalation systems ranged between 20.4 and 23.0°C. They are within the range suggested by the respective testing guidelines.
- Air flow rate: The air flows were constantly maintained in the desired range.
- Air change rate: An air change of about 20 times per hour can be calculated by dividing the supply air flow through the volume of each inhalation system.
- Method of particle size determination: The particle size analysis was carried out with a cascade impactor. Equipment for particle size analysis: Stack sampler Marple 298 (New Star Environmental, Inc., Roswell, Georgia 30075, USA) ; Vacuum compressed air pump (Millipore Corporation, Billerica, MA 01821, USA) ; Limiting orifice 3 L/min (Millipore Corporation, Billerica, MA 01821, USA) ; Sampling probe internal diameter 6.9 mm ; Balance Sartorius MSA 6.6S-000-DF (Sartorius AG, Göttingen, Germany). The calculation of the particle size distribution was carried out in the Laboratory for Inhalation Toxicology of the Experimental Toxicology and Ecology of BASF SE on the basis of mathematical methods for evaluating particle measurements (OECD guidance document No. 39).
To determine the particle size distribution in the submicrometer range, each test atmosphere was measured with the Scanning Mobility Particle Sizer (SMPS; Grimm Aerosol Technik GmbH & Co KG, Ainring, Germany). The SMPS system comprises an Electrostatic Classifier (Model Vienna U-DMA) which separates the particles into known size fractions, and a Condensation Particle Counter (CPC) which measures particle count concentrations. The DMA was equipped with Am-241 neutralizer. The sampling duration was about 7 minutes. As a rule 10 repeats were measured for each exposure concentration.
- Treatment of exhaust air: Exhaust air was filtered and conducted into the exhaust air of the building.

TEST ATMOSPHERE
- Brief description of analytical method used: The concentrations of the inhalation atmospheres were determined by gravimetrical measurements of filter samples in all test groups. Control group was not sampled. This analytical method was judged to be valid because the test substances did not possess an appreciable vapor pressure.
- Samples taken from breathing zone: yes

Duration of treatment / exposure:

14 days

Frequency of treatment:

14 days, 6 h per day

Dose / conc.:

0 mg/m³ air

Remarks:

Test Group 0 - air control

Dose / conc.:

7.56 mg/m³ air (analytical)

Remarks:

SD: ± 1.88 mg/m³ ; target concentration: 8 mg/m³: Test group 7

No. of animals per sex per dose:

5 male rats per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

ethylmethanesulphonate
- Justification for choice of positive control(s):
- Route of administration: oral gavage which guarantees systemic distribution of the compound and thus exposure to all assessed tissues
- Doses / concentrations: 300mg/kg body weight

Tissues and cell types examined:

Comet assay:
1. Bone marrow
2. Liver
3. Lung
4. Nasal mucosa

Bronchoalveolar lavage fluid (BAL): for cytology and total protein and enzyme levels.

Details of tissue and slide preparation:

COMET ASSAY
Samples were minced or aspirated in cold mincing buffer to produce a cell suspension. Cell suspensions were diluted as necessary and kept cold until they were processed further. At least three comet slides were prepared per sample. An aliquot of cell suspension was mixed with low melting point agarose, layered onto microscope slides precoated with normal melting point agarose, and covered with an additional layer of low melting point agarose. After the agarose had solidified, slides were lysed in cold working high salt lysing solution and maintained cold for at least 1 hour. At least two comet slides were removed from lysis for electrophoresis. Slides were rinsed with 0.4M Tris buffer and submerged in alkaline electrophoresis buffer for 20 minutes at 1 to 10°C to unwind the DNA. After unwinding, slides were electrophoresed in the same buffer at 1 to 10°C for 40 minutes at a constant voltage of 0.7V/cm. The buffer level was adjusted as necessary at the start of electrophoresis to achieve a starting current of 300±10 mA. After electrophoresis, the slides were neutralized with 0.4M Tris buffer, rinsed in ethanol, and air dried. The air-dried comet slides were stored at room temperature at a RH of ≤60% until shipment with desiccant to Helix3 for analysis.

Slides were stained with SYBR Gold™ stain and unless precluded by poor cell density and/or poor sample/slide quality, 150 cells per sample (75 cells per slide, if possible) were scored using the Komet© Image Analysis System (Andor Technology, Northern Ireland). The image analysis version and settings were documented in the raw data. For each sample ghosts defined as comets with heavily diffused tail and a non-discernable head that cannot be accurately measured by image analysis were counted in parallel with the image analysis scoring. Slides were scored without knowledge of the sample treatment group.

Evaluation criteria:

Criteria for a Valid Test
a. Where no statistically significant (p<0.05) response in DNA damage as measured by %Tail is detected at any test article dose concentration, the concurrent positive control must induce a statistically significant increase in the same genotoxic endpoint when compared to the concurrent vehicle control.

b. The concurrent negative control must be considered acceptable for addition to the Helix3 historical control database by providing a sufficient dynamic range to detect a statistically significant positive effect.

Criteria for a Positive Response
An experienced scientific investigator classifies a test article as positive, equivocal, or negative for inducing genotoxicity based on the results of the statistical analysis and the biological relevance of the results, taking into consideration the appropriateness of the concurrent control data and the reproducibility of the results in any repeat experiments.

The test article may be classified as positive for inducing genotoxicity if the following criteria are met:

a. a statistically significant increase in DNA damage is detected at one or more dose concentrations and

b. a statistically significant dose dependent response is detected in the same tissue

If cytotoxicity is detected in the same tissue and dose concentration(s) at which a significant increase in DNA damage is detected, cytotoxicity may be considered a confounding factor in the determination of genotoxicity. Where cytotoxicity may be a confounding factor and/or when cytotoxicity is present in all doses tested, a repeat study including lower non-cytotoxic doses may be conducted to evaluate for the presence of genotoxicity in the absence of cytotoxicity.

A test article may be classified as equivocal for inducing genotoxicity if either criteria (a) or (b) are met, but not both. If the results are equivocal and/or the biological relevance of the results are unclear, a repeat study at the same doses may be con

Statistics:

Mean values and standard deviations were calculated. In addition, the median of the values from each slide was determined and for each animal the mean of the median from the slides were calculated. To be consistent with historical control comet data generated at Helix3, the individual animal mean %Tail values was calculated as the mean of the total cells scored. The following statistical analyses were carried out, additionally. For each test, a 95% confidence Interval (P<0.05) was used to determine statistical significance:

Parameter: Statistical test
%Tail and %LMW data distribution: Shapiro-Wilk, test group 0 only

%Tail and %LMW equality of variances: Bartlett test (2-tailed; test groups 0 and 1 to 3; test groups 0 and 4 to 6)

Normally distributed data with equal variances: Dunnett (2-tailed; test groups 1 to 3 compared with test group 0; test groups 4 to 6 compared with test group 0); Line fit trend test (2-tailed; test groups 0 and 1 to 3; test groups 0 and 4 to 6)

Non-normally distributed data or unequal variances: Steel ((2-tailed; test groups 1 to 3 compared with test group 0; test groups 4 to 6 compared with test group 0); Kendall rank trend test (2-tailed; test groups 0 and 1 to 3; test groups 0 and 4 to 6)

%Tail and %LMW References and Positive control only: Fisher’s F-test (2-tailed; test group 7 compared with test group 0; test group 8 compared with test group 0; test group 9 compared with test group 0); student’s t-test for equal variances or Welch’s t-test for unequal variances (1-tailed; test group 7 compared with test group 0; test group 8 compared with test group 0; test group 9 compared with test group 0)

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

COMET ASSAY
- in the nasal epithelium tissue:
The assessment of the DNA damage in the nasal epithelium showed that neither Zinc oxide T0420 nor Zinc oxide T0421 showed a potential to induce DNA damage (see table in any other info on results). The mean % tail DNA of test groups treated with Zinc oxide T0420 ranged between 14.6 – 15.9% (mean of mean values) and 8.3 - 9.3% (mean of medians) and for Zinc oxide T0421 the range was between 15.9 to 16.6% (mean of means) and 8.8 to 10.8% (mean of medians). The values were not statistically significant as compared to the corresponding air control value (16.6 and 10.7% mean of mean and medians, respectively). A dose response was not observed as determined via the two-tailed trend test. Micro-scaled Zinc oxide T0242 showed a lower level of % tail DNA in the comet assay as compared to the air control values. The mean % tail DNA values were 13.4% (mean of means) and 7.8% (mean of medians). The difference between the mean of mean values was statistically significant. However, the difference between the mean of medians was not. Similarly, Zinc sulfate monohydrate also showed lower tail intensity values (15.4 and 7.6% mean of means and medians, respectively) as compared to the air control. However, in this case the drop in the mean of medians was statistically significant but not the mean of means. The group treated with the positive control (EMS) showed a distinct, statistically significant and biologically relevant increase in the mean % DNA tail intensity values (34.9 and 32.3% mean of means and medians, respectively).
The % tail intensity values (mean of means) of all groups (except for the positive control group) were below the upper range (23.1%) of the historical control data range (mean of means).
- in the lung tissue:
The assessment of the DNA damage in the lung tissue also did not show a biologically relevant alteration of the level of observed DNA damage after treatment with either Zinc oxide T0420 or Zinc oxide T0421 as compared to the air control values (The % tail intensity values (mean of means) of all groups (except for the positive control group) were below the upper range (15.3%) of the historical control data range (mean of means) (see see table in any other info on results). The mean % tail DNA of test groups treated with Zinc oxide T0420 ranged between 9.9 – 11.5% (mean of mean values) and 4.9 - 6.9% (mean of medians) and for Zinc oxide T0421 the range was between 8.2 to 10.8% (mean of means) and 4.2 to 5.3% (mean of medians). The values were not statistically significant as compared to the corresponding air control value (11.3 and 5.6% mean of mean and medians, respectively). A dose response was not observed as determined via the two-tailed trend test with T0420, however, a negative trend (dose related decrease in the % tail intensity values) was observed in the mean of mean values of the test groups treated with T0421. This effect was not, however, observed in the mean of median values. Micro-scaled Zinc oxide T0242 showed a similar level of % tail DNA in the comet assay as compared to the air control values. The mean % tail DNA values were 8.6% (mean of means) and 4.0% (mean of medians) and not statistically significant as compared to the air control values. Zinc sulfate monohydrate showed slightly lower tail intensity values (8.3 and 4.1% mean of means and medians, respectively) as compared to the air control. The drop in the mean of means was statistically significant but not the mean of medians. The group treated with the positive control (EMS) showed a distinct, statistically significant and biologically relevant increase in the mean % DNA tail intensity values (52.8 and 52.4% mean of means and medians, respectively).
The % tail intensity values (mean of means) of all groups (except for the positive control group) were below the upper range (15.3%) of the historical control data range (mean of means).
- in the liver:
The % tail DNA intensity in the liver tissue (see table in any other info on results) ranged between 6.9 – 8.7% (mean of means) and 2.7 to 3.8% (mean of medians) for test groups treated with various concentrations of T0420. The mean of mean value of the test group treated with 8 mg/m3 (8.7 ± 1.08%) was statistically higher than the respective air control value (6.6 ± 1.15%). Furthermore, a concentration related trend was observed in the groups treated with 0.5, 2.0 and 8.0 mg/m3 Zinc oxide T0420. However, the mean of median value at 8.0 mg/m3 (3.8 ± 1.19%) was not statistically higher than its respective air control value (2.8 ± 1.00) and the trend observed when using the mean of means was not observed when using the mean of medians. The mean % tail DNA of test groups treated with Zinc oxide T0421 ranged between 7.7 – 8.7% (mean of mean values) and 3.1-3.6% (mean of medians). The values were not statistically significant as compared to the corresponding air control value (6.6 and 2.8% mean of mean and medians, respectively). A dose response was not observed as determined via the two-tailed trend test. Micro-scaled Zinc oxide T0242 showed a similar level of % tail DNA in the comet assay as compared to the air control values. The mean % tail DNA values were 7.6% (mean of means) and 3.2% (mean of medians) and not statistically significant as compared to the air control values. Similarly, Zinc sulfate monohydrate also did not show significantly altered tail intensity values (7.5 and 3.1% mean of means and medians, respectively) as compared to the air control. The group treated with the positive control (EMS) showed a distinct, statistically significant and biologically relevant increase in the mean % DNA tail intensity values (32.1 and 30.7% mean of means and medians, respectively).
The % tail intensity values (mean of means) of all groups (except for the positive control group) were below the upper range (16.9%) of the historical control data range (mean of means).
- in the bone marrow:
The assessment of the DNA damage in the bone showed that neither Zinc oxide T0420 nor Zinc oxide T0421 showed a potential to induce DNA damage (see table in any other info on results). The mean % tail DNA of test groups treated with Zinc oxide T0420 ranged between 6.1 - 6.6% (mean of mean values) and 2.7 – 2.9% (mean of medians) and for Zinc oxide T0421 the range was between 5.2 to 6.6% (mean of means) and 2.6 to 2.9% (mean of medians). The values were not statistically significant as compared to the corresponding air control value (6.2 and 2.9% mean of mean and medians, respectively). A dose response was not observed as determined via the two-tailed trend test. Micro-scaled Zinc oxide T0242 showed a similar level of % tail DNA in the comet assay as compared to the air control values. The mean % tail DNA values were 5.4% (mean of means) and 2.2% (mean of medians) and not statistically significant as compared to the air control values. Similarly, Zinc sulfate monohydrate also showed similar and not statistically significant tail intensity values (6.0 and 2.5% mean of means and medians, respectively) as compared to the air control. The group treated with the positive control (EMS) showed a distinct, statistically significant and biologically relevant increase in the mean % DNA tail intensity values (32.4 and 30.6% mean of means and medians, respectively).
The % tail intensity values (mean of means) of all groups (except for the positive control group) were below the upper range (11.0%) of the historical control data range (mean of means).

CYTOTOXICITY:
The assessment of the potential of the test and reference substances to induce cytotoxicity in the LMW DNA diffusion assay did not show any significant increases in the percentage of diffused low molecular weight DNA in any of the tested tissues and doses (see table in any other info on results), except for micro-scaled Zinc oxide T0242, which induced a statistically higher diffusible LMW DNA (12.6%) as compared to the corresponding air control (8.6%). The positive control (EMS), however, induced statistically significant increases in the amount of diffused LMW DNA in all examined organs.
The comparison of the obtained LMW DNA data with the historical control data showed that all values from the nasal epithelium (including the air control values; 26.8 - 36.8%) were above the maximum value obtained in the historical control data for this tissue (8.0%). In the lung all values (8.4 - 12.6%), except for the positive control group, were below the maximum value of the historical control data range for the lung (17.6%). In the liver the values were similarly below the maximum historical control data range (13.6%), except for the low dose group of T0420, which had a value of 15.2% and the positive control group (38.2%). All bone marrow values (12.0 – 16.8% for the test and reference substances and 34.6% for the positive control group) were also above the upper limit of the historical control data (6.8%).


TOXICITY
- General toxicity: No Signs of general toxicity. No impairment of body weight gain.
- Local effects: In lavage, the exposure of single concentration of reference substance 1 (T0242) and reference substance 2 (zinc sulfate monohydrate) caused significantly increases in most of the lavage parameters, indicating inflammation process in the lung (see table ' Changes in mean total protein and enzyme levels in BAL' in any other info on results)

Table. Results of the comet assay in the nasal epithelium

Test group	Test Substance	Concentration (mg/m^3)	% Tail intensity ± SD
			Mean of means	Mean of medians
0	Air control	0	16.6 ± 2.64	10.7 ± 2.89
1	Zinc Oxide T0420	0.5	14.6 ± 3.62	9.3 ± 2.64
2		2.0	15.9 ± 3.60	9.0 ± 2.86
3		8.0	14.8 ± 3.00	8.3 ± 1.69
4	Zinc Oxide T0421	0.5	16.6 ± 5.28	10.8 ± 5.05
5		2.0	15.9 ± 4.73	9.9 ± 4.03
6		8.0	16.0 ± 3.34	8.8 ± 3.91
7	Zinc Oxide T0242	8.0	13.4 ± 2.67^#	7.8 ± 2.13
8	Zinc sulfate monohydrate	18.0	15.4 ± 1.89	7.6 ± 1.28^$
9	EMS	300 mg/kg b.w.	34.9 ± 3.63^*	32.3 ± 4.12^*

^#: statistically significant P=0.048 (decrease)
^$: statistically significant P=0.031 (decrease)
^*: statistically significant P<0.001 (increase)

 

Table. Results of the comet assay in the lung

Test group	Test Substance	Concentration (mg/m^3)	% Tail intensity ± SD
			Mean of means	Mean of medians
0	Air control	0	11.3 ± 1.93	5.6 ± 1.50
1	Zinc Oxide T0420	0.5	11.5 ± 3.25	6.9 ± 4.15
2		2.0	9.9 ± 1.62	4.9 ± 1.19
3		8.0	10.1 ± 2.70	5.4 ± 2.89
4	Zinc Oxide T0421	0.5	10.8 ± 3.68^1	5.3 ± 2.67
5		2.0	9.0 ± 1.54^1	4.5 ± 1.34
6		8.0	8.2 ± 1.58^1	4.2 ± 1.60
7	Zinc Oxide T0242	8.0	8.6 ± 2.85	4.0 ± 1.60
8	Zinc sulfate monohydrate	18.0	8.3 ± 1.69^#	4.1 ± 1.51
9	EMS	300 mg/kg b.w.	52.8 ± 6.84^*	52.4 ± 7.84^*

^#: statistically significant P=0.015 (decrease)
^*: statistically significant P<0.001 (increase)
¹: negative trend statistically significant P=0.047

 

Table. Results of the comet assay in the liver

Test group	Test Substance	Concentration (mg/m^3)	% Tail intensity ± SD
			Mean of means	Mean of medians
0	Air control	0	6.6 ± 1.15	2.8 ± 1.00
1	Zinc Oxide T0420	0.5	7.9 ± 0.49^1	3.2 ± 0.53
2		2.0	6.9 ± 1.30^1	2.7 ± 0.38
3		8.0	8.7 ± 1.08^#1	3.8 ± 1.19
4	Zinc Oxide T0421	0.5	8.7 ± 0.91	3.1 ± 0.80
5		2.0	7.8 ± 1.05	3.6 ± 0.73
6		8.0	7.7 ± 2.25	3.1 ± 0.83
7	Zinc Oxide T0242	8.0	7.6 ± 2.03	3.2 ± 1.11
8	Zinc sulfate monohydrate	18.0	7.5 ± 2.03	3.1 ± 1.26
9	EMS	300 mg/kg b.w.	32.1 ± 2.55^*	30.7 ± 3.07^*

^#: statistically significant P=0.016
^*: statistically significant P<0.001
¹: positive trend statistically significant P=0.032

 

Table. Results of the comet assay in the bone marrow

Test group	Test Substance	Concentration (mg/m^3)	% Tail intensity ± SD
			Mean of means	Mean of medians
0	Air control	0	6.2 ± 1.00	2.9 ± 0.66
1	Zinc Oxide T0420	0.5	6.6 ± 1.02	2.9 ± 0.52
2		2.0	6.5 ± 0.78	2.8 ± 0.76
3		8.0	6.1 ± 0.30	2.7 ± 0.42
4	Zinc Oxide T0421	0.5	6.0 ± 0.60	2.9 ± 0.68
5		2.0	6.6 ± 1.63	2.9 ± 0.67
6		8.0	5.2 ± 0.80	2.6 ± 0.75
7	Zinc Oxide T0242	8.0	5.4 ± 0.55	2.2 ± 0.54
8	Zinc sulfate monohydrate	18.0	6.0 ± 0.83	2.5 ± 0.37
9	EMS	300 mg/kg b.w.	32.4 ± 4.84^*	30.6 ± 5.53^*

^*: statistically significant P<0.001

Table. Results of the LMW DNA diffusion assay

Test group	Test Substance	Concentration (mg/m³)	Group Mean ± SD (%)
			Nasal epithelium	Lung	Liver	Bone marrow
0	Air control	0	30.0 ± 9.19	8.6 ± 1.52	12.6 ± 5.37	14.8 ± 2.17
1	Zinc Oxide T0420	0.5	26.8 ± 6.76	11.6 ± 3.29	15.2 ± 6.02	13.6 ± 2.07
2		2.0	33.8 ± 8.98	11.4 ± 2.51	13.2 ± 3.35	14.0 ± 3.54
3		8.0	35.8 ± 16.40	10.6 ± 1.52	13.4 ± 6.95	13.8 ± 5.02
4	Zinc Oxide T0421	0.5	31.6 ± 8.62	11.8 ± 3.56	12.6 ± 6.80	12.0 ± 3.54
5		2.0	36.8 ± 15.67	8.4 ± 3.36	10.0 ± 4.30	16.8 ± 5.89
6		8.0	34.6 ± 14.24	10.4 ± 2.61	11.0 ± 3.74	15.2 ± 5.40
7	Zinc Oxide T0242	8.0	29.0 ± 11.05	12.6 ± 4.22^€	11.8 ± 4.09	15.6 ± 5.98
8	Zinc sulfate monohydrate	18.0	33.0 ± 8.80	12.0 ± 5.92	10.6 ± 7.64	14.6 ± 6.66
9	EMS	300 mg/kg b.w.	39.8 ± 4.49^*	26.8 ± 13.37^#	38.2 ± 7.05^$	34.6 ± 6.73^$

^*: statistically significant P=0.032
^€: statistically significant P=0.041
^#: statistically significant P=0.019
^$: statistically significant P=0.032

 

Table. Changes in mean total protein and enzyme levels in BAL (x-fold of concurrent control) in male rats after inhalation exposure to Zinc oxide T0242 and zinc sulfate monohydrate on 14 consecutive days

Analyte	Gr. 7 8 mg/m^3	Gr8 18 mg/m^3
Total Protein	7.6**	2.4**
GGT	4.2**	3.9**
LDH	13.2**	4.6**
ALP	19.1**	4.3**
NAG	2.4**	1.5

GGT = γ-Glutamyl-transferase; LDH = Lactate dehydrogenase; ALP = Alkaline phosphatase; NAG = β-N-Acetyl glucosaminidase, One-sided Wilcoxon-test: * : p £ 0.05; ** : p £ 0.01

Conclusions:

In this study, male Wistar rats were whole-body exposed to dust aerosols of T0420 and T0421 at target concentrations of 0.5, 2 and 8 mg/m³, as well as to 8 mg/m³ miconsize zinc oxide or 18 mg/m³ zinc sulfate monohydrate for 6 hours daily on 14 consecutive days. A concurrent control group was exposed to conditioned air.

The tested atmospheric were met and they were maintained throughout the study. Cascade impactor measurement of both substances showed particle sizes within the respirable range. There were no signs of toxicity, nor were there any impairment of body weight gain. In the range finding 14-day study (BASF project no.: 36I0050/20I005) animals of the mid dose groups were nominally exposed to 12 mg/m3. The actual assessment of test substance concentrations, however, revealed an exposure to 10.9 and 10.8 mg/m3 for T0420 and T0421, respectively. At this dose level significant histopathological alterations (grade 2 degeneration/regeneration, olfactory epithelium) as well as alterations indicative of inflammatory responses in the lung were observed, which could have had a confounding impact on the outcome of the comet assays results. Thus, the top exposure concentration was slightly reduced to 8 mg/m3. At this level in the lavage fluid, several parameters were increased in a concentration related manner in animals exposed to T0420 and T0421, indicating inflammation process in the lung. These findings were comparable with those observed previously in the range finding 14-day inhalation study with these two test materials. Similar findings were observed in animals exposed to the reference substances.


LMW DNA diffusion assay
In all test groups using the test or reference substances, the quantification of the diffused low molecular weight DNA as a measure for cytotoxicity did not show any indication of excessive cytotoxicity as compared to the respective air control value, which could have hampered the interpretation of the comet assay data. The significantly increased value observed for the lung using micro-scaled Zinc oxide T0242 was not high enough to interfere the comet data evaluation. The positive control group, however, showed significant increases in the LMW DNA value. This is, however, expected from the positive control considering the amount of DNA damage it has induced. The fact that all diffusible LMW DNA values from bone marrow and nasal epithelium samples were above the historical control data, does not have an impact on the data interpretation, since the air control values were also above the historical control data range and furthermore, the high LMW DNA values did not have an impact on the % tail intensities, which were all within the historical control data range for these two organs.

Comet assay
The study is considered as valid, since all concurrent negative control data are acceptable for addition to the historical data base. Furthermore, the positive control group showed that the test is able to detect positive response in all assessed tissues.
The assessment of the comet assay in the various tissues did not show and biologically relevant indication of a genotoxic potential in any of the test or reference substances. The significant concentration dependent increase observed in the mean of mean values obtained from the liver of animals treated with T0420 is not considered as relevant, since the same values were not statistically significant, when the mean of medians was used instead of mean of means. This indicates that the observed effect is more likely due to the greater impact of individual values on the group mean value when using the mean of means. The current OECD guideline, therefore, also recommends the use of mean of medians. The statistically significant decreases in the observed in the lung and nasal epithelium using Zinc oxide T0421, micro-scaled Zinc oxide T0242 and Zinc sulfate are not considered as biologically relevant effects, since once again the statistical significance is only observed in either mean of mean or mean of median calculations.

CONCLUSION
In conclusion it can be stated that under the described circumstances neither Zinc oxide T0420 nor Zinc oxide T0421 showed a genotoxic potential in the nasal epithelium, lung liver and bone marrow of rats exposed for 14 days via inhalation.

Executive summary:

In this study two nanosized test substances Zinc oxide T0420 and Zinc oxide T0421 were assessed for their genotoxic potential using the alkaline comet assay after a 14-day exposure period via inhalation. This was a multisite study, where the in-life phase, necropsy, as well as the examination of the lung lavage fluid was performed by the test facility. The processing of the isolated tissues and the comet assay was performed by the test site (Helix 3 Inc.). The target tissues addressed in this study were the nasal epithelium, the lung, the liver as well as the bone marrow.

The concentrations used in this study was based on a separately performed dose range finding assay (BASF Project no.: 36I0050/20I005). The three concentrations ,selected based on the dose range finding study, were 0.5, 2.0 and 8.0 mg/m³.  Wistar rats were exposed whole-body to the indicated concentrations of each test substance for a 6 h period per day for 14 days. In addition, for comparison, a micro-scaled Zinc oxide as well as well as a soluble Zinc salt (Zinc sulfate monohydrate) were tested in parallel under the same conditions at a single (equimolar) concentration. Ethylmethane sulfonate (EMS) was used as a positive control and applied orally at a single time point.

During the exposure period, the animals were observed for signs of toxicity before, during and after the exposure. Body weight was determined once weekly. The following mean concentrations and particle size distribution were determined.

In animals exposed to test item 1, concentration-related increases of lavage parameters were observed, as well as in animals exposed to test item 2. The exposure of single concentration of reference substance 1 and 2 caused significantly increases in most of the lavage parameters. The changes in lavage fluid demonstrate the toxicity in the lungs, which were comparable with the range finding study.

The assessment of the target tissues in the comet assay did not show any biologically relevant increases in the % tail intensity of the analyzed tissues under the indicated conditions. The positive control group showed a distinct and statistically significant increase in all analyzed tissues.

Thus, under the indicated circumstances, the two test substances as well as the reference substances (micro-scaled Zinc oxide and Zinc sulfate monohydrate) are considered as non-genotoxic in this assay.

 

Reference 2

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 December 2021 to June 2022

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study presented herein is a guideline study with a major deficiency under GLP conditions. Only one concentration level was tested, which precludes an evaluation of dose-response relationships.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

adopted 29 July 2016

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Specific details on test material used for the study:

Expiry date: 30 June 2022

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Wistar rats, Crl:WI(Han)
Rats were selected since this rodent species is recommended in the respective test guidelines. Wistar rats were selected since there is extensive experience available in the laboratory with this strain of rats.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland (Sulzfeld/Germany)
- Age at study initiation: approx. 7 weeks
- Weight at study initiation: The weight variation of the animals used did not exceed +/- 20 percent of the mean weight of each sex.
- Assigned to test groups randomly: yes: All animals were randomized before the start of the pre-exposure period (according to weight).
- Fasting period before study: No
- Housing: 5 rats per cage, Typ 2000P ca. 2065 cm2 (polysulfone cages) supplied by TECNIPLAST, Germany. Dust-free wooden bedding
- Diet: milled/ mouse and rat maintenance diet, GLP, 12 mm pellets, Granovit AG, Kaiseraugst, Switzerland; ad libitum
- Water: tap water; ad libitum
- Acclimation period: +/- 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 22-24°C
- Humidity: 45-65%
- Air changes: 15 air changes per hour
- Photoperiod: 12 hrs dark / 12 hrs light

Route of administration:

inhalation: aerosol

Vehicle:

- Vehicle(s)/solvent(s) used: clean air

Details on exposure:

TYPE OF INHALATION EXPOSURE: whole-body

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Generation of the inhalation atmospheres via a solid particle generators (brush-generator; BASF SE, Ludwigshafen, Germany) & Aerosol mixing tube (stainless steel; BASF SE, Ludwigshafen, Germany). Whole body exposure systems were used. The animals were kept singly in wire cages located in a glass steel inhalation chamber, volume of 1.1 m³ or 1.4 m³(BASF SE).
- Method of holding animals in test chamber: Whole body exposure systems. The animals were kept singly in wire cages located in a glass steel inhalation chamber, volume of 1.1 m³ or 1.4 m³(BASF SE). The chambers were located in exhaust hoods in an air conditioned room.
- Source and rate of air: Conditioned air from the central air conditioning system, compressed and exhaust air. Compressed air was produced by an oil-free compressor (HT 6, Josef Mehrer GmbH & Co KG, Germany). For this purpose, air is filtered by an inlet air strainer and introduced into the compressor. After passing through an second ultra filter (SMF 5/3, 108 mm, Donalson), the compressed air (15 bar) is stored in a storage of 1500 or 5000 L. The compressed air is conducted to the laboratories via pipes, where the pressure is reduced to 5 - 6 bar. In the laboratory, the compressed air can be taken as required.
- Method of conditioning air: Conditioned air from the central air conditioning system provides cold air of about 15°C. This cold air passes through an activated charcoal filter, is adjusted to room temperature of 20 to 24°C and passes through a second particle filter (H13 (HEPA) Camfil Farr, Germany). The so generated conditioned air was used to generate inhalation atmospheres.
- System of generating particulates/aerosols: The particles/aerosol was generated via a solid particle generator (brush-generator; BASF SE, Ludwigshafen, Germany) and an aerosol mixing tube (stainless steel; BASF SE, Ludwigshafen, Germany), according to the following method: For each concentration the dust aerosol was generated with the dust generator and compressed air inside a mixing stage; mixed with conditioned dilution air and passed into the inhalation system.
- Temperature, humidity, pressure in air chamber: Daily mean relative humidities in the inhalation systems ranged between 40.7 and 50.8 %. Daily mean temperatures in the inhalation systems ranged between 20.4 and 23.0°C. They are within the range suggested by the respective testing guidelines.
- Air flow rate: The air flows were constantly maintained in the desired range.
- Air change rate: An air change of about 20 times per hour can be calculated by dividing the supply air flow through the volume of each inhalation system.
- Method of particle size determination: The particle size analysis was carried out with a cascade impactor. Equipment for particle size analysis: Stack sampler Marple 298 (New Star Environmental, Inc., Roswell, Georgia 30075, USA) ; Vacuum compressed air pump (Millipore Corporation, Billerica, MA 01821, USA) ; Limiting orifice 3 L/min (Millipore Corporation, Billerica, MA 01821, USA) ; Sampling probe internal diameter 6.9 mm ; Balance Sartorius MSA 6.6S-000-DF (Sartorius AG, Göttingen, Germany). The calculation of the particle size distribution was carried out in the Laboratory for Inhalation Toxicology of the Experimental Toxicology and Ecology of BASF SE on the basis of mathematical methods for evaluating particle measurements (OECD guidance document No. 39).
To determine the particle size distribution in the submicrometer range, each test atmosphere was measured with the Scanning Mobility Particle Sizer (SMPS; Grimm Aerosol Technik GmbH & Co KG, Ainring, Germany). The SMPS system comprises an Electrostatic Classifier (Model Vienna U-DMA) which separates the particles into known size fractions, and a Condensation Particle Counter (CPC) which measures particle count concentrations. The DMA was equipped with Am-241 neutralizer. The sampling duration was about 7 minutes. As a rule 10 repeats were measured for each exposure concentration.
- Treatment of exhaust air: Exhaust air was filtered and conducted into the exhaust air of the building.

TEST ATMOSPHERE
- Brief description of analytical method used: The concentrations of the inhalation atmospheres were determined by gravimetrical measurements of filter samples in all test groups. Control group was not sampled. This analytical method was judged to be valid because the test substances did not possess an appreciable vapor pressure.
- Samples taken from breathing zone: yes

Duration of treatment / exposure:

14 days

Frequency of treatment:

14 days, 6 h per day

Dose / conc.:

0 mg/m³ air

Remarks:

Test Group 0 - air control

Dose / conc.:

17.82 mg/m³ air (analytical)

Remarks:

SD: ± 0.90 mg/m³ ; target concentration: 18 mg/m³: Test group 8

No. of animals per sex per dose:

5 male rats per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

ethylmethanesulphonate
- Justification for choice of positive control(s):
- Route of administration: oral gavage which guarantees systemic distribution of the compound and thus exposure to all assessed tissues
- Doses / concentrations: 300mg/kg body weight

Tissues and cell types examined:

Comet assay:
1. Bone marrow
2. Liver
3. Lung
4. Nasal mucosa

Bronchoalveolar lavage fluid (BAL): for cytology and total protein and enzyme levels.

Details of tissue and slide preparation:

COMET ASSAY
Samples were minced or aspirated in cold mincing buffer to produce a cell suspension. Cell suspensions were diluted as necessary and kept cold until they were processed further. At least three comet slides were prepared per sample. An aliquot of cell suspension was mixed with low melting point agarose, layered onto microscope slides precoated with normal melting point agarose, and covered with an additional layer of low melting point agarose. After the agarose had solidified, slides were lysed in cold working high salt lysing solution and maintained cold for at least 1 hour. At least two comet slides were removed from lysis for electrophoresis. Slides were rinsed with 0.4M Tris buffer and submerged in alkaline electrophoresis buffer for 20 minutes at 1 to 10°C to unwind the DNA. After unwinding, slides were electrophoresed in the same buffer at 1 to 10°C for 40 minutes at a constant voltage of 0.7V/cm. The buffer level was adjusted as necessary at the start of electrophoresis to achieve a starting current of 300±10 mA. After electrophoresis, the slides were neutralized with 0.4M Tris buffer, rinsed in ethanol, and air dried. The air-dried comet slides were stored at room temperature at a RH of ≤60% until shipment with desiccant to Helix3 for analysis.

Slides were stained with SYBR Gold™ stain and unless precluded by poor cell density and/or poor sample/slide quality, 150 cells per sample (75 cells per slide, if possible) were scored using the Komet© Image Analysis System (Andor Technology, Northern Ireland). The image analysis version and settings were documented in the raw data. For each sample ghosts defined as comets with heavily diffused tail and a non-discernable head that cannot be accurately measured by image analysis were counted in parallel with the image analysis scoring. Slides were scored without knowledge of the sample treatment group.

Evaluation criteria:

Criteria for a Valid Test
a. Where no statistically significant (p<0.05) response in DNA damage as measured by %Tail is detected at any test article dose concentration, the concurrent positive control must induce a statistically significant increase in the same genotoxic endpoint when compared to the concurrent vehicle control.

b. The concurrent negative control must be considered acceptable for addition to the Helix3 historical control database by providing a sufficient dynamic range to detect a statistically significant positive effect.

Criteria for a Positive Response
An experienced scientific investigator classifies a test article as positive, equivocal, or negative for inducing genotoxicity based on the results of the statistical analysis and the biological relevance of the results, taking into consideration the appropriateness of the concurrent control data and the reproducibility of the results in any repeat experiments.

The test article may be classified as positive for inducing genotoxicity if the following criteria are met:

a. a statistically significant increase in DNA damage is detected at one or more dose concentrations and

b. a statistically significant dose dependent response is detected in the same tissue

If cytotoxicity is detected in the same tissue and dose concentration(s) at which a significant increase in DNA damage is detected, cytotoxicity may be considered a confounding factor in the determination of genotoxicity. Where cytotoxicity may be a confounding factor and/or when cytotoxicity is present in all doses tested, a repeat study including lower non-cytotoxic doses may be conducted to evaluate for the presence of genotoxicity in the absence of cytotoxicity.

A test article may be classified as equivocal for inducing genotoxicity if either criteria (a) or (b) are met, but not both. If the results are equivocal and/or the biological relevance of the results are unclear, a repeat study at the same doses may be con

Statistics:

Mean values and standard deviations were calculated. In addition, the median of the values from each slide was determined and for each animal the mean of the median from the slides were calculated. To be consistent with historical control comet data generated at Helix3, the individual animal mean %Tail values was calculated as the mean of the total cells scored. The following statistical analyses were carried out, additionally. For each test, a 95% confidence Interval (P<0.05) was used to determine statistical significance:

Parameter: Statistical test
%Tail and %LMW data distribution: Shapiro-Wilk, test group 0 only

%Tail and %LMW equality of variances: Bartlett test (2-tailed; test groups 0 and 1 to 3; test groups 0 and 4 to 6)

Normally distributed data with equal variances: Dunnett (2-tailed; test groups 1 to 3 compared with test group 0; test groups 4 to 6 compared with test group 0); Line fit trend test (2-tailed; test groups 0 and 1 to 3; test groups 0 and 4 to 6)

Non-normally distributed data or unequal variances: Steel ((2-tailed; test groups 1 to 3 compared with test group 0; test groups 4 to 6 compared with test group 0); Kendall rank trend test (2-tailed; test groups 0 and 1 to 3; test groups 0 and 4 to 6)

%Tail and %LMW References and Positive control only: Fisher’s F-test (2-tailed; test group 7 compared with test group 0; test group 8 compared with test group 0; test group 9 compared with test group 0); student’s t-test for equal variances or Welch’s t-test for unequal variances (1-tailed; test group 7 compared with test group 0; test group 8 compared with test group 0; test group 9 compared with test group 0)

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

COMET ASSAY
- in the nasal epithelium tissue:
The assessment of the DNA damage in the nasal epithelium showed that neither Zinc oxide T0420 nor Zinc oxide T0421 showed a potential to induce DNA damage (see table in any other info on results). The mean % tail DNA of test groups treated with Zinc oxide T0420 ranged between 14.6 – 15.9% (mean of mean values) and 8.3 - 9.3% (mean of medians) and for Zinc oxide T0421 the range was between 15.9 to 16.6% (mean of means) and 8.8 to 10.8% (mean of medians). The values were not statistically significant as compared to the corresponding air control value (16.6 and 10.7% mean of mean and medians, respectively). A dose response was not observed as determined via the two-tailed trend test. Micro-scaled Zinc oxide T0242 showed a lower level of % tail DNA in the comet assay as compared to the air control values. The mean % tail DNA values were 13.4% (mean of means) and 7.8% (mean of medians). The difference between the mean of mean values was statistically significant. However, the difference between the mean of medians was not. Similarly, Zinc sulfate monohydrate also showed lower tail intensity values (15.4 and 7.6% mean of means and medians, respectively) as compared to the air control. However, in this case the drop in the mean of medians was statistically significant but not the mean of means. The group treated with the positive control (EMS) showed a distinct, statistically significant and biologically relevant increase in the mean % DNA tail intensity values (34.9 and 32.3% mean of means and medians, respectively).
The % tail intensity values (mean of means) of all groups (except for the positive control group) were below the upper range (23.1%) of the historical control data range (mean of means).
- in the lung tissue:
The assessment of the DNA damage in the lung tissue also did not show a biologically relevant alteration of the level of observed DNA damage after treatment with either Zinc oxide T0420 or Zinc oxide T0421 as compared to the air control values (The % tail intensity values (mean of means) of all groups (except for the positive control group) were below the upper range (15.3%) of the historical control data range (mean of means) (see see table in any other info on results). The mean % tail DNA of test groups treated with Zinc oxide T0420 ranged between 9.9 – 11.5% (mean of mean values) and 4.9 - 6.9% (mean of medians) and for Zinc oxide T0421 the range was between 8.2 to 10.8% (mean of means) and 4.2 to 5.3% (mean of medians). The values were not statistically significant as compared to the corresponding air control value (11.3 and 5.6% mean of mean and medians, respectively). A dose response was not observed as determined via the two-tailed trend test with T0420, however, a negative trend (dose related decrease in the % tail intensity values) was observed in the mean of mean values of the test groups treated with T0421. This effect was not, however, observed in the mean of median values. Micro-scaled Zinc oxide T0242 showed a similar level of % tail DNA in the comet assay as compared to the air control values. The mean % tail DNA values were 8.6% (mean of means) and 4.0% (mean of medians) and not statistically significant as compared to the air control values. Zinc sulfate monohydrate showed slightly lower tail intensity values (8.3 and 4.1% mean of means and medians, respectively) as compared to the air control. The drop in the mean of means was statistically significant but not the mean of medians. The group treated with the positive control (EMS) showed a distinct, statistically significant and biologically relevant increase in the mean % DNA tail intensity values (52.8 and 52.4% mean of means and medians, respectively).
The % tail intensity values (mean of means) of all groups (except for the positive control group) were below the upper range (15.3%) of the historical control data range (mean of means).
- in the liver:
The % tail DNA intensity in the liver tissue (see table in any other info on results) ranged between 6.9 – 8.7% (mean of means) and 2.7 to 3.8% (mean of medians) for test groups treated with various concentrations of T0420. The mean of mean value of the test group treated with 8 mg/m3 (8.7 ± 1.08%) was statistically higher than the respective air control value (6.6 ± 1.15%). Furthermore, a concentration related trend was observed in the groups treated with 0.5, 2.0 and 8.0 mg/m3 Zinc oxide T0420. However, the mean of median value at 8.0 mg/m3 (3.8 ± 1.19%) was not statistically higher than its respective air control value (2.8 ± 1.00) and the trend observed when using the mean of means was not observed when using the mean of medians. The mean % tail DNA of test groups treated with Zinc oxide T0421 ranged between 7.7 – 8.7% (mean of mean values) and 3.1-3.6% (mean of medians). The values were not statistically significant as compared to the corresponding air control value (6.6 and 2.8% mean of mean and medians, respectively). A dose response was not observed as determined via the two-tailed trend test. Micro-scaled Zinc oxide T0242 showed a similar level of % tail DNA in the comet assay as compared to the air control values. The mean % tail DNA values were 7.6% (mean of means) and 3.2% (mean of medians) and not statistically significant as compared to the air control values. Similarly, Zinc sulfate monohydrate also did not show significantly altered tail intensity values (7.5 and 3.1% mean of means and medians, respectively) as compared to the air control. The group treated with the positive control (EMS) showed a distinct, statistically significant and biologically relevant increase in the mean % DNA tail intensity values (32.1 and 30.7% mean of means and medians, respectively).
The % tail intensity values (mean of means) of all groups (except for the positive control group) were below the upper range (16.9%) of the historical control data range (mean of means).
- in the bone marrow:
The assessment of the DNA damage in the bone showed that neither Zinc oxide T0420 nor Zinc oxide T0421 showed a potential to induce DNA damage (see table in any other info on results). The mean % tail DNA of test groups treated with Zinc oxide T0420 ranged between 6.1 - 6.6% (mean of mean values) and 2.7 – 2.9% (mean of medians) and for Zinc oxide T0421 the range was between 5.2 to 6.6% (mean of means) and 2.6 to 2.9% (mean of medians). The values were not statistically significant as compared to the corresponding air control value (6.2 and 2.9% mean of mean and medians, respectively). A dose response was not observed as determined via the two-tailed trend test. Micro-scaled Zinc oxide T0242 showed a similar level of % tail DNA in the comet assay as compared to the air control values. The mean % tail DNA values were 5.4% (mean of means) and 2.2% (mean of medians) and not statistically significant as compared to the air control values. Similarly, Zinc sulfate monohydrate also showed similar and not statistically significant tail intensity values (6.0 and 2.5% mean of means and medians, respectively) as compared to the air control. The group treated with the positive control (EMS) showed a distinct, statistically significant and biologically relevant increase in the mean % DNA tail intensity values (32.4 and 30.6% mean of means and medians, respectively).
The % tail intensity values (mean of means) of all groups (except for the positive control group) were below the upper range (11.0%) of the historical control data range (mean of means).

CYTOTOXICITY:
The assessment of the potential of the test and reference substances to induce cytotoxicity in the LMW DNA diffusion assay did not show any significant increases in the percentage of diffused low molecular weight DNA in any of the tested tissues and doses (see table in any other info on results), except for micro-scaled Zinc oxide T0242, which induced a statistically higher diffusible LMW DNA (12.6%) as compared to the corresponding air control (8.6%). The positive control (EMS), however, induced statistically significant increases in the amount of diffused LMW DNA in all examined organs.
The comparison of the obtained LMW DNA data with the historical control data showed that all values from the nasal epithelium (including the air control values; 26.8 - 36.8%) were above the maximum value obtained in the historical control data for this tissue (8.0%). In the lung all values (8.4 - 12.6%), except for the positive control group, were below the maximum value of the historical control data range for the lung (17.6%). In the liver the values were similarly below the maximum historical control data range (13.6%), except for the low dose group of T0420, which had a value of 15.2% and the positive control group (38.2%). All bone marrow values (12.0 – 16.8% for the test and reference substances and 34.6% for the positive control group) were also above the upper limit of the historical control data (6.8%).


TOXICITY
- General toxicity: No Signs of general toxicity. No impairment of body weight gain.
- Local effects: In lavage, the exposure of single concentration of reference substance 1 (T0242) and reference substance 2 (zinc sulfate monohydrate) caused significantly increases in most of the lavage parameters, indicating inflammation process in the lung (see table ' Changes in mean total protein and enzyme levels in BAL' in any other info on results)

Table. Results of the comet assay in the nasal epithelium

Test group	Test Substance	Concentration (mg/m^3)	% Tail intensity ± SD
			Mean of means	Mean of medians
0	Air control	0	16.6 ± 2.64	10.7 ± 2.89
1	Zinc Oxide T0420	0.5	14.6 ± 3.62	9.3 ± 2.64
2		2.0	15.9 ± 3.60	9.0 ± 2.86
3		8.0	14.8 ± 3.00	8.3 ± 1.69
4	Zinc Oxide T0421	0.5	16.6 ± 5.28	10.8 ± 5.05
5		2.0	15.9 ± 4.73	9.9 ± 4.03
6		8.0	16.0 ± 3.34	8.8 ± 3.91
7	Zinc Oxide T0242	8.0	13.4 ± 2.67^#	7.8 ± 2.13
8	Zinc sulfate monohydrate	18.0	15.4 ± 1.89	7.6 ± 1.28^$
9	EMS	300 mg/kg b.w.	34.9 ± 3.63^*	32.3 ± 4.12^*

^#: statistically significant P=0.048 (decrease)
^$: statistically significant P=0.031 (decrease)
^*: statistically significant P<0.001 (increase)

 

Table. Results of the comet assay in the lung

Test group	Test Substance	Concentration (mg/m^3)	% Tail intensity ± SD
			Mean of means	Mean of medians
0	Air control	0	11.3 ± 1.93	5.6 ± 1.50
1	Zinc Oxide T0420	0.5	11.5 ± 3.25	6.9 ± 4.15
2		2.0	9.9 ± 1.62	4.9 ± 1.19
3		8.0	10.1 ± 2.70	5.4 ± 2.89
4	Zinc Oxide T0421	0.5	10.8 ± 3.68^1	5.3 ± 2.67
5		2.0	9.0 ± 1.54^1	4.5 ± 1.34
6		8.0	8.2 ± 1.58^1	4.2 ± 1.60
7	Zinc Oxide T0242	8.0	8.6 ± 2.85	4.0 ± 1.60
8	Zinc sulfate monohydrate	18.0	8.3 ± 1.69^#	4.1 ± 1.51
9	EMS	300 mg/kg b.w.	52.8 ± 6.84^*	52.4 ± 7.84^*

^#: statistically significant P=0.015 (decrease)
^*: statistically significant P<0.001 (increase)
¹: negative trend statistically significant P=0.047

 

Table. Results of the comet assay in the liver

Test group	Test Substance	Concentration (mg/m^3)	% Tail intensity ± SD
			Mean of means	Mean of medians
0	Air control	0	6.6 ± 1.15	2.8 ± 1.00
1	Zinc Oxide T0420	0.5	7.9 ± 0.49^1	3.2 ± 0.53
2		2.0	6.9 ± 1.30^1	2.7 ± 0.38
3		8.0	8.7 ± 1.08^#1	3.8 ± 1.19
4	Zinc Oxide T0421	0.5	8.7 ± 0.91	3.1 ± 0.80
5		2.0	7.8 ± 1.05	3.6 ± 0.73
6		8.0	7.7 ± 2.25	3.1 ± 0.83
7	Zinc Oxide T0242	8.0	7.6 ± 2.03	3.2 ± 1.11
8	Zinc sulfate monohydrate	18.0	7.5 ± 2.03	3.1 ± 1.26
9	EMS	300 mg/kg b.w.	32.1 ± 2.55^*	30.7 ± 3.07^*

^#: statistically significant P=0.016
^*: statistically significant P<0.001
¹: positive trend statistically significant P=0.032

 

Table. Results of the comet assay in the bone marrow

Test group	Test Substance	Concentration (mg/m^3)	% Tail intensity ± SD
			Mean of means	Mean of medians
0	Air control	0	6.2 ± 1.00	2.9 ± 0.66
1	Zinc Oxide T0420	0.5	6.6 ± 1.02	2.9 ± 0.52
2		2.0	6.5 ± 0.78	2.8 ± 0.76
3		8.0	6.1 ± 0.30	2.7 ± 0.42
4	Zinc Oxide T0421	0.5	6.0 ± 0.60	2.9 ± 0.68
5		2.0	6.6 ± 1.63	2.9 ± 0.67
6		8.0	5.2 ± 0.80	2.6 ± 0.75
7	Zinc Oxide T0242	8.0	5.4 ± 0.55	2.2 ± 0.54
8	Zinc sulfate monohydrate	18.0	6.0 ± 0.83	2.5 ± 0.37
9	EMS	300 mg/kg b.w.	32.4 ± 4.84^*	30.6 ± 5.53^*

^*: statistically significant P<0.001

Table. Results of the LMW DNA diffusion assay

Test group	Test Substance	Concentration (mg/m³)	Group Mean ± SD (%)
			Nasal epithelium	Lung	Liver	Bone marrow
0	Air control	0	30.0 ± 9.19	8.6 ± 1.52	12.6 ± 5.37	14.8 ± 2.17
1	Zinc Oxide T0420	0.5	26.8 ± 6.76	11.6 ± 3.29	15.2 ± 6.02	13.6 ± 2.07
2		2.0	33.8 ± 8.98	11.4 ± 2.51	13.2 ± 3.35	14.0 ± 3.54
3		8.0	35.8 ± 16.40	10.6 ± 1.52	13.4 ± 6.95	13.8 ± 5.02
4	Zinc Oxide T0421	0.5	31.6 ± 8.62	11.8 ± 3.56	12.6 ± 6.80	12.0 ± 3.54
5		2.0	36.8 ± 15.67	8.4 ± 3.36	10.0 ± 4.30	16.8 ± 5.89
6		8.0	34.6 ± 14.24	10.4 ± 2.61	11.0 ± 3.74	15.2 ± 5.40
7	Zinc Oxide T0242	8.0	29.0 ± 11.05	12.6 ± 4.22^€	11.8 ± 4.09	15.6 ± 5.98
8	Zinc sulfate monohydrate	18.0	33.0 ± 8.80	12.0 ± 5.92	10.6 ± 7.64	14.6 ± 6.66
9	EMS	300 mg/kg b.w.	39.8 ± 4.49^*	26.8 ± 13.37^#	38.2 ± 7.05^$	34.6 ± 6.73^$

^*: statistically significant P=0.032
^€: statistically significant P=0.041
^#: statistically significant P=0.019
^$: statistically significant P=0.032

 

Table. Changes in mean total protein and enzyme levels in BAL (x-fold of concurrent control) in male rats after inhalation exposure to Zinc oxide T0242 and zinc sulfate monohydrate on 14 consecutive days

Analyte	Gr. 7 8 mg/m^3	Gr8 18 mg/m^3
Total Protein	7.6**	2.4**
GGT	4.2**	3.9**
LDH	13.2**	4.6**
ALP	19.1**	4.3**
NAG	2.4**	1.5

GGT = γ-Glutamyl-transferase; LDH = Lactate dehydrogenase; ALP = Alkaline phosphatase; NAG = β-N-Acetyl glucosaminidase, One-sided Wilcoxon-test: * : p £ 0.05; ** : p £ 0.01

Conclusions:

In this study, male Wistar rats were whole-body exposed to dust aerosols of T0420 and T0421 at target concentrations of 0.5, 2 and 8 mg/m³, as well as to 8 mg/m³ miconsize zinc oxide or 18 mg/m³ zinc sulfate monohydrate for 6 hours daily on 14 consecutive days. A concurrent control group was exposed to conditioned air.

The tested atmospheric were met and they were maintained throughout the study. Cascade impactor measurement of both substances showed particle sizes within the respirable range. There were no signs of toxicity, nor were there any impairment of body weight gain. In the range finding 14-day study (BASF project no.: 36I0050/20I005) animals of the mid dose groups were nominally exposed to 12 mg/m3. The actual assessment of test substance concentrations, however, revealed an exposure to 10.9 and 10.8 mg/m3 for T0420 and T0421, respectively. At this dose level significant histopathological alterations (grade 2 degeneration/regeneration, olfactory epithelium) as well as alterations indicative of inflammatory responses in the lung were observed, which could have had a confounding impact on the outcome of the comet assays results. Thus, the top exposure concentration was slightly reduced to 8 mg/m3. At this level in the lavage fluid, several parameters were increased in a concentration related manner in animals exposed to T0420 and T0421, indicating inflammation process in the lung. These findings were comparable with those observed previously in the range finding 14-day inhalation study with these two test materials. Similar findings were observed in animals exposed to the reference substances.


LMW DNA diffusion assay
In all test groups using the test or reference substances, the quantification of the diffused low molecular weight DNA as a measure for cytotoxicity did not show any indication of excessive cytotoxicity as compared to the respective air control value, which could have hampered the interpretation of the comet assay data. The significantly increased value observed for the lung using micro-scaled Zinc oxide T0242 was not high enough to interfere the comet data evaluation. The positive control group, however, showed significant increases in the LMW DNA value. This is, however, expected from the positive control considering the amount of DNA damage it has induced. The fact that all diffusible LMW DNA values from bone marrow and nasal epithelium samples were above the historical control data, does not have an impact on the data interpretation, since the air control values were also above the historical control data range and furthermore, the high LMW DNA values did not have an impact on the % tail intensities, which were all within the historical control data range for these two organs.

Comet assay
The study is considered as valid, since all concurrent negative control data are acceptable for addition to the historical data base. Furthermore, the positive control group showed that the test is able to detect positive response in all assessed tissues.
The assessment of the comet assay in the various tissues did not show and biologically relevant indication of a genotoxic potential in any of the test or reference substances. The significant concentration dependent increase observed in the mean of mean values obtained from the liver of animals treated with T0420 is not considered as relevant, since the same values were not statistically significant, when the mean of medians was used instead of mean of means. This indicates that the observed effect is more likely due to the greater impact of individual values on the group mean value when using the mean of means. The current OECD guideline, therefore, also recommends the use of mean of medians. The statistically significant decreases in the observed in the lung and nasal epithelium using Zinc oxide T0421, micro-scaled Zinc oxide T0242 and Zinc sulfate are not considered as biologically relevant effects, since once again the statistical significance is only observed in either mean of mean or mean of median calculations.

CONCLUSION
In conclusion it can be stated that under the described circumstances neither Zinc oxide T0420 nor Zinc oxide T0421 showed a genotoxic potential in the nasal epithelium, lung liver and bone marrow of rats exposed for 14 days via inhalation.

Executive summary:

In this study two nanosized test substances Zinc oxide T0420 and Zinc oxide T0421 were assessed for their genotoxic potential using the alkaline comet assay after a 14-day exposure period via inhalation. This was a multisite study, where the in-life phase, necropsy, as well as the examination of the lung lavage fluid was performed by the test facility. The processing of the isolated tissues and the comet assay was performed by the test site (Helix 3 Inc.). The target tissues addressed in this study were the nasal epithelium, the lung, the liver as well as the bone marrow.

The concentrations used in this study was based on a separately performed dose range finding assay (BASF Project no.: 36I0050/20I005). The three concentrations ,selected based on the dose range finding study, were 0.5, 2.0 and 8.0 mg/m³.  Wistar rats were exposed whole-body to the indicated concentrations of each test substance for a 6 h period per day for 14 days. In addition, for comparison, a micro-scaled Zinc oxide as well as well as a soluble Zinc salt (Zinc sulfate monohydrate) were tested in parallel under the same conditions at a single (equimolar) concentration. Ethylmethane sulfonate (EMS) was used as a positive control and applied orally at a single time point.

During the exposure period, the animals were observed for signs of toxicity before, during and after the exposure. Body weight was determined once weekly. The following mean concentrations and particle size distribution were determined.

In animals exposed to test item 1, concentration-related increases of lavage parameters were observed, as well as in animals exposed to test item 2. The exposure of single concentration of reference substance 1 and 2 caused significantly increases in most of the lavage parameters. The changes in lavage fluid demonstrate the toxicity in the lungs, which were comparable with the range finding study.

The assessment of the target tissues in the comet assay did not show any biologically relevant increases in the % tail intensity of the analyzed tissues under the indicated conditions. The positive control group showed a distinct and statistically significant increase in all analyzed tissues.

Thus, under the indicated circumstances, the two test substances as well as the reference substances (micro-scaled Zinc oxide and Zinc sulfate monohydrate) are considered as non-genotoxic in this assay.

 

Reference 3

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

data not available

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Remarks:

Used in EU risk assessment report for zinc metal.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

not specified

Remarks:

information on GLP not available

Type of assay:

mammalian erythrocyte micronucleus test

Species:

rat

Strain:

not specified

Sex:

not specified

Details on test animals or test system and environmental conditions:

data not available

Route of administration:

oral: feed

Vehicle:

data not available

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

continous

Post exposure period:

data not available

Dose / conc.:

0.05 other: %

Dose / conc.:

0.2 other: %

Dose / conc.:

1 other: %

No. of animals per sex per dose:

data not available

Control animals:

yes

Positive control(s):

data not available

Tissues and cell types examined:

Bone marrow cells examined for all types of chromosome and chromatid-type aberrations and hyperdiploid cells.

Details of tissue and slide preparation:

data not available

Evaluation criteria:

data not available

Statistics:

data not available

Sex:

not specified

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

not specified

Remarks on result:

not determinable

Additional information on results:

- Zinc monoglycerolate was negative in this assay.

Conclusions:

The information summarised in this study record was taken from the EU Risk Assessment report for zinc (EU RAR 2004) where it has been thoroughly reviewed. Due to the unavailability of the primary reference, the study was rated with reliability 4 for formal reasons. However, the results of this study will be used in the hazard assessment of the zinc category substances in a weight of evidence approach.

In a micronucleus study (Windebank et al., 1995) comparable to OECD guideline study 474, rats were fed 0.05, 0.2 and 1% zinc monoglycerolate in a purified diet. Zinc monoglycerolate was negative in this assay.

No conclusion can be drawn due to the insufficient documentation.

Reference 4

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

not specified

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

The test material was administered via intraperitoneal injections, which is considered to be a non-physiological exposure route and bears only very limited value for hazard assessment purposes. The test material was only poorly characterised, since information on the purity, source, manufacturer, and physical appearance are not provided. Furthermore, details on the test material preparation and dosing volume are missing. Information on terminal body weights, clinical signs, and mortalities are not reported. The description of the test animals is insufficient, since information on the sex, housing, water supply, and test group randomisation are missing. Moreover, the animals are kept at higher temperatures than usually recommended by in vivo genotoxicity guidelines (28±2°C vs. 22±3°C). Further, the number of animals scored for chromosomal aberrations was lower than recommended by the test guideline in force at that time (5 vs. 10). The description of the treatment with the metaphase arresting agent lacks details, since the time point of treatment is not specified. Moreover, the description of the metaphase preparation lacks details. Historical control data is not included. Further, evaluation and scoring criteria are not specified.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

no guideline followed

Principles of method if other than guideline:

Animals were exposed to the test material intraperitoneally and sacrificed after treatment. Chromosome preparations were made from the bone marrow cells and the metaphase cells were analysed for chromosome aberrations.

GLP compliance:

not specified

Remarks:

publication

Type of assay:

mammalian bone marrow chromosome aberration test

Species:

mouse

Strain:

Swiss

Sex:

not specified

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 8 - 10 weeks
- Weight at study initiation: 25 g (average)
- Diet: Standard balanced diet (Hindustan Lever Limited, India); ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature: 28 ± 2°C
- Humidity: 60 ± 5%
- Photoperiod: 12 hrs dark / 12 hrs light

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: glass-distilled water

Details on exposure:

No details provided

Duration of treatment / exposure:

Acute treatment: one day
Repeated treatment: 8, 16 and 24 d

Frequency of treatment:

Acute treatment: one single injection
Repeated treatment: every alternate day

Post exposure period:

Acute treatment: 24 h
Repeated treatment: Not reported

Dose / conc.:

7.5 mg/kg bw/day

Remarks:

acute treatment; 1/4 LD50

Dose / conc.:

10 mg/kg bw/day

Remarks:

acute treatment; 1/3 LD50

Dose / conc.:

15 mg/kg bw/day

Remarks:

acute treatment; 1/2 LD50

Dose / conc.:

2 other: mg/kg bw

Remarks:

repeated treatment; 1/15 LD50

Dose / conc.:

3 other: mg/kg bw

Remarks:

repeated treatment; 1/10 LD50

No. of animals per sex per dose:

5 mice per group

Control animals:

yes

Positive control(s):

Cyclophosphamide
- Route of administration: intraperitoneal
- Doses / concentrations: 25 mg/kg bw

Tissues and cell types examined:

bone marrow erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: 1/4 - 1/2 LD50 (acute treatment); 1/15 - 1/10 LD50 (repeated treatment)

CA ASSAY
For acute treatment, mice received a single intraperitoneal injection of zinc sulfate and were sacrificed 24 h later. For the repeated treatment, the mice received test material injections on alternate days and were sacrificed on days 8, 16, and 24. Negative and positive control mice received intraperitoneal injections of isotonic saline and cyclophosphamide (25 mg/kg bw), respectively, and were sacrificed after 24 h.
Bone marrow preparations were made by standard colchicine (2 mg/kg, ip), hypotonic (0.075 M KCl), fixative (1:3 glacial acetic acid/ethanol) flame-drying schedule*. Slides were coded and stained with Giemsa. From each of the animals, 60 metaphases were scored for chromosomal aberrations, and 1000 cells for mitotic index. Types of aberration were recorded separately, strictly in accordance with the schedule of Tice (1987)*. All aberrations (chromatid gap, chromosome gap, chromatid break, chromosome break, and rearrangements) were considered equal regardless of the number of breakages involved. Percentages of aberrant metaphase cells and number of aberrations per cell (excluding gaps) were computed.

*References:
- Sharma, A.K. and Sharma, A., 1980. Chromosome techniques: Theory and Practice, 3rd Ed, Butterworth, London.
- Tice, R.R., Boucher, R., Luke, C.A., and Shelby, D., 1987. Environ. Mol. Mutagen 9, 235.

Evaluation criteria:

not specified

Statistics:

A one-tailed trend test was performed for the determination of dose response and a two-way ANOVA test followed by Duncan's multiple-range test was carried out to detect significant differences among different concentrations and sampling times on the clastogenicity of test material.
Level of significance was established at α = 0.05 for all statistical analysis.

Sex:

not specified

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

not specified

Remarks on result:

not determinable because of methodological limitations

Additional information on results:

CA ASSAY
- The chromosomal aberration frequency was statistically significantly increased after zinc chloride treatment (please refer to tables 1 and 2 in the field ‘any other information on results incl. tables’). The number and proportion of cells with chromosomal aberration was clearly elevated above the negative control group value.
- The increase in the CA frequency was calculated to be dose and treatment duration dependent (please refer to table 3 in the field ‘any other information on results incl. tables’).
- The positive control substance, cyclophosphamide, induced a marked increase in the proportion of cells with aberrations and the proportion of chromosomal aberrations per cells.
- The mitotic index did not show statistically significant variation in all the sets.

Table 1 : Chromosomal Aberrations (CA) recorded in the bone-marrow cells following acute treatment

Concentration (mg/kg bw)	Total CA/300 Cells					% DC		CA/Cell	MI
	G'	G"	B'	B"	RA	DC	X ± SEM	X ±SEM	X ±SD
0	9	0	1	0	0	1	0.33 ± 0.74	0.0034 ± 0.007	2.41 ± 0.57
7.5	25	0	28	0	4	22	7.33 ± 2.78	0.0800 ± 0.025	1.83 ± 0.70
10.0	28	0	20	0	7	34	11.32 ± 5.93	0.1168 ± 0.061	1.52 ± 1.11
15.0	28	0	42	0	5	44	14.66 ± 1.79	0.157 ± 0.022	1.60 ± 0.65
Cyclophosphamide- 25 mg/kg bw	39	2	282	2	15	119	43.60 ± 9.53	1.1880 ± 0.343	1.76 ± 0.44

Z = 6.16, P < 0.001, 1-tailed trend test

Table 2: Chromosomal Aberrations (CA) recorded in the bone-marrow cells following repeated treatment

Concentration (mg/kg bw)	Total CA/300 Cells					% DC		CA/Cell	MI
	G'	G"	B'	B"	RA	DC	X ± SEM	X ±SEM	X ±SD
8 days
2.0	6	2	0	0	1	1	0.34 ± 0.74	0.0034 ± 0.007	2.27 ± 0.29
3.0	35	0	19	0	0	18	6.00 ± 3.48	0.0632 ± 0.036	2.23 ± 1.06
16 days
2.0	20	1	4	1	4	9	3.01 ± 2.73	0.0334 ± 0.033	2.21 ± 0.87
3.0	34	0	27	0	5	27	8.99 ±.2.79	0.1200 ± 0. 044	1.58 ± 0.24
24 days
2.0	10	1	10	1	12	19	6.30 ± 2.16	0.0732 ± 0.025	1. 92 ± 0.56
3.0	25	0	44	0	7	48	16.98 ± 2.74	0.1732 ± 0. 033	2.13 ± 0.51
Negative control	9	0	1	0	0	1	0.33 ± 0.74	0.0034 ± 0.007	2.41 ± 0.57

G', G" = Chromatid and chromosome gap

B', B" = Chromatid and chromosome break

RA = Rearrangement

DC = Damaged cell

MI = Mitotic index

X = Mean of five animals

Table 3: Anova table showing effects of dose and duration on clastogenicity in mice following chronic treatment

Source of Variation	Degree of freedom	Sum of squares	Mean sum of squares	F
Error	2	0.0212	0.0106	13.68*
Duration	2	0.0054	0.0027	3.48
Dose	4	0.0031	0.00121775

* Significant at F = 005 level

Conclusions:

Gupta, T. et al. (1991) conducted a micronucleus assay in Swiss albino mice after both single and repeated intraperitoneal injections of zinc chloride. In the acute exposure experiment, the mice received a single intraperitoneal injection of zinc chloride at dose levels of 7.5, 10, and 15 mg/kg bw. The mice were sacrificed 24 hours after the treatment. In the repeated dose experiment, the test material was administered, at dose levels of 2 and 3 mg/kg bw, every alternate day and the mice were sacrificed on day 8, 16, or 24. Test animals assigned to negative and positive control groups received intraperitoneal injections of isotonic and cyclophosphamide, respectively. Afterwards, the bone marrow cells were obtained and stained with Giemsa. A total of 60 metaphases per animal were scored for chromosomal aberrations and 1000 cells per animal were scored for the determination of the mitotic index.

The chromosomal aberration frequency was statistically significantly increased at all zinc chloride doses tested, when compared to the negative control group. However, in the repeated exposure experiment, the low dose group showed relevant increases in the proportion of cells with chromosomal aberration and chromosomal aberrations per cell only at exposure durations longer exceeding eight days. The response was calculated to be related to the dose level and exposure duration. The mitotic index was not statistically significantly altered but was, in some sets, reduced by up to 37%. The authors considered zinc chloride to be a potent clastogen.

No conclusion can be drawn due to reporting and methodological deficiencies.

The test material was administered via intraperitoneal injections, which is considered to be a non-physiological exposure route and bears only very limited value for hazard assessment purposes. The test material was only poorly characterised, since information on the purity, source, manufacturer, and physical appearance are not provided. Furthermore, details on the test material preparation and dosing volume are missing. Information on terminal body weights, clinical signs, and mortalities are not reported. The description of the test animals is insufficient, since information on the sex, housing, water supply, and test group randomisation are missing. Moreover, the animals are kept at higher temperatures than usually recommended by in vivo genotoxicity guidelines (28±2°C vs. 22±3°C). Further, the number of animals scored for chromosomal aberrations was lower than recommended by the test guideline in force at that time (5 vs. 10). The description of the treatment with the metaphase arresting agent lacks details, since the time point of treatment is not specified. Moreover, the description of the metaphase preparation lacks details. Historical control data is not included. Further, evaluation and scoring criteria are not specified.

Reference 5

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2009-10-05 to 2013-12-19

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

The study presented herein is considered to be acceptable with restrictions. The experiment included in the study were not performed under GLP conditions, which was basically due to the fact that an OECD validated test guideline was not available at that time. The tail intensity was determined only using a single slide per animal; replicates were not included. Furthermore, the responses observed showed in several cases a huge variability. Thus, the robustness of the data may be limited. Moreover, the description of the methodology lacks detailed information on cell lysis, neutralisation, and electrophoresis. The positive controls were run only in an independent experiment, concurrent positive control animals were not included. Cytotoxicity was not sufficiently investigated and evaluated. The only parameters useful for an evaluation of cytotoxicity are the LDH activity and protein level in BAL fluid. Evaluation criteria are not specified. Historical control data are not included. Only one dose level was tested, which precludes an evaluation of dose-response relationships.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

A 14-day repeated dose inhalation toxicity study (Creutzenberg, 2013) was conducted to establish exposure dose-response relationships of the microscaled zinc oxide in rats after subacute nose-only exposure according to the OECD Guideline 412 in compliance with GLP. In the framework of this study, potential site of contact genotoxicity was evaluated as well.

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP certificate signed on 2011-08-15

Type of assay:

mammalian comet assay

Species:

rat

Strain:

other: Crl:WU

Details on species / strain selection:

Wistar rats are commonly used in subchronic and chronic inhalation toxicity studies. They fulfil the criteria stated by a U.S. EPA Workshop (Vu et al., 1996)* such as (i) a low background rate of neoplasia, (ii) a low background rate of pulmonary disease, (iii) longevity, and (iv) a history of laboratory use. In this study the specified Wistar strain is preferred to the Fischer strain because young Fischer rats available in Germany sporadically show a slight latent inflammation of lungs which might interfere with the scheduled examinations.

*References:
- Vu, V., Barrett, J.C, Roycroft, J., Schuman, L., Dankovic, D. Workshop report: Chronic inhalation toxicity and carcinogenicity testing of respirable fibrous particles. Reg Tox Pharm 24, 202-212 (1996)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland (Sulzfeld/Germany)
- Age at study initiation: approx. 8 weeks
- Weight at study initiation: approx. 230 g (males), approx. 165 g (females)
- Assigned to test groups randomly: yes, under following basis: randomized and grouped by the PROVANTIS system on body weight basis
- Fasting period before study: No
- Housing: 2 rats per cage, absorbing softwood bedding
- Diet: Ssniff "V1534" (ssniff Spezialdiäten GmbH; Soest, Germany); ad libitum
- Water: tap water; ad libitum
- Acclimation period: 1 d followed by 3 weeks of training in nose-only tubes without exposure

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2°C
- Humidity: 55 ± 15%
- Air changes: Fully airconditioned
- Photoperiod: 12 hrs dark / 12 hrs light

Route of administration:

inhalation: aerosol

Vehicle:

- Vehicle(s)/solvent(s) used: clean air

Details on exposure:

TYPE OF INHALATION EXPOSURE: nose only

GENERATION OF TEST ATMOSPHERE CHAMBER DESCRIPTION
- Exposure apparatus: Flow-past nose-only exposure system, individually exposure of each rat, exhaled air is immediately exhausted
- Method of holding animals in test chamber: Individual acrylic tubes
- Source and rate of air: Pressurized air, 1 L/min
- System of generating particulates/aerosols: Feeding system and high-pressure, high-velocity pressurized air dispersion with computerized control
- Temperature, humidity, pressure in air chamber: 22 ± 2°C, 55 ± 15%,
- Air flow rate: 1 L/min
- Method of particle size determination: Cascade impactor/ Marple impactor
- Treatment of exhaust air: Disposal in compliance with local, federal and state regulations

TEST ATMOSPHERE
- Brief description of analytical method used: Gravimetrically by filter samples, feed back loop to actual aerosol concentrations measured by an aerosol photometer
- Samples taken from breathing zone: Yes

Duration of treatment / exposure:

14 days (10 days exposure)

Frequency of treatment:

5 consecutive days per week, 6 h per day

Post exposure period:

1 and 14 days

Dose / conc.:

8.24 mg/m³ air (analytical)

Remarks:

SD: 1.27 mg/m³ (gravimetric analysis); 8.04 ± 0.75 mg/m³ (photometer); target concentration: 8 mg/m³

No. of animals per sex per dose:

5 male rats per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

Potassium bromate-treated L5178Y/TK+/- mouse lymphoma cells served as a technical positive control to assess both activity of the used hOGG1 enzyme batch and appropriate performance of slide preparation, electrophoresis, and staining.

Tissues and cell types examined:

BAL (comet assay) and lung epithelial (immunohistochemistry) cells

Details of tissue and slide preparation:

COMET ASSAY
DNA-strand breaks and oxidative DNA damage (8-OH-dG) were analysed in BAL cells of 5 males per group on day 1 and day 14 post exposure using the hOGG1-modified comet assay under non-GLP conditions.
BAL cell suspensions of male rats treated with clean air or the test and particulate reference items served as a new model system. Preparation time points for the comet assay were limited to two time points, 24 h after the last exposure (day 1 post exposure) and 14 days after the last exposure.
Two aliquots of each BAL cell suspension (a minimum of 20.000 cells in a volume of 1 mL) per animal of clean air or test item-exposed animals were centrifuged, resuspended in pre-heated 0.75% low melting agarose, applied to agarose pre-coated slides, and lysed overnight at 4°C to liberate the DNA. Based on the limited cell number, only two slides per animal could be generated. One of the two slides per animal was subsequently incubated for 10 min with 0.16 U/mL of hOGG1. The other slide only received enzyme buffer. In both cases, DNA-unwinding and electrophoresis was done on ice in 4°C cold electrophoresis buffer (pH > 13). DNA was finally stained with ethidium bromide and analysed using the Comet Assay III Software (Perceptive Instruments, UK). As the main end point tail intensity (proportion of DNA in tail) (tail length and tail moment determinations were included as supplemental information) of 100 nuclei per animal and slide treatment (with or without hOGG1 incubation) was determined (in total, 500 nuclei per group and slide treatment). An increase in TI (TL, TM) on the hOGG1-treated slides, as compared to the slides treated with enzyme buffer only, is indicative for the occurrence of the oxidative base lesion 8-OHdG.
Percent heavily damaged nuclei on the comet assay slides, both without and with hOGG1 incubation, were used as a surrogate endpoint for the cell integrity of the isolated BAL cells. Nuclei with a TI of ≥ 90 % were defined as “heavily damaged”. The percentage of “heavily damaged nuclei” was retrospectively calculated from the number of nuclei with a TI ≥ 90 % within the fraction of 100 nuclei, evaluated per animal. These nuclei were then retrospectively excluded from statistical analysis. Hedgehogs, which could not be scored by the image-analysis system, were a priori excluded from analysis. This is a generally agreed approach for evaluation of comet assay slides.
The ability of the hOGG1-modified comet assay to detect oxidative DNA-damage in BAL cells in vivo was independently confirmed in the same rat strain (not part of this study) by i.p. injection of 250 mg/kg bw KBrO3, with isolation of BAL cells 3 h after application.

IMMUNOHISTOCHEMICAL DETECTION OF OXIDATIVE DNA DAMAGE
Immunohistochemical detection of 8-OH-dG in lung tissue as marker of oxidative DNA damage was performed on samples prepared for histopathology (cf. section 7.5.2: Creutzenberg, 2013). Formalin-fixed tissue of the terminal bronchioles and lung parenchymal cells were examined for the formation of 8-OH-dG by an antibody labelling technique.

Evaluation criteria:

not specified

Statistics:

The median derived mean TI was used for statistical analysis (mean derived TI was included as supplemental data). The Mann-Whitney Rank Sum Test was performed to compare the treatment groups with the concurrent negative controls. To compare the hOGG1-untreated slides with the hOGG1-treated slides, the paired t-test was used, as the tested samples were directly linked to each other and represent a “before and after” situation. The results obtained in the immunohistochemical analysis of 8-OH-dG positive nuclei were tested for statistical significance using the Tukey's studentised range test. Differences between groups/slides were considered statistically significant at p ≤ 0.05.

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

COMET ASSAY
- The exposure to microscaled zinc oxide did not result in statistically significantly increased median derived mean tail intensities, both with and without hOGG1 treatment, in BAL cells examined 1 and 14 days post exposure (please refer to table 1 in the field ‘any other information on results incl. tables’).
- The technical positive control cultures showed a statistically significant increase in the tail intensity after hOGG1 treatment, when compared to cells without hOGG1 treatment. Thus, the activity and the appropriate performance of the comet assay were demonstrated. Moreover, the in vivo positive control rats, tested in an independent experiment, showed a clear and statistically significant increase in the tail intensity with and without hOGG1 treatment, when compared to negative controls. Thus, the general sensitivity with regards to general and oxidative DNA damage was demonstrated.
- The number of heavily damaged (≥ 90%) nuclei was in general low and in most cases did not correlate with the respective TI values of the preparation.
- Hedgehogs were only sporadically observed.
- In an independent experiment performed in 2020, the positive reference for general DNA damage, EMS, induced a marked increase in the tail intensity, when compared to concurrent negative controls (please refer to table 3 in the field ‘any other information on results incl. tables’)

IMMUNOHISTOCHEMICAL DETECTION OF OXIDATIVE DNA DAMAGE
- At post exposure days 1 and 14, the number of 8-OH-dG positive nuclei per area was not statistically significantly increased in microscaled zinc oxide exposed animals, when compared to the vehicle control group (please refer to table 2 in the field ‘any other information on results incl. tables’).

TOXICITY
- General toxicity: Signs of general toxicity were not evident.
- Local effects: A strong acute response characterised by cell infiltration and enzymatic response as well as histopathological alteration characterised by bronchiolo-alveolar hyperplasia and mononuclear infiltration.
- Notably, the LDH activity and the total protein level in the BAL fluid were markedly and statistically significantly increased 24 hours after exposure to microscaled zinc oxide.

Table 1. hOGG1-modified Comet assay, DNA-damage in BAL cells after 1 and 14 days of recovery.

	Tail intensity [%] Mean ± SD^1		Tail intensity [%] Mean ± SD^1
Post exposure day	1		14
Treatment	hOGG1		hOGG1
	without	with	without	with
Vehicle control: Clean air	0.6 ± 0.98	1.1 ± 1.67	0.5 ± 0.28	0.5 ± 0.31
Microscaled ZnO: 8 mg/m³	0.2 ± 0.08	0.2 ± 0.11	0.4 ± 0.29	2.3 ± 1.98
Positive control: KBrO3	1.3 ± 1.10	7.8 ± 3.83#	0.5 ± 0.22	7.9 ± 2.51##

#/## Significantly different from samples incubated without hOGG1: p ≤ 0.05, Student’s t-test for paired values.

* Significantly different from vehicle control: p ≤ 0.05, Mann-Whitney Rank Sum Test.

¹Mean derived from medians of approx. 100 nuclei per animal and 5 animals per group.

 

Table 2. 8-OH-dG positive nuclei in terminal bronchioles and lung parenchymal cells

	8_OH-dG
	Positive nuclei per 10,000 μm² Mean ± SD
Post exposure day	1	14
Vehicle control: Clean air	6.92 ± 1.34	7.63 ± 0.78
Microscaled ZnO: 8 mg/m³	9.28 ± 1.34	7.74 ± 0.74

Table 3. In vivo control values rfom independent experiment performed by the same toxicological laboratory in 2020.

Group and treatment	Animal number	Sacrifice day	Median Tail Intensity [%]		Arithmetic Mean Animals	Group Mean ± SD
			Slide A	Slide B
Vehicle control	1201	1	0.08	0.06	0.07	0.32 ± 0.237
	1202	1	0.22	0.19	0.20
	1203	1	0.08	0.28	0.18
	52	2	0.69	0.47	0.58
	107	3	0.62	0.71	0.66
	110	3	0.20	0.29	0.24
Positive control: EMS	2	1	11.14	12.21	11.67	11.72 ± 1.335
	3	1	13.56	14.43	14.00
	4	2	12.01	9.27	10.64
	5	2	10.30	9.34	9.82
	6	3	12.82	11.21	12.01
	7	3	13.41	11.54	12.47

Conclusions:

Creutzenberg (2013) investigated on the potential site-of-contact genotoxicity of microscaled zinc oxide in male Wistar rats after repeated nose-only inhalation. Five rats were exposed to microscaled zinc oxide aerosol at a concentration level of 8.24 mg/m³ (analytical) for 5 days/week and 6 hours/day over a total period of two weeks. Vehicle control (clean air) rats were run concurrently. At post exposure days 1 and 14, BAL cells were obtained and tested for DNA damage using the modified alkaline comet assay. In this type of comet assay, lysed cells were either treated with buffer or with hOGG1 enzyme to detect general and oxidative DNA damage. A total of 100 cells per animal were scored for the tail intensity (% DNA in tail). As technical positive control served potassium bromate-treated L5178Y/TK+/- mouse lymphoma cells. Moreover, in an independent experiment, rats exposed to potassium bromate (250 mg/kg bw) via intraperitoneal injection served as in vivo positive controls. Apart from the comet assay, the authors examined the formation of 8-OH-dG in formalin-fixed tissue of the terminal bronchioles and lung parenchymal cells via immunohistochemistry.

Wistar rats exposed to microscaled zinc oxide did not show signs of general toxicity. However, local adverse effects in the lung were evident 24 hours after the exposure to microscaled zinc oxide. Notably, the LDH activity and total protein level in BAL fluid was markedly and statistically significantly increased in animals exposed at a concentration level of 8 mg/m³ indicating marked cytotoxicity. However, all these effects were reversible and returned to controls during the 14-day recovery period.

The BAL cells of rats exposed to microscaled zinc oxide did not show a statistically significant increase in the tail intensity after 1 and 14 days of the final treatment, when compared to the concurrent vehicle control. Moreover, the hOGG1 treatment did not reveal an increase of oxidative DNA damage. In contrast, the independent in vivo positive controls showed marked and statistically significantly both with and without hOGG1 treatment, when compared negative controls. Furthermore, independently generated positive control data from rats exposed to EMS showed a marked increase in the tail intensity, when compared to concurrent negative controls, in a conventional alkaline comet without hOGG1 treatment. Thus, the general sensitivity of the test system was demonstrated. Moreover, the technical positive control demonstrated the activity of the used hOGG1 enzyme and the appropriate performance of the comet assay methodology.
In addition, terminal bronchioles and lung parenchymal cells from microscaled zinc oxide exposed rats did not show statistically significantly increased numbers of 8-OH-dG positive nuclei, when compared to the vehicle control values. Thus, the lack of microscaled zinc oxide-induced oxidative DNA damage observed in the comet assay was further substantiated by the data obtained in the immunohistochemical assay using lung sections.

The study presented herein is considered to be acceptable with restrictions.

The experiment included in the study were not performed under GLP conditions, which was basically due to the fact that an OECD validated test guideline was not available at that time. The tail intensity was determined only using a single slide per animal; replicates were not included. Furthermore, the responses observed showed in several cases a huge variability. Thus, the robustness of the data may be limited. Moreover, the description of the methodology lacks detailed information on cell lysis, neutralisation, and electrophoresis. The positive controls were run only in an independent experiment, concurrent positive control animals were not included. Cytotoxicity was not sufficiently investigated and evaluated. The only parameters useful for an evaluation of cytotoxicity are the LDH activity and protein level in BAL fluid. Evaluation criteria are not specified. Historical control data are not included. Only one dose level was tested, which precludes an evaluation of dose-response relationships.

The test material, microscaled zinc oxide, is considered to have no DNA damaging potential based on the absence of biological relevant increases in the median derived mean tail intensity under the conditions tested.

Reference 6

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2009-10-05 to 2013-12-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted on July 21, 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP certificate signed on 2011-08-15

Type of assay:

mammalian erythrocyte micronucleus test

Species:

rat

Strain:

other: Crl:WU

Details on species / strain selection:

Wistar rats are commonly used in subchronic and chronic inhalation toxicity studies. They fulfil the criteria stated by a U.S. EPA Workshop (Vu et al., 1996)* such as (i) a low background rate of neoplasia, (ii) a low background rate of pulmonary disease, (iii) longevity, and (iv) a history of laboratory use. In this study the specified Wistar strain is preferred to the Fischer strain because young Fischer rats available in Germany sporadically show a slight latent inflammation of lungs which might interfere with the scheduled examinations.

*References:
- Vu, V., Barrett, J.C, Roycroft, J., Schuman, L., Dankovic, D. Workshop report: Chronic inhalation toxicity and carcinogenicity testing of respirable fibrous particles. Reg Tox Pharm 24, 202-212 (1996)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland (Sulzfeld/Germany)
- Age at study initiation: approx. 8 weeks
- Weight at study initiation: approx. 230 g (males), approx. 165 g (females)
- Assigned to test groups randomly: yes, under following basis: randomized and grouped by the PROVANTIS system on body weight basis
- Fasting period before study: No
- Housing: 2 rats per cage, absorbing softwood bedding
- Diet: Ssniff "V1534" (ssniff Spezialdiäten GmbH; Soest, Germany); ad libitum
- Water: tap water; ad libitum
- Acclimation period: 1 d followed by 3 weeks of training in nose-only tubes without exposure

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2°C
- Humidity: 55 ± 15%
- Air changes (per hr): Fully airconditioned
- Photoperiod: 12 hrs dark / 12 hrs light

Route of administration:

inhalation: aerosol

Vehicle:

- Vehicle(s)/solvent(s) used: clean air

Details on exposure:

TYPE OF INHALATION EXPOSURE: nose only

GENERATION OF TEST ATMOSPHERE CHAMBER DESCRIPTION
- Exposure apparatus: Flow-past nose-only exposure system, individually exposure of each rat, exhaled air is immediately exhausted
- Method of holding animals in test chamber: Individual acrylic tubes
- Source and rate of air: Pressurized air, 1 L/min
- System of generating particulates/aerosols: Feeding system and high-pressure, high-velocity pressurized air dispersion with computerized control
- Temperature, humidity, pressure in air chamber: 22 ± 2°C, 55 ± 15%,
- Air flow rate: 1 L/min
- Method of particle size determination: Cascade impactor/ Marple impactor
- Treatment of exhaust air: Disposal in compliance with local, federal and state regulations

TEST ATMOSPHERE
- Brief description of analytical method used: Gravimetrically by filter samples, feed back loop to actual aerosol concentrations measured by an aerosol photometer
- Samples taken from breathing zone: Yes

Duration of treatment / exposure:

14 days (10 days exposure)

Frequency of treatment:

5 consecutive days per week, 6 h per day

Post exposure period:

1 day

Dose / conc.:

8.24 mg/m³ air (analytical)

Remarks:

SD: 1.27 mg/m³ (gravimetric analysis); 8.04 ± 0.75 mg/m³ (photometer); target concentration: 8 mg/m³

No. of animals per sex per dose:

5 male and 5 female rats per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide (CP) monohydrate
- Route of administration: oral
- Doses / concentrations: single dose of 20 mg/kg (dissolved in tap water)
- Group size: 5 rats

Tissues and cell types examined:

bone marrow tissue - polychromatic erythrocytes (PCE)

Details of tissue and slide preparation:

From suspensions of bone marrow tissue, cleaned of nucleated cells by a cellulose column procedure, smears were prepared on slides, fixed for 10 minutes in absolute methanol and stained with May-Grünwald- and Giemsa-solution. The slides were analyzed microscopically under 630-1000 x magnification. Microscopic analysis was conducted on a blind basis. Micronuclei were counted in 2000 polychromatic erythrocytes (PCE) per animal. The ratio of polychromatic to normochromatic erythrocytes (NCE) was determined in 500 red blood cells.

Evaluation criteria:

The micronucleus assay is judged as valid if the clean air controls demonstrate low spontaneous frequencies of micronucleus induction and if the positive controls demonstrate significantly higher frequencies of micronucleus induction as compared to the clean air controls. In addition, in test item treated animals the PCE ratio should not fall below 20% of the clean air control values to avoid unspecific effects due to excessive cytotoxicity in the bone marrow.
In the mammalian erythrocyte micronucleus test a genotoxic effect is claimed if a dose-related increase in the number of polychromatic erythrocytes or a statistically significant increase in the number of micronucleated cells in a single dose group at a single sampling time is observed, but biological relevance of the results is considered first.

Statistics:

Differences between groups were considered statistically significant at p < 0.05. Data were analyzed using analysis of variance. If the group means differ significantly by the analysis of variance the means of the treated groups were compared with the means of the control groups using Dunnett's test.
The statistical evaluation of the histopathological findings was done with the two-tailed Fisher test by the P.L.A.C.E.S. system.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

MN ASSAY
- Under the conditions of this assays, microscaled ZnO did not significantly enhance the number of micronuclei in polychromatic erythrocytes of the bone marrow (after 14 days of inhalation exposure) of both male and female Wistar rats.
- The positive control CP significantly induced micronucleus formation in PCE of the bone marrow, with higher rates in male than in female animals.
- As assessed by differential cell counting of bone marrow smears, microscaled ZnO in the given doses (8.0 mg/m3) did not significantly influence red blood cell formation in male Wistar rats after 14 days of inhalation exposure. However, there was a high fluctuation rate in PCE numbers, in part, perhaps based on the combination of the micronucleus test with the hOGG1-modified comet assay ex vivo in the male animals. In female Wistars rats, number of PCE/500 RBC was significantly reduced to 124±23.1 (p ≤0.01) by the positive control CP, as compared to 179±18.0 for the clean air control.

- General toxicity: Signs of general toxicity were not evident.
- Local effects: A strong acute response characterised by cell infiltration and enzymatic response as well as histopathological alteration characterised by bronchiolo-alveolar hyperplasia and mononuclear infiltration.
- Notably, the LDH activity and the total protein level in the BAL fluid were markedly and statistically significantly increased 24 hours after exposure to microscaled zinc oxide.

Table 1. Red blood cell formation and micronucleus induction in bone marrow of rats, after 14 days of exposure to clean air, cyclophosphamide, and microscaled ZnO.

Treatment group	Concentration, sampling time	PCE/500 RBC	PCE:NCE	MN/2000 PCE	% MN PCE
Negative control, clean air	24 h	♂ 86    ♀ 179	♂ 0.22    ♀ 0.56	♂ 5.8    ♀ 5.4	♂ 0.29   ♀ 0.27
Positive control, CP	20 mg/kg b.w., p.o., 24 h	♂ 67       ♀ 124**	♂ 0.16       ♀ 0.33**	♂ 55.4**     ♀ 22.0**	♂ 2.77**   ♀ 1.10**
Microscaled ZnO	8.0 mg/m^3,24 h	♂ 109    ♀ 173	♂ 0.29    ♀ 0.53	♂ 3.2     ♀ 2.0	♂ 0.16   ♀ 0.10

PCE: Polychromatic erythrocytes; NCE: Normochromatic erythrocytes; RBC: Red blood cells; MN: Micronuclei; % MN PCE: Percent micronucleated PCE; Significantly different from negative controls:*:P≤0.05, **:P≤0.01, Mann-Whitney Rank Sum Test; (*): P ≤0.05, Student’st-Test.

Conclusions:

A 14-day repeated dose inhalation toxicity study (Creutzenberg, 2013) was conducted with microscaled zinc oxide in rats exposed subacutely via nose-only exposure according to the OECD Guideline 412 in compliance with GLP. In the framework of this study also systemic clastogenic and aneugenic effects were investigated in male and female rats using the bone marrow micronucleus assay according to OECD Guideline 474. Five rats per sex and group were exposed at a concentration level of 8.24 mg/m³ (analyitcal) with microscaled zinc oxide. Fresh air treated animals served as concurrent control. Positive control animals were orally exposed to 20 mg/kg cyclophosphamide monohydrate.

There was no treatment-related reduction of body weights in any test group; no clinical signs were detected. However, high dose rats showed acute adverse local effects in the lungs. In male rats, the test item and the reference substances did not mediate significant repression of red blood cell formation. In females, the PCE/NCE was significantly reduced in the positive control. There was no evidence of a significantly enhanced mean frequency of micronucleated erythrocytes due to microscaled zinc oxide exposure in males or females, as compared to the vehicle control groups (clean air) at any dose level. The positive and vehicle controls gave valid results. Notably, exposure of the target organ was not demonstrated.

Inhaled microscaled zinc oxide is considered non-mutagenic in immature bone marrow erythrocytes (PCE) of Wistar rats under the conditions tested.

Reference 7

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

data not available

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Remarks:

Used in EU risk assessment report for zinc metal.

Qualifier:

no guideline followed

Guideline:

other: Single cell gel electrophoresis (SCGE)/ Comet assay by Singh et al. (1988)*. *Reference: Singh NP, McCoy MT, Tice RR and Schneider EL. (1988). Exp. Cell Res. 175: 184-91.

Principles of method if other than guideline:

The experiment and the evaluation of the slides were performed according to an internationally accepted protocol for the comet assay in vivo.

GLP compliance:

not specified

Remarks:

publication

Type of assay:

mammalian comet assay

Species:

mouse

Strain:

Swiss

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: National Institute of Nutrition, Hyderabad, India
- Weight at study initiation: 25 - 30 g
- Assigned to test groups randomly: Yes
- Housing: Housed individually in polypropylene cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 d


ENVIRONMENTAL CONDITIONS
- Temperature: 26 ± 2°C
- Humidity: 50 - 70%
- Photoperiod: 12 hrs light / 12 hrs dark

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Water
- Justification for choice of solvent/vehicle: Soluble in water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: ZnSO4 was dissolved in distilled water

Duration of treatment / exposure:

Single dose

Frequency of treatment:

Once

Post exposure period:

7 d

Dose / conc.:

5.7 other: mg/kg bw

Dose / conc.:

8.55 other: mg/kg bw

Dose / conc.:

11.4 other: mg/kg bw

Dose / conc.:

14.25 other: mg/kg bw

Dose / conc.:

17.1 other: mg/kg bw

Dose / conc.:

19.95 other: mg/kg bw

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Route of administration: i.p.
- Justification for choice of positive control(s): Clear effect on genotoxicity
- Doses / concentrations: 25 mg/kg bw

Tissues and cell types examined:

peripheral blood leukocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Based on the LD50 value of test material


SAMPLING TIMES: at 24, 48, 72, 96 h and first wk post-treatment


DETAILS OF SLIDE PREPARATION: Fully frosted slides were covered with 140 mL of 0.75% RMA and allowed to polymerize at 4 °C for 2 min. Then, 20 mL of whole blood was mixed with 110 mL of 0.5% of LMA and 110 mL of sample mixture was layered on the top of RMA and allowed to polymerize at 4 °C for 10 min. A third layer of 110 mL LMA was added and allowed to polymerize for 10 min at 4 °C. Then the slides were immersed in freshly prepared, cold lysing solution in a coplin jar and kept overnight at 4 °C. After lysis, the slides were immersed in alkaline buffer for 25 min to allow unwinding of DNA to occur. Electrophoresis was conducted for 30 min at 25 V (0.66 v/cm) adjusted to 300 mA by raising or lowering the buffer level in the tank. During electrophoresis, in contrast to the undamaged tightly packed DNA, DNA fragments if any, due to DNA damage migrate into the gel. Slides were then drained, placed on a tray and washed slowly with three changes, 5 min. each, of neutralization buffer (0.4 M Tris, pH 7.5). The slides were then dehydrated in absolute methanol for 10min. and left at room temperature to dry.


METHOD OF ANALYSIS: Slides were stained with 50 µL of ethidium bromide were analyzed at 400x magnification using a fluorescent microscope equipped with an excitation filter of 515-560 nm and a barrier filter of 590 nm

Evaluation criteria:

Images of 75 randomly selected cells (25 cells from each of 3 replicate slides) were analysed for each sample. Cells with increased DNA damage display increased migration of the DNA from the nucleus towards the anode. The amount of DNA able to migrate and to a lesser extent, the distance of migration are indications of the number of strand breaks present in that cell.

Statistics:

- One way and two way ANOVA
- Student’s t-test

Sex:

male

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

not specified

Remarks on result:

not determinable

Additional information on results:

COMET ASSAY
- Significant DNA damage was observed at all zinc sulfate doses tested, when compared to controls. The response was considered to be clearly dose-dependent. A gradual decrease in the tail-lengths from 48 h post-treatment onwards was observed indicating a time dependent decrease in the DNA damage.

Conclusions:

A study was conducted by Banu et al. (2001) to determine the in vivo genotoxic effect of zinc sulfate in mouse peripheral blood leukocytes using alkaline single cell gel electrophoresis.
 
Mice were administered orally with doses of 5.70, 8.55, 11.40, 14.25, 17.10 and 19.95 mg/kg bw of zinc sulfate. The samples of whole blood were collected at 24, 48, 72, 96 h and first wk post-treatment and the assay was carried out to determine single strand DNA breaks as represented by comet tail-lengths.

According to the authors, significant DNA damage was observed at all zinc sulfate doses tested, when compared to controls. The response was considered to be clearly dose-dependent. A gradual decrease in the tail-lengths from 48 h post-treatment onwards was observed indicating a time dependent decrease in the DNA damage. Under the test conditions, zinc sulfate was considered as potent genotoxic agent capable of inducing DNA damage.

No conclusion can be drawn due to the insufficient documentation.

Reference 8

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

not specified

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

Used in risk assessment report for zinc sulphate, acceptable for assessment. However, the study shows shortcomings with regard to reporting and the methodology applied. The test material is insufficiently characterised, since information on purity, source, manufacturer, and physical appearance are missing. The test material was not tested up to toxic (no toxic effects reported) or cytotoxic doses. Thus, the exposure of the bone marrow was not demonstrated. The number of animals in the solvent control group was low (n=3). Furthermore, the number of cells scored for chromosomal aberration (n=50 per animal) was too low, since no chromosomal aberrations were detected in both solvent control and test material-exposed animals. General toxicity was either not sufficiently investigated or not sufficiently described.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

A chromosomal aberration assay was conducted in rats to evaluate the genotoxic potential of zinc sulfate.

GLP compliance:

no

Remarks:

pre-GLP study

Type of assay:

mammalian bone marrow chromosome aberration test

Species:

rat

Strain:

Sprague-Dawley

Remarks:

CD

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: obtained from a closed colony (random-bred)
- Age at study initiation: 10 -12 weeks
- Weight at study initiation: 280 - 350 g
- Housing: 1 - 5 rats per cage
- Diet: commercial 4% fat diet; ad libitum
- Water: ad libitum
- Acclimation period: 4 - 11 days

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 0.85% saline

Duration of treatment / exposure:

1 (acute study) or 5 (subacute study) days

Frequency of treatment:

daily

Post exposure period:

6 (acute and subacute study), 24 (acute study), and 48 (acute study) hours

Dose / conc.:

2.75 other: mg/kg bw

Dose / conc.:

27.5 other: mg/kg bw

Dose / conc.:

275 other: mg/kg bw

No. of animals per sex per dose:

3 (vehicle) - 5 (all other groups) rats per group

Control animals:

yes, concurrent vehicle

Positive control(s):

triethylenemelamine
- Justification for choice of positive control(s): known mutagen
- Route of administration: intraperitoneal injection
- Doses / concentrations: 0.3 mg/kg bw

Tissues and cell types examined:

bone marrow cells arrested in metaphase

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: The LD5 was determined and selected as top dose.

TREATMENT AND SAMPLING TIMES:
In the acute study, a total of 69 animals received a single oral gavage of the test material (at the given doses), solvent, or positive control substance. and were killed 6, 24, and 48 hours after the administration. In the subacute study, the test animals received five doses 24 hours apart. Six hours after the final administration, rats were sacrificed. Four hours after the last compound administration, and two hours prior killing, each animal was given colcemid (4 mg/kg bw) intraperitoneally. Animals were euthanised by using CO2 and bone marrow cells were sampled from the femur.

DETAILS OF SLIDE PREPARATION:
The bone marrow cell suspensions were treated with a hypotonic solution, fixed in 3:1 methanol/glacial acetic acid, and stained with 5% Giemsa solution.

METHOD OF ANALYSIS:
The preparations were examined via bright field microscopy. The chromosomes of each cell were counted, and only diploid cells were analysed. They were scored for chromatid gaps and breaks, chromosome gaps and breaks, reunions, cells with greater than ten aberrations, polyploidy, pulverisation, and any other chromosomal aberrations which were observed. Fifty metaphase spreads were scored per animal. Mitotic indices were obtained by counting at least 500 cells and the ratio of the number of cells in mitosis/the number of cells observed was expressed as the mitotic index.

Evaluation criteria:

not specified

Statistics:

not specified

Sex:

male

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

not determinable because of methodological limitations

Additional information on results:

CA ASSAY
- Acute study: There were no chromosomal aberrations observed in the test groups. The negative control group animals also exhibited no chromosomal aberrations. The expected severe chromosomal damage due to the action of the positive control compound was seen. The mitotic indices were within normal limits.

- Subacute study: There were no aberrations observed in either the test compound groups or the negative control group. The mitotic indices were within normal limits.

Conclusions:

The clastogenic potential of zinc sulfate was assessed in Sprague Dawley rats using the bone marrow chromosome aberration test (Litton Bionetics Inc., 1974). Groups of five male rats were exposed to zinc sulfate at doses of 2.75, 27.5, and 275 mg/kg bw via gavage. In the acute study part, the animals received a single treatment and were sacrificed 6, 24, and 48 hours later. Animals assigned to the subacute study received daily treatments on five consecutive days. A vehicle control group was run concurrently. Test animals of the positive control group received a single intraperitoneal injection of triethylenemelamine. Prior to sacrifice, the animals received an intraperitoneal injection of colcemid. Afterwards, femoral bone marrow cells were harvested and stained with Giemsa. A total of 50 metaphases per animal were scored for the presence of chromosomal aberrations. In addition, the mitotic index was determined based on 500 cells scored for their mitotic state.

Zinc sulfate did not induce any chromosomal aberration in bone marrow cells of rats exposed via gavage under the conditions tested. Moreover, the mitotic indices were comparable to the vehicle control values indicating the absence of cytotoxic effects.

No conclusion can be drawn due to reporting and methodological deficiencies.

The test material is insufficiently characterised, since information on purity, source, manufacturer, and physical appearance are missing. The test material was not tested up to toxic (no toxic effects reported) or cytotoxic doses. Thus, the exposure of the bone marrow was not demonstrated. The number of animals in the solvent control group was low (n=3). Furthermore, the number of cells scored for chromosomal aberration (n=50 per animal) was too low, since no chromosomal aberrations were detected in both solvent control and test material-exposed animals. General toxicity was either not sufficiently investigated or not sufficiently described.

Reference 9

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

not specified

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

No conclusion can be drawn due to reporting and methodological deficiencies. The test material is insufficiently characterised, since information on purity, source, manufacturer, and physical appearance are missing. The test material was not tested up to toxic (no toxic effects reported). The positive control group did not demonstrate the sensitivity of the test system, since the values obtained were generally comparable to and not statistically significantly different from the concurrent vehicle control groups. General toxicity was either not sufficiently investigated or not sufficiently described. Historical negative control data is only specified as mean without ranges. The results presented in the tables are only limitedly retraceable.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

A dominant lethal assay was conducted in rats to evaluate the genotoxic potential of zinc sulphate.

GLP compliance:

no

Remarks:

pre-GLP study

Type of assay:

rodent dominant lethal assay

Species:

rat

Strain:

Sprague-Dawley

Remarks:

CD

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: males and females obtained from a closed colony (random-bred)
- Age at study initiation: 10 -12 weeks
- Weight at study initiation: 280 - 350 g
- Housing: 1 - 5 rats per cage
- Diet: commercial 4% fat diet; ad libitum
- Water: ad libitum
- Acclimation period: 4 - 11 days

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 0.85% saline

Duration of treatment / exposure:

1 (acute study) or 5 (subacute study) days

Frequency of treatment:

daily

Post exposure period:

7 - 8 weeks

Dose / conc.:

2.75 other: mg/kg bw

Dose / conc.:

27.5 other: mg/kg bw

Dose / conc.:

275 other: mg/kg bw

No. of animals per sex per dose:

ten male rats per group

Control animals:

yes, concurrent vehicle

Positive control(s):

triethylenemelamine
- Justification for choice of positive control(s): known mutagen
- Route of administration: intraperitoneal injection
- Doses / concentrations: 0.3 mg/kg bw

Tissues and cell types examined:

germ cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: The LD5 was determined and selected as top dose.

TREATMENT AND SAMPLING TIMES:
Zinc sulfate was administered orally by intubation in both the acute study (1 dose) and the subacute study (1 dose per day for 5 days). Following treatment, the males were sequentially mated to 2 females per week for 8 weeks (7 weeks in the subacute study). Two virgin female rats were housed with a male for 5 consecutive days. These two females were removed and housed in a cage until sacrifice. After a two-day rest, two new females were introduced to the cage. This procedure was repeated until study termination. The females were sacrificed after at 14 days after separation from the male, and at necropsy the uterus was examined for early deaths, late foetal deaths, and total implantations. Each male was mated with two females per week, and this provided for an adequate number of implantations per group per week (200 minimum) for negative controls, even if there was a fourfold reduction in fertility of implantations. Corpora lutea, early foetal deaths, late foetal deaths, and total implantations per uterine horn were recorded.

DETAILS OF SLIDE PREPARATION:

METHOD OF ANALYSIS:

OTHER:

Evaluation criteria:

not specified

Statistics:

- Fertility index: The number of pregnant female/number of mated females with the chi-square was used to compare each treatment to the control. Armitage's trend was used for linear proportions to test whether the fertility index was linearly related to arithmetic or log dose.
- Total number of implantations: The t-test was used to determine significant differences between average number of implantations per pregnant female for each treatment compared to control. Regression techniques were used to determine whether the average number of implantations per female was related to arithmetic or log dose.
- Total number of corpora lutea: The t-test was used to determine significant differences between average number of corpora lutea per pregnant female for each treatment compared to the control.
- Preimplantation loss and dead implants: Freeman-Tukey transformation was used on the preimplantation losses for each female and then the t-test was used to compare each treatment to control. Regression technique was used to determine whether the average number of preimplantation losses per female was related to the arithmetic or log dose.
- One or more dead implants: The proportion of females with one or more dead implants was computed, each treatment compared to control by chi-square test and Armitage's trend used for linear proportions.
- Dead implants per total implants: Dead implants per total implants: were computed for each female and used Freeman-Tukey arc-sine transformation on data for each treatment to control.
- In order to take variation between males into account, a nested model was used. An analysis across weeks was also performed.
- The statistical reports give the level of significance using both a one-tailed and two tailed test.

Sex:

male

Vehicle controls validity:

not specified

Negative controls validity:

not examined

Positive controls validity:

not specified

Remarks on result:

not determinable because of methodological limitations

Additional information on results:

DOMINANT LETHAL ASSAY
- Acute study: In general, the fertility index was lower in the experimental groups throughout the weeks, reaching significance in week 4. Significant decreases in the average implantations at week 2 and corpora lutea at week 4 were seen in the low dose group when compared to the vehicle control group. Significant increase in pre-implantation losses were seen at weeks 1 and 2 in the low dose group. No significant differences were seen in the above parameters when the positive control was compared with the vehicle control group; however, the positive control group showed a significant increase in average resorptions at week 2. This showed again in the proportion of females with one or more dead implants and in dead implants to total implants.
- Subacute study: In general, significant differences between the vehicle control and experimental groups were shown in a few instances at various weeks throughout the parameters. However, no strong indications were seen.

Conclusions:

In a dominant lethal assay (Litton Bionetics Inc., 1974), zinc sulfate was evaluated for its ability to induce mutations resulting from chromosomal aberrations in germ cells of orally exposed Sprague Dawley rats. Groups of five male rats were exposed to zinc sulfate at doses of 2.75, 27.5, and 275 mg/kg bw via gavage. In the acute study part, the animals received a single treatment and were sacrificed 6, 24, and 48 hours later. Animals assigned to the subacute study received daily treatments on five consecutive days. A vehicle control group was run concurrently. Test animals of the positive control group received a single intraperitoneal injection of triethylenemelamine. Each male was mated with two virgin females on five consecutive day. This procedure was repeated for 7 (subacute study) to 8 (acute study) weeks with a two-days break between each mating interval with fresh females. After 14 days, the mated females were sacrificed and scored for number of implantations, corpora lutea, pre-implantation losses, and resorptions (dead implants) as well as the proportion of females with one or more dead implants, two or more dead implants, and dead implants among total implants. In addition, the fertility index was calculated.

When compared to concurrent vehicle controls, the acute zinc sulfate groups showed statistically significantly increased fertility indices at weeks four and six. The observed response was, however, restricted to only these two out of eight weeks and the response did not show a positive dose-response relationship. Similarly, in the subacute study, a statistically significant reduction of the fertility index was observed at the third week of mating. However, no such effect was observed in the high dose group or at other mating intervals. Thus, the findings with regard to fertility are considered to be of an incidental nature. The average preimplantation loss per pregnant female was sporadically statistically significantly increased in the zinc sulfate experimental groups. In the acute study, the average preimplantation losses per pregnant female was statistically significantly increased in the low dose group at week 1 and 2 as well as in the intermediate dose group at week seven. However, these findings are considered to be chance findings, since these effects were only of a sporadic nature and not observed in the high dose group. The remaining alterations showing statistical significance are considered to be not biologically relevant.

No conclusion can be drawn due to reporting and methodological deficiencies.

The test material is insufficiently characterised, since information on purity, source, manufacturer, and physical appearance are missing. The test material was not tested up to toxic doses (no toxic effects reported). The positive control group did not demonstrate the sensitivity of the test system, since the values obtained were generally comparable to and not statistically significantly different from the concurrent vehicle control groups. General toxicity was either not sufficiently investigated or not sufficiently described. Historical negative control data is only specified as mean without ranges. The results presented in the tables are only limitedly retraceable.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In this dossier, the endpoint genetic toxicity is not addressed by substance-specific information, but instead by a weight of evidence approach based on collected information for all zinc substances of the zinc category. The assessment of the genotoxic properties of zinc and its substances is related to the assumption that once inorganic zinc compounds or zinc metal become bioavailable, this will be in the form of the divalent zinc cation. Further assuming that the anion of such inorganic zinc compounds can be regarded as “inert” with regard to genetic toxicity, the subsequent discussion focuses on the zinc cation. This assessment is restricted to those studies which are relevant from a regulatory point of view and fulfil the relevance reliability and adequacy criteria as e.g. laid down in the ECHA Guidance on information requirements and chemical safety assessment, Chapter R.4: Evaluation of available information. In contrast, studies reporting DNA damage in bacteria, induction of SCE in mammalian cells or tests in inappropriate test systems such as yeasts are considered to contribute far less to the overall assessment and are therefore not further discussed.

 

 

1 In vitro

1.1 bacterial reverse mutation assays

 

The bacterial reverse mutation assay by Taublova (2010) was conducted in four indicator Salmonella typhimurium strains TA98, TA100, TA1535 and TA 1537 and one indicator Escherichia coli WP2 uvrA strain with zinc hydrogen phosphate. The plates were treated with the test material suspended in dimethylsulfoxide using the plate incorporation method at doses of 50 -5000 µg/plate. No significant increases in the frequency of revertant colonies were recorded at any dose level. The test material was considered to be non-mutagenic under the conditions of this test. The study is well documented, conducted under GLP and meets generally accepted scientific principles and thus considered reliable without restriction (RL1).

 

The bacterial reverse mutation assay by Taublova (2010) was conducted in four indicator Salmonella typhimurium strains TA98, TA100, TA1535 and TA 1537 and one indicator Escherichia coli WP2 uvrA strain with zinc dinitrate hexahydrate. The plates were treated with the test material suspended in dimethylsulfoxide using the plate incorporation method at doses of 50 -5000 µg/plate. No significant increases in the frequency of revertant colonies were recorded at any dose level. The test material was considered to be non-mutagenic under the conditions of this test. The study is well documented, conducted under GLP and meets generally accepted scientific principles and thus considered reliable without restriction (RL1).

 

 

Li, et al. (2012) performed a Salmonella reverse mutation assay in order to evaluate the mutagenic potential ZnO MPs. The assay was performed according to the plate incorporation method both in presence and absence of a metabolic activation system. Three independent experiments were performed. According to the authors, ZnO MPs did not induce a significant increase in the revertant colony number in any of the strains tested, when compared to the solvent control. The positive controls elicit a significant response as compared to the solvent controls. The test material is insufficiently characterised, since information on purities and impurities is missing. Information on confounding factors, i.e. precipitation and cytotoxicity, in the main experiments is not provided. No confirmatory experiment was performed. Historical control data is missing. Based on the above-mentioned shortcomings the reference is suitable for the weight of evidence analysis (RL2).

 

The study by Marzin et al. (1985) was conducted to determine the potential mutagenicity of zinc sulfate using bacterial reverse mutation assay (e.g. Ames test). Salmonella typhimurium strain TA102 was treated with the test material using the plate incorporation method at six dose levels (10, 30, 100, 300, 1,000 and 3,000 nM/plate) in triplicate. Cytotoxicity was observed at 3,000 nM. No significant increases in the frequency of revertant colonies were recorded at any dose level. The test material was considered to be non-mutagenic under the conditions of this test. The test used only one tester strain instead of 4, as foreseen by OECD 471 (1983). Due to significant methodological deficiencies, the reference was considered not reliable (RL3).

 

In a bacterial reverse mutation assay by Litton Bionetics (1976), three strains of Salmonella typhimurium were treated with zinc oxide at unknown concentrations. No significant increases in the frequency of his+ revertant colonies were recorded therefore the test material was considered to be non-mutagenic under the conditions of this test. The information summarised in this study record was taken from the EU Risk Assessment report for zinc (EU RAR 2004) where it has been thoroughly reviewed. Due to the unavailability of the primary reference, the study was rated with reliability 4 for formal reasons. However, the results of this study will be used in the hazard assessment of the zinc category substances in a weight of evidence approach.

 

The study by Crebelli et al. (1985) was conducted to determine the potential mutagenicity of Zinc oxide using bacterial reverse mutation assay. Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 were treated with the test material using the plate incorporation method at dose levels ranging from 1,000 to 5,000 μg/plate in triplicate in presence and absence of a metabolic activation system. No significant increases in the frequency of his+ revertant colonies were recorded at the dose range tested. The test material was considered to be non-mutagenic under the conditions of this test. However, no conclusion can be drawn on the results presented, due to major deficiencies in reporting and the methodology. The description of the results is restricted to the general outcome of the test (Activity: negative). The authors did not provide any information on individual plate counts or revertant colony means and standard deviations. The authors did not discuss or have not examined confounding factors, such as cytotoxicity, test material precipitation, or pH and osmolality effects of the test material. The methodology is only poorly described, since nearly all essential details, including incubation time or number of cells treated, are not specified. The concentration levels tested are only specified as range; however, information on the number and levels of all concentrations tested are missing. The type of negative control used is not specified.

Historical control data are not included. Positive control cultures were not run for the strains TA 1535 and TA 1537 in absence of the metabolic activation system. Based on the above-mentioned shortcomings the reference is considered not reliable and therefore disregarded for the hazard assessment

 

The study by Jones et al. (1994) was conducted to determine the potential mutagenicity of zinc monoglycerolate using bacterial reverse mutation assay. Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 were treated with the test material using the plate incorporation method at dose levels ranging from 50 to 5,000 μg/plate in triplicate in presence and absence of a metabolic activation system. No toxicity observed up to 5000 ug/plate. No significant increases in the frequency of his+ revertant colonies were recorded at the dose range tested. The test material was considered to be non-mutagenic under the conditions of this test. The information summarised in this study record was taken from the EU Risk Assessment report for zinc (EU RAR 2004) where it has been thoroughly reviewed. Due to the unavailability of the primary reference, the study was rated with reliability 4 for formal reasons. However, the results of this study will be used in the hazard assessment of the zinc category substances in a weight of evidence approach.

 

Gocke et al. 1981 conducted a study to determine the potential mutagenicity of zinc sulfate using bacterial reverse mutation assay. Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA1538 strain were treated with the test material in Vogel-Bonner (VB) & ZLM medium (modified minimal medium for E. coli) at minimal five dose levels, up to 3,600 μg/plate, both with and without activation by the S9 liver fraction from Aroclor-pretreated rats. The positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. No significant increases in the frequency of revertant colonies were observed for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. Zinc sulfate was considered to be non-mutagenic under the conditions of this test. However, no conclusion can be drawn, due to major deficiencies in reporting and methodology. The test material is only poorly characterised, since information on the purity, manufacturer, and physical appearance are missing. Information on the vehicle used and test material preparation is not provided. The description of the results is restricted to the general outcome of the test (Activity: negative). However, the authors did not provide any information on individual plate counts or revertant colony means and standard deviations. The authors did not discuss confounding factors, such as cytotoxicity, test material precipitation, or pH and osmolality effects of the test material on the culture medium. The methodology is only poorly described, since nearly all essential details, including incubation time or number of cells treated, are not specified. The concentration level tested is not explicitly specified. The authors tested a list of chemicals and stated only that the Ames assay was performed ”…usually up to 3600 μg/plate for non-toxic and soluble compounds…”. Thus, the actual concentrations levels tested remain unclear. Results of negative and positive control cultures were neither shown nor discussed. The type of negative control used is not specified. Historical control data are not included. Benzo(a)pyrene was the only positive control substance tested. Based on the above-mentioned shortcomings the reference is considered not reliable and therefore disregarded for the hazard assessment.

 

 

1.2 in vitro mammalian mutagenicity tests (TK/HPRT)

 

Mouse lymphoma L5178Y/TK± cells in suspension culture were treated for 4 hours with different concentrations of zinc oxide with and without S9 -mix (Ziemann 2011). Medium, medium with soy lecithine, methyl methanesulfonate and cyclophosphamide were used as negative control, vehicle control, positive control without S9 -mix and positive control with S9 -mix, respectively. Zinc oxide induced increases of MF with and without S9 -mix in the presence of S9 -mix. The increase mutant colonies were primarily characterised as small colonies, indicating a clastogenic event. The significantly increased MF was always linked to pronounced cytotoxicity. For this reason and the limited significance of the test system for particles, the test results should more likely be judged as questionable in L5178Y/TK cells under the conditions and restrictions of this assay, implying a possible false positive result. Consequently, the results are interpreted as equivocal, as the clastogenic findings were confounded with cytotoxicity. This guideline study is considered reliable without restriction (RL1).

 

Amacher and Paillet (1980) tested zinc chloride for induction of tk mutations in mouse lymphoma cells. Treatment was only for 3 hrs in the absence of metabolic activation. Concentrations ranged from 1.21 -12.13 μg/mL, but there is no indication what levels of toxicity (if any) these treatments induced. There were no increases in mutant frequency as a result of these treatments. However, at the time this study was conducted there was no optimisation of the assay to detect small colony mutants (which would be expected to be representative of clastogenic or aneugenic modes of action), and no continuous 24 hr treatment in the absence of metabolic activation, as is now routine. Zinc dichloride was found to be non-mutagenic under the test conditions. This publication is well documented, meets generally accepted scientific principles and was therefore considered acceptable for assessment (RL2).

 

In an OECD 476 guideline study by Adams et al. (1994), zinc monoglycerolate was tested at concentrations of 1-15 μg/ml without metabolic activation and 1 -30 μg/ml with metabolic activation in mouse lymphoma cells. The results showed the following positive results: without metabolic activation from 10 μg/ml with metabolic activation from 15 μg/ml. The information summarised in this study record was taken from the EU Risk Assessment report for zinc (EU RAR 2004) where it has been thoroughly reviewed. Due to the unavailability of the primary reference, the study was rated with reliability 4 for formal reasons. However, the results of this study will be used in the hazard assessment of the zinc category substances in a weight of evidence approach.

 

 

1.3 in vitro mammalian clastogenicity assays (CA)

 

The in vitro mammalian chromosome aberration test was performed by Ziemann (2011) with V79 cells to assess the potential of microscaled ZnO to induce structural (and numerical) chromosome aberrations in somatic mammalian cells according to the OECD guideline 473 in compliance with GLP. V79 Chinese hamster lung fibroblast cells were exposed to the test substance for 4 h both in the absence and presence of metabolic activation system. An initial experiment on cytotoxicity was conducted to determine the concentrations of the test substance to be used in main experiment. For the particulate microscaled ZnO one concentration per main experiment was used, with 10 μg/mL for the short-term experiment without S9-mix, 12.5 μg/mL for the short-term experiment with S9-mix, and 3.0 μg/mL for the long term experiment without S9-mix, respectively. There was a decrease in mitotic index for microscaled ZnO, primarily without S9-mix. Test performance and activity of the metabolizing system were satisfactory. Irrespective of its cytotoxic potential in the absence of S9-mix, the microscaled ZnO, under all treatment modalities, did not increase significantly the aberration frequency, as compared to the respective vehicle controls. Besides some gaps, which were also evident in the negative and vehicle controls, a sum of only 4 chromosome breaks was observed in all 3 main experiments. The aberration frequencies (without gaps) in no case exceeded 5 % and they all fell within the range of the historical negative controls. Under the test conditions, microscaled ZnO is considered not to induce structural chromosome aberrations in cultured mammalian somatic cells. This guideline and GLP compliant study is reliable without restriction (RL1).

 

The study by Someya et al. (2008) was conducted to investigate the ability of zinc oxide and zinc dichloride to induce chromosome aberrations in human dental pulp cells. Human dental pulp cells (D824 cells) treated with the test material, were evaluated for chromosome aberrations at up to 3 dose levels, together with vehicle and positive controls. Rat liver (5%) post mitochondrial supernatant mixture was used as the exogenous metabolic activator. Ability to induce chromosome aberrations was examined in cells treated with test material for 3 and 30 h. The test material induced weak chromosome aberrations in the presence or absence of exogenous metabolic activation. The percentages of cells with polyploid or endoreduplication were not enhanced by test material. The authors concluded that the findings suggest that the results of genetic-toxicity tests for zinc are equivocal, probably depending on the zinc compounds tested and the type of cells used. The study is well documented, meets generally accepted scientific principles and was therefore considered acceptable for assessment (RL2).

 

Hikiba et al. (2005) tested zinc oxide in an in vitro chromosomal aberration test in SHE cells. Zinc oxide was dissolved in 0.1N hydrochloric acid and incubated at concentration of 0, 60, 120 and 180 mM (corresponding to concentrations of 4.8, 9.6 and 19.2mg/L) for 24 hrs. The highest concentration caused 91% cytotoxicity, thus only 2 concentration could be evaluated. A statistically significant increase in chromosome breaks was observed in the mid concentration (P<0.05) at acceptable cytotoxicity. No positive control was used. No explanation was provided why zinc oxide was dissolved in HCl prior testing. No confirmatory experiment was performed and the results were obtained from a single culture. Experimental conditions and chromosome preparation are insufficiently described.

 

In an in vitro chromosomal aberration test (similar to OECD 473 guideline study), Akhurst et al. (1994) tested zinc monoglycerolate at concentrations of 5 -20 μg/ml without metabolic activation and 10-40 μg/ml with metabolic activation in human lymphocytes. Zinc monoglycerolate was positive with metabolic activation at 30 and 40 μg/ml. The information summarised in this study record was taken from the EU Risk Assessment report for zinc (EU RAR 2004) where it has been thoroughly reviewed. Due to the unavailability of the primary reference, the study was rated with reliability 4 for formal reasons. However, the results of this study will be used in the hazard assessment of the zinc category substances in a weight of evidence approach.

 

A chromosome aberration test was conducted on human lymphocyte cultures to determine the mutagenic potential of zinc chloride (Deknudt & Deminatti, 1978). The concentration of zinc chloride inhibiting mitotic activity was found to be 3 X 10-3 M. Three subtoxic doses i.e., 1.5 X 10-3, 3 X 10-5 and 3 X 10-4 M were taken for the study. Human lymphocytes were obtained from a healthy donor and cultured for 48 or 72 h in Ham's F 10 medium. The test material was added to 48 and 72 h cultures at 0 and 24 h after initiation. Chromosome preparations were prepared and 100 well-spread metaphases from each culture were evaluated for the presence of numerical and structural aberrations. Chromosome aberrations (dicentric chromosomes) were observed at the lowest concentration of 3 X 10-5 M of the test material. However, the results were found to be insignificant when compared to only controls in chi-square analysis. The information summarised in this study record was taken from the EU Risk Assessment report for zinc (EU RAR 2004) where it has been thoroughly reviewed. Due to the unavailability of the primary reference, the study was rated with reliability 4 for formal reasons. However, the results of this study will be used in the hazard assessment of the zinc category substances in a weight of evidence approach.

 

In a cytogenetic study (Litton Bionetics Inc., 1974), human embryonic lung cells (Wl-38) were exposed to zinc sulfate. The cells were treated at zinc sulfate concentration levels of 0.1, 1.0, and 10 μg/plate. Vehicle (saline) and positive (triethylenemelamine) control cultures were run concurrently. A total of 100 cells were scored per concentration level. In addition, the mitotic index was determined for cytotoxicity evaluations. Zinc sulfate did not induce an increase of the proportion of cells with chromosomal damage in human embryonic lung cells (Wl-38), when compared to vehicle control cultures. The mitotic index was the same for zinc sulfate-exposed cells and vehicle control cultures. The positive control cultures showed marked increased in the proportion of cells with chromosomal damage. However, no conclusion can be drawn on the results presented due to major deficiencies with regards to reporting and the methodology applied. The test material is insufficiently characterised, since information on purity, source, manufacturer, and physical appearance are missing. The number of cells scored for chromosomal damage was very low (n=100). The vehicle control and lowest dose did not show any cells with chromosomal damage. The test material was not tested up to toxic concentrations. Evaluation and scoring criteria are not specified. Historical control data was not included. The authors did not discuss confounding factors, such as test material precipitation, or pH and osmolality effects of the test material on the culture medium. Based on the above-mentioned shortcomings the reference is considered not reliable.

 

 

1.4 in vitro mammalian clastogenicity assays (MN)

 

Kononenko et al. (2017) examined potential chromosomal damage induced by ZnO NPs, ZnO macroparticles (MPs), and ZnCl2 treatment in MDCK cells. The chromosomal damage was evaluated in a cytokinesis-block micronucleus test. Cell viability and cytostatic effects were examined concurrently by analysis of the nuclear division index and scoring of apoptotic or necrotic cells. Moreover, cytotoxicity was investigated utilising the MTT, NRU, and trypan blue exclusion assays. In further experiments, the authors examined ROS generation, ZnO NP dissolution, and the activity of catalase and glutathione-S-transferase (GST) after test material treatment. According to the authors, cell viability and proliferation were not affected under the conditions tested in the micronucleus test. Chromosomal damage was only increased after treatment with ZnO NPs at concentrations of 61 and 123 μM, when compared to untreated controls. Moreover, the test materials induced statistically significant cytotoxicity at concentration of 184 μM in the MTT and NRU assay, whereas the trypan blue exclusion assay indicated considerable cytotoxicity at concentrations of 360 μM and above. GST activity was statistically significantly decreased only after ZnO NP treatment, whereas catalase activity was reduced in treatment with both ZnO NPs and ZnCl2. However, catalase activity was only slightly impaired after ZnCl2 treatment. Acellular measurement of ROS indicated no significant in ROS levels in cell culture media after the 24 h incubation with either ZnO NPs, ZnO MPs, or ZnCl2. No conclusion can be drawn due to reporting and methodological deficiencies. The test item is insufficiently characterised, since information on the purity, impurity elements and surface modifications are missing. Cytotoxicity was not evaluated using parameters recommend by the current test guideline (OECD TG 487, 2016) and the results of the NDI calculation and apoptosis/necrosis evaluation are not shown. The micronucleus test was performed in a cell line, which is not recommended by the current test guideline. Moreover, details on the cell lines, including cell doubling time, are missing. The description of the micronucleus test lacks some details. Furthermore, the authors did not state on precipitation and distribution of ZnO NPs during the treatment. Potential effects on pH or osmolality of the culture medium induced by ZnO NPs were not discussed. Information on evaluation criteria is unclear or missing. Exposure concentrations are stated in μg/mL; however, the cell line is composed of adherent cells, and thus, it would be appropriate to specify exposure concentrations in μg/cm². Cellular uptake of the test material was not investigated. Historical control data is not provided. The micronucleus frequency was only slightly increased and micronucleus frequencies show considerable variability within groups. Based on the above-mentioned shortcomings the reference is considered not reliable and therefore disregarded for the hazard assessment.

Roszak et al. (2016) investigated on cytogenetic damage in BEAS-2B (human bronchial epithelial cells) cells after 48-exposue to ZANO® (ZnO nanosized) and Pharma B (fine ZnO) using the CBMN assay. After 6-h of exposure, cytokinesis was blocked by a cytochalasin B treatment. Each replicate was scored for the number of micronuclei present in 1000 binucleated cells. The experiment was repeated independently with adjusted concentrations. Concurrently, cytostasis was calculated from 500 cells per replicate. In addition, cytotoxicity was evaluated in a separate experiment by counting manually propidium iodide stained cells. According to the authors, both ZnO particle types showed a very steep decreasing dose-response curve in the concentration range of 1.5-3.0 μg/cm². Moreover, nanosized ZnO particles were found to be more cytotoxic in presence of BSA, whereas fine ZnO showed weaker cytotoxic effects in the presence of BSA. Based on the cytotoxicity experiment, a concentration range of 0.75 to 2.5 μg/cm² was used in the micronucleus tests. In the micronucleus test, the first experiment gave a negative outcome under any conditions tested. In contrast, in the second experiment, exposure to nanosized ZnO particles resulted in significantly increased micronucleus frequency at concentrations of 1.5 μg/cm² under all conditions tested. The increase showed, however, no concentration related response. The fine ZnO NP resulted in significant and concentration related increases of the micronucleus frequency, when treated at concentrations of >1.25 and > 1.5 μg/cm² suspended in water or water and BSA, respectively. No conclusion can be drawn due to reporting and methodological deficiencies. The micronucleus test was performed in a cell line, which is not recommended by the current test guideline or the test guideline followed. Moreover, details on the cell lines, including cell doubling time, passages, and mycoplasma infection, are missing. Description of exposure conditions lacks some details since information on cell density and temperature are not provided. The authors did not state on precipitation and distribution of ZnO NPs during the treatment. Potential effects on pH or osmolality of the culture medium induced by ZnO NPs were not discussed. Information on evaluation criteria is unclear or missing. Exposure to cytochalasin B during the particle exposure could have interfered with cellular NP uptake. Historical control data is missing. Cellular uptake and dissolution of the test material were not investigated. Notably, the results of the two independent experiments show great variability and equivocal results. In experiment 1, all treatments resulted in negative findings, whereas the second experiment showed positive results for both ZnO particle types and combinations with different vehicles used. Confirmatory experiment and experiments with other treatment schedules (according to the test guideline) were not performed. Based on the above-mentioned shortcomings the reference is considered not reliable and was disregarded for the hazard assessment.

 

 

1.5 in vitro DNA damage assays (Comet assay)

 

Kononenko et al. (2017) investigated on the DNA damaging capacity of three different test materials in in the MDCK (Madin-Darby Canine Kidney) cell line. The DNA damage was evaluated in a conventional alkaline comet assay. Moreover, cytotoxicity was investigated utilising the MTT, NRU, and trypan blue

exclusion assays. In further experiments, the authors examined ROS generation, ZnO NP dissolution, and the activity of catalase and glutathione-S-transferase (GST) after test material treatment. According to the authors, the comet assay results indicated a statistically significant increase in double- and single-strand DNA breaks in cells exposed to ZnO NP at concentrations of 61 and 123 μM, whereas ZnO macroparticles (MPs) and ZnCl2 did not. Moreover, the test materials induced statistically significant cytotoxicity at concentration of 184 μM in the MTT and NRU assay, whereas the trypan blue exclusion assay indicated considerable cytotoxicity at concentrations of 360 μM and above. GST activity was statistically significantly decreased only after ZnO NP treatment, whereas catalase activity was reduced in treatment with both ZnO NPs and ZnCl2. However, catalase activity was only slightly impaired after ZnCl2 treatment. Acellular measurement of ROS indicated no significant in ROS levels in cell culture media after the 24 h incubation with either ZnO NPs, ZnO MPs, or ZnCl2. No conclusion can be drawn due to reporting and methodological deficiencies. The test item is insufficiently characterised, since information on the purity, impurity elements and surface modifications are missing. Description of test material preparation and comet methodology lacks some details. Only 60 cells were scored for DNA damage per experiment, the number of experiments was, however, comparatively high (n=6). Cytotoxicity was not tested in parallel. The comet assay was performed in a cell line which is non-standard for genotoxicity testing. Details on the cell lines, including cell doubling time, are missing. The authors do not state removal and disposition of particles after exposure. Remaining ZnO NPs could have interfered with electrophoresis and comet scoring. Furthermore, the author did not state on precipitation and distribution of ZnO NPs during the treatment. Potential effects on pH or osmolality of the culture medium induced by ZnO NPs were not discussed. Information on comet assay evaluation criteria is unclear or missing. The authors do not state on scorable vs. non-scorable cells and hedgehog occurrence is not stated. Exposure concentrations are stated in μM and μg/mL; however, the cell line is composed of adherent cells, and thus, it would be appropriate to specify exposure concentrations in μg/cm². Moreover, laboratory’s proficiency in preparing and analysing comets’ characteristics in comet assays is a prerequisite for reliable data. It was shown that there is huge variability in tail DNA intensities even within negative or positive controls (Tavares, A.M. et al. (2014); Nanogenotox Programme). Cellular uptake was not examined. Based on the above-mentioned shortcomings the reference is considered not reliable and therefore disregarded for the hazard assessment.

 

 

1.6 Other information

 

Several studies were identified which do not fulfil the relevance, reliability and adequacy criteria as foreseen by the ECHA Guidance on information requirements. DNA damage in bacteria, induction of SCE in mammalian cells, or tests in yeasts or drosophila are no longer recommended as part of regulatory testing by many agencies worldwide and there are no up-to-date OECD guidelines for their conduct. Other studies used study designs with unclear or questionable informative value for the endpoint heritable gene mutation, such as e.g. the host mediated assay. All references in the following summary entries are more than 30 years of age and do not comply with today’s standards in genetic toxicity testing. Interpretation of the relevance of both positive and negative results from such tests is therefore unclear and was not used for the current assessment.

The studies are discussed below in brief for information purposes only:

 

In a test series by Suzuki et al. (1987), a sister chromatid exchange (SCE), an unscheduled DNA synthesis (UDS) and a cell transformation assay was conducted in Syrian hamster embryo cells with zinc oxide. Results were mixed, showing positive as well as ambiguous results.

 

Syrian hamster embryo (SHE) cell assay was conducted by DiPaolo and Casto (1979), to evaluate the morphologic transformation inducing ability of zinc chloride, using hamster embryo cells (HEC). At the highest concentrations (20 μg/mL), cloning efficiency was reduced by 20 to 25% relative to the control. No morphological transformations were observed in the test cultures. Under the test conditions, the test material was found to be non-transforming at 20 μg/mL.

 

A host mediated assay was conducted by Litton Bionetics (1974), showing a weakly positive response with zinc sulphate.

A Bacillus subtilis recombination assay (rec-assay) was conducted using H17 Rec+ (rec+ arg try) and M45 Rec- (rec45 arg try) strains exposed to zinc chloride (Kada et al. 1980). The test material did not show an increased lethal activity on Rec- in comparison with Rec+ cells and therefore did not cause cellular damage.

 

A preliminary screening of zinc sulphate was carried out to determine the potential of zinc sulfate for induction of gene conversion at trp locus and reverse mutation at the ilv locus in the D7 strain of Saccharomyces cerevisiae (Singh et al. 1983). Weak positive responses were observed for both induction of gene conversion and reverse mutation in the D7 strains of Saccharomyces cerevisiae.

 

Siebert et al. (1970) conducted a study to determine the potential of zinc sulfate to induce mitotic gene conversion using diploid strain D4 of Saccharomyces cerevisiae. The test material showed inability to induce mitotic gene conversion. Convertants per 10E6 survivors at the loci ade2 & trp5 was 45.7 & 2.9, respectively.

 

Rossman et al. (1984) conducted a study to determine the potential of the test material for induction of λ prophage in E coli WP2 (λ) with zinc chloride. Under the test conditions, 3.2 mM of the test material caused a two-fold increase in induction of λ prophage over control (reported as +/-).

 

 

2 In vivo

2.1 Chromosomal aberration tests

 

Gupta et al. (1991) conducted a chromosomal aberration assay in Swiss albino mice after both single and repeated intraperitoneal injections of zinc chloride. In the acute exposure experiment, the mice received a single intraperitoneal injection of zinc chloride at dose levels of 7.5, 10, and 15 mg/kg bw. The mice were sacrificed 24 hours after the treatment. In the repeated dose experiment, the test material was administered, at dose levels of 2 and 3 mg/kg bw, every alternate day and the mice were sacrificed on day 8, 16, or 24. Test animals assigned to negative and positive control groups received intraperitoneal injections of isotonic and cyclophosphamide, respectively. Afterwards, the bone marrow cells were obtained and stained with Giemsa. A total of 60 metaphases per animal were scored for chromosomal aberrations and 1000 cells per animal were scored for the determination of the mitotic index. The chromosomal aberration frequency was statistically significantly increased at all zinc chloride doses tested, when compared to the negative control group. However, in the repeated exposure experiment, the low dose group showed relevant increases in the proportion of cells with chromosomal aberration and chromosomal aberrations per cell only at exposure durations longer exceeding eight days. The response was calculated to be related to the dose level and exposure duration. The mitotic index was not statistically significantly altered but was, in some sets, reduced by up to 37%. The authors considered zinc chloride to be a potent clastogen. No conclusion can be drawn due to reporting and methodological deficiencies. The test material was administered via intraperitoneal injections, which is considered to be a non-physiological exposure route and bears only very limited value for hazard assessment purposes. The test material was only poorly characterised, since information on the purity, source, manufacturer, and physical appearance are not provided. Furthermore, details on the test material preparation and dosing volume are missing. Information on terminal body weights, clinical signs, and mortalities are not reported. The description of the test animals is insufficient, since information on the sex, housing, water supply, and test group randomisation are missing. Moreover, the animals are kept at higher temperatures than usually recommended by in vivo genotoxicity guidelines (28±2°C vs. 22±3°C). Further, the number of animals scored for chromosomal aberrations was lower than recommended by the test guideline in force at that time (5 vs. 10). The description of the treatment with the metaphase arresting agent lacks details, since the time point of treatment is not specified. Moreover, the description of the metaphase preparation lacks details. Historical control data is not included. Further, evaluation and scoring criteria are not specified.

 

Deknudt and Gerber (1979) investigated on the potential of zinc (zinc chloride) to induce chromosome aberrations in bone marrow cells of C57BL mice. Male C57BL mice were exposed to the test material daily via standard and low-calcium diet for one month. The metaphase bone marrow cells were obtained and stained using lacto-orcein. A total of 50 cells per animal (a total of 500 from each group) were analysed for chromosomal aberrations. The body weights of mice were statistically significantly reduced after zinc exposure. Male mice kept on standard diet in combination with zinc showed terminal body weights reduced by 33.4%, when compared to mice fed with standard diet only. Moreover, exposure to a low calcium diet in combination with zinc resulted in terminal body weights reduced by 44.7% and 32.7%, when compared to corresponding control mice and mice fed on low calcium in combination with zinc, respectively. Notably, according to the authors, all mice kept on low calcium diet were weak, seemed anaemic and had brittle femurs. The serum calcium level was statistically significantly reduced only in animals exposed to standard diet in combination with zinc. The number of dicentrics as well as the number of cells carrying structural

aberrations was statistically significantly increased in mice kept on a low calcium diet in combination with zinc. No conclusion can be drawn due to reporting and methodological deficiencies. The positive finding was only observed only in mice kept on an extreme low calcium diet and presence of increased general toxicity as characterised by marked body weight reductions, weakness, anaemic appearance, and brittle femurs. In contrast, mice kept on standard diet did not show effects on the chromosomal aberration frequency. Further, the mitotic index or equivalent cytotoxicity parameters were not evaluated. Thus, the effects observed cannot be clearly assigned to be caused by genotoxicity. Moreover, the experiments were restricted to a single zinc dose level, which precludes the evaluation of dose-response relationships. The test material was insufficiently characterised, since information on the purity, source, manufacturer, and physical appearance are missing. Historical control data are not provided. Furthermore, evaluation and scoring criteria are not specified. A positive control group was not included. It is not explicitly stated whether the chromosome aberration frequency is calculated including or excluding gaps. The results are only presented as group means, individual data are, however, missing. The food consumption and actual test material intake are not specified. Information on water consumption is missing. The description of the test animals lacks details, since information on the source, housing, environmental conditions, and water availability are missing. Based on the above-mentioned shortcomings the reference is considered not reliable and therefore disregarded for the hazard assessment.

 

Voroshilin et al. (1978) investigated on chromosomal aberrations in bone marrow cells of rats, which were exposed to zinc oxide via inhalation. Noninbred female white rats were exposed to zinc oxide aerosols at concentration levels of 0.1 and 0.5 mg/m³ continuously for a total period of five months. Negative control animals were run concurrently. A total of 200 metaphases per group were scored for chromosomal aberrations. According to the authors, the chromosomal aberration and hyperdiploid frequency were statistically significantly increased in rats exposed to zinc oxide aerosol at both concentration levels tested. No conclusion can be drawn due to reporting and methodological deficiencies. The test material was only poorly characterised, since information on the source, purity, manufacturer, and physical appearance are missing. Furthermore, all essential information on the aerosol generation and characterisation are missing. The concentration level of zinc oxide in the test atmosphere was not analytically verified. The number of test animals and metaphases per animal scored for chromosomal aberrations are not specified. The authors provided no information on body weight development, clinical signs, food/water consumption, mortality, or any other parameter potentially indicating general toxicity. Moreover, nearly all relevant information on the test animals, i.e. strain, age, body weights, source, housing, environmental conditions, and food/water supply, are missing. Cytotoxicity was not examined. The methodology of the assay is insufficiently described, since information on cell sampling and slide preparation are not included. Only two dose groups were included which impedes a robust evaluation of potential concentration-response relationships. Historical control data are not included. Evaluation and scoring criteria are not specified. Based on the above-mentioned shortcomings the reference is considered not reliable and therefore disregarded for the hazard assessment.

 

The clastogenic potential of zinc sulfate was assessed in Sprague Dawley rats using the bone marrow chromosome aberration test (Litton Bionetics Inc., 1974). Groups of five male rats were exposed to zinc sulfate at doses of 2.75, 27.5, and 275 mg/kg bw via gavage. In the acute study part, the animals received a single treatment and were sacrificed 6, 24, and 48 hours later. Animals assigned to the subacute study received daily treatments on five consecutive days. A vehicle control group was run concurrently. Test animals of the positive control group received a single intraperitoneal injection of triethylenemelamine. Prior to sacrifice, the animals received an intraperitoneal injection of colcemid. Afterwards, femoral bone marrow cells were harvested and stained with Giemsa. A total of 50 metaphases per animal were scored for the presence of chromosomal aberrations. In addition, the mitotic index was determined based on 500 cells scored for their mitotic state. Zinc sulfate did not induce any chromosomal aberration in bone marrow cells of rats exposed via gavage under the conditions tested. Moreover, the mitotic indices were comparable to the vehicle control values indicating the absence of cytotoxic effects. No conclusion can be drawn due to reporting and methodological deficiencies. The test material is insufficiently characterised, since information on purity, source, manufacturer, and physical appearance are missing. The test material was not tested up to toxic (no toxic effects reported) or cytotoxic doses. Thus, the exposure of the bone marrow was not demonstrated. The number of animals in the solvent control group was low (n=3). Furthermore, the number of cells scored for chromosomal aberration (n=50 per animal) was too low, since no chromosomal

aberrations were detected in both solvent control and test material-exposed animals. General toxicity was either not sufficiently investigated or not sufficiently described.

 

 

2.2 Micronucleus assays

 

A 14-day repeated dose inhalation toxicity study (Creutzenberg, 2013) was conducted with microscaled zinc oxide in rats exposed subacutely via nose-only exposure according to the OECD Guideline 412 in compliance with GLP. In the framework of this study also systemic clastogenic and aneugenic effects were investigated in male and female rats using the bone marrow micronucleus assay according to OECD Guideline 474. Five rats per sex and group were exposed at a concentration level of 8 mg/m³ with microscaled zinc oxide. Fresh air treated animals served as concurrent control. Positive control animals were orally exposed to 20 mg/kg cyclophosphamide monohydrate. There was no treatment-related reduction of body weights in any test group; no clinical signs were detected. However, high dose rats showed adverse local effects in the lungs. In male rats, the test item and the reference substances did not mediate significant repression of red blood cell formation. In females, the PCE/NCE was significantly reduced in the positive control. There was no evidence of a significantly enhanced mean frequency of micronucleated erythrocytes due to microscaled zinc oxide exposure in males or females, as compared to the vehicle control groups (clean air) at any dose level. The positive and vehicle controls gave valid results. Notably, exposure of the target was not demonstrated. Inhaled microscaled zinc oxide is considered non-mutagenic in immature bone marrow erythrocytes (PCE) of Wistar rats under the conditions tested.

 

Li et al. (2012) examined on the capacity of ZnO NP and ZnO MP to induce chromosomal damage in peripheral blood erythrocytes of orally treated mice. The mice received a single gavage of the test materials and blood samples were taken after 24, 48, and 72 h. The chromosomal damage in erythrocytes was examined via a micronucleus test. Moreover, the authors investigated on the adsorption and biodistribution of the test material after a single oral gavage. According to the authors, mice orally exposed to ZnO NPs and ZnO MPs showed no significant differences in the frequency of micronucleated PCEs, when compared to the solvent control group. Moreover, the ratio of PCEs among total erythrocytes was not statistically significantly different between groups. In contrast, the positive control group showed both significantly increased micronucleated PCE frequencies and depressed PCE to total erythrocytes ratios. The analysis of the adsorption and biodistribution after oral treatment revealed rapid absorption in the serum and biodistribution to liver, spleen, and kidneys. In contrast, the lung, heart, brain, and testes showed no increased zinc levels after ZnO NP and ZnO MP treatment. No conclusion can be drawn due to reporting and methodological deficiencies. The test material is insufficiently characterised, since information on purity, and impurity elements is missing. Further, the description of the test material preparation lacks details. The description of the test animals lacks details, including body weights at study initiation, test group randomisation, and housing. Moreover, the description of the micronucleus test methodology lacks details. The number of cells scored for chromosomal damage (1000) was lower than recommended by the OECD TG 474 (≥4000). Furthermore, the number of cells scored to determine the ratio of PCEs among total erythrocytes was lower than requested by OECD TG 474. Information on clinical signs is missing. Evaluation and scoring criteria are not specified. Data from individuals is not specified. Exposure of the target organ was not discussed. However, the analysis of the serum showed rapidly increased zinc levels after a single oral treatment with ZnO NPs and ZnO MPs at 2.5 g/kg bw. Historical control data is missing. Based on the above-mentioned shortcomings the reference is considered not reliable and therefore disregarded for the hazard assessment.

Gocke et al. (1981) evaluated zinc sulfate for its clastogenic and aneugenic potential in bone marrow erythrocytes of NMRI mice treated via intraperitoneal injections. Two mice per sex and group received two intraperitoneal injections of zinc sulfate at dose level of 28.8, 57.5, and 86.3 mg/kg at 0- and 24-hours. Six hours after the final treatment, the mice were euthanised and the bone marrow cells were harvested. Subsequently, bone marrow smears were prepared and 1000 polychromatic erythrocytes per mouse were scored for the occurrence of micronuclei. A negative control group was run concurrently.

NMRI mice exposed to zinc sulfate, at three different dose levels, via intraperitoneal injections did not show a statistically significant increase in the micronucleus formation rate, when compared to the concurrent negative control group. No conclusion can be drawn due to reporting and methodological deficiencies. The test material was administered via intraperitoneal injections, which is a non-physiological exposure route with only limited value for hazard assessment purposes. The test material is only poorly characterised, since information on the purity, manufacturer, and physical appearance are missing. Information on the and test material preparation are not provided. The animal number was low, and thus, the robustness of the data is questionable. One animal of the high dose group died prematurely; the cause for the death was, however, not discussed. The authors did not examine general toxicity and cytotoxic effects. It was not demonstrated the test material reached the target organ. The harvest of the bone marrow cells, six hours after the final treatment, appears to early in consideration of exposure and micronucleus formation kinetics. The dose range and top dose selected were not justified. Historical control data is not provided. Evaluation and scoring criteria are not specified. Based on the above-mentioned shortcomings the reference is considered not reliable and therefore disregarded for the hazard assessment.

 

In a micronucleus study (Windebank et al., 1995) comparable to OECD guideline study 474, rats were fed 0.05, 0.2 and 1% zinc monoglycerolate in a purified diet. Zinc monoglycerolate was negative in this assay. The information summarised in this study record was taken from the EU Risk Assessment report for zinc (EU RAR 2004) where it has been thoroughly reviewed. Due to the unavailability of the primary reference, the study was rated with reliability 4 for formal reasons. However, the results of this study will be used in the hazard assessment of the zinc category substances in a weight of evidence approach.

 

 

2.3 Rodent dominant lethal assay

 

In a dominant lethal assay (Litton Bionetics Inc., 1974), zinc sulfate was evaluated for its ability to induce mutations resulting from chromosomal aberrations in germ cells of orally exposed Sprague Dawley rats. Groups of five male rats were exposed to zinc sulfate at doses of 2.75, 27.5, and 275 mg/kg bw via gavage. In the acute study part, the animals received a single treatment and were sacrificed 6, 24, and 48 hours later. Animals assigned to the subacute study received daily treatments on five consecutive days. A vehicle control group was run concurrently. Test animals of the positive control group received a single intraperitoneal injection of triethylenemelamine. Each male was mated with two virgin females on five consecutive day. This procedure was repeated for 7 (subacute study) to 8 (acute study) weeks with a two-days break between each mating interval with fresh females. After 14 days, the mated females were sacrificed and scored for number of implantations, corpora lutea, pre-implantation losses, and resorptions (dead implants) as well as the proportion of females with one or more dead implants, two or more dead implants, and dead implants among total implants. In addition, the fertility index was calculated. When compared to concurrent vehicle controls, the acute zinc sulfate groups showed statistically significantly increased fertility indices at weeks four and six. The observed response was, however, restricted to only these two out of eight weeks and the response did not show a positive dose-response relationship. Similarly, in the subacute study, a statistically significant reduction of the fertility index was observed at the third week of mating. However, no such effect was observed in the high dose group or at other mating intervals. Thus, the findings with regard to fertility are considered to be of an incidental nature. The average preimplantation loss per pregnant female was sporadically statistically significantly increased in the zinc sulfate experimental groups. In the acute study, the average preimplantation losses per pregnant female was statistically significantly increased in the low dose group at week 1 and 2 as well as in the intermediate dose group at week seven. However, these findings are considered to be chance findings, since these effects were only of a sporadic nature and not observed in the high dose group. The remaining alterations showing statistical significance are considered to be not biologically relevant. No conclusion can be drawn due to reporting and methodological deficiencies. The test material is insufficiently characterised, since information on purity, source, manufacturer, and physical appearance are missing. The test material was not tested up to toxic doses (no toxic effects reported). The positive control group did not demonstrate the sensitivity of the test system, since the lethals were comparable to the lethals of the concurrent vehicle control groups. General toxicity was either not sufficiently investigated or not sufficiently described. Historical negative control data is only specified as mean without ranges. The results presented in the tables are only limitedly retraceable.

 

 

2.4 in vivo DNA damage (Comet assay)

 

Hornarvar (2022) investigated on the genotoxic potential of micro-scaled Zinc oxide (T0242) and Zinc sulfate monohydrate in male Wistar rats after a whole-body inhalation exposure. Five rats per group were exposed to micro-scaled Zinc oxide (T0242) and Zinc sulfate monohydrate at respective target concentration levels of 8 mg/m³ and 18 mg/m³ for 6 hours/day on 14 consecutive days. Vehicle control (clean air) rats were run concurrently. Approximately 0.5-1 hour after the last exposure, the animals were sacrificed, and tissues were prepared for the Comet assay and BALF analysis. Bone marrow, liver, lung and nasal mucosa are tested for DNA damage using the Comet assay. A total of 150 cells per tissue sample were scored for the tail intensity (%DNA in tail). As positive controls, sample tissues of animals administered orally at a single time point Ethyl methanesulfonate (EMS), were scored.

There were no signs of general toxicity, nor were there any impairment of body weight gain. Excessive cytotoxicity was not detected. The exposure of single concentration of the micro-scaled Zinc oxide (T0242) and Zinc sulfate monohydrate caused significantly increases in the lavage parameters gamma-glutamyl-transferase; lactate dehydrogenase; alkaline phosphatase and beta-N-acetyl glucosaminidase (only for Zinc oxide (T0242)). The assessment of the target tissues in the comet assay did not show any biologically relevant increases in the % tail intensity of the analyzed tissues. The positive control group showed a distinct and statistically significant increase in all analyzed tissues. The study is considered valid, since negative control values were acceptable for addition to the historical control data base. Thus, under the conditions tested, micro-scaled Zinc oxide and Zinc sulfate monohydrate are considered as non-genotoxic in this assay.

 

Creutzenberg (2013) investigated on the potential site-of-contact genotoxicity of microscaled zinc oxide in male Wistar rats after repeated nose-only inhalation. Five rats were exposed to microscaled zinc oxide aerosol at a concentration level of 8 mg/m³ for 5 days/week and 6 hours/day over a total period of two weeks. Vehicle control (clean air) rats were run concurrently. At post exposure days 1 and 14, BAL cells were obtained and tested for DNA damage using the modified alkaline comet assay. In this type of comet assay, lysed cells were either treated with buffer or with hOGG1 enzyme to detect general and oxidative DNA damage. A total of 100 cells per animal were scored for the tail intensity (% DNA in tail). As technical positive control served potassium bromate-treated L5178Y/TK+/- mouse lymphoma cells. Moreover, in an independent experiment, rats exposed to potassium bromate (250 mg/kg bw) via intraperitoneal injection served as in vivo positive controls. Apart from the comet assay, the authors examined the formation of 8-OH-dG in formalin-fixed tissue of the terminal bronchioles and lung parenchymal cells via immunohistochemistry. Wistar rats exposed to microscaled zinc oxide did not show signs of general toxicity. However, the local adverse effects in the lung were evident in the highest dose group. Notably, the LDH activity and total protein level in BAL fluid was markedly and statistically significantly increased in animals exposed at a concentration level of 8 mg/m³ indicating marked cytotoxicity. The BAL cells of rats exposed to microscaled zinc oxide did not show a statistically significant increase in the tail intensity after 1 and 14 days of the final treatment, when compared to the concurrent vehicle control. Moreover, the hOGG1 treatment did not reveal an increase of oxidative DNA damage. In contrast, the independent in vivo positive controls showed marked and statistically significantly increase of oxidative DNA damage both with and without hOGG1 treatment, when compared negative controls. Furthermore, independently generated positive control data from rats exposed to EMS showed a marked increase in the tail intensity, when compared to concurrent negative controls, in a conventional alkaline comet without hOGG1 treatment. Thus, the general sensitivity of the test system was demonstrated. Moreover, the technical positive control demonstrated the activity of the used hOGG1 enzyme and the appropriate performance of the comet assay methodology. In addition, terminal bronchioles and lung parenchymal cells from microscaled zinc oxide exposed rats did not show statistically significantly increased numbers of 8-OH-dG positive nuclei, when compared to the vehicle control values. Thus, the lack of microscaled zinc oxide-induced oxidative DNA damage observed in the comet assay was further substantiated by the data obtained in the immunohistochemical assay using lung sections. The study presented herein is considered to be acceptable with restrictions. The experiment included in the study were not performed under GLP conditions, which was basically due to the fact that an OECD validated test guideline was not available at that time. The tail intensity was determined only using a single slide per animal; replicates were not included. Furthermore, the responses observed showed in several cases a huge variability. Thus, the robustness of the data may be limited. Moreover, the description of the methodology lacks detailed information on cell lysis, neutralisation, and electrophoresis. The positive controls were run only in an independent experiment, concurrent positive control animals were not included. Cytotoxicity was not sufficiently investigated and evaluated. The only parameters useful for an evaluation of cytotoxicity are the LDH activity and protein level in BAL fluid. Evaluation criteria are not specified. Historical control data are not included. Only one dose level was tested, which precludes an evaluation of dose-response relationships. The test material, microscaled zinc oxide, is considered to have no DNA damaging potential based on the absence of biological relevant increases in the median derived mean tail intensity under the conditions tested (RL2).

 

A study was conducted by Banu et al. (2001) to determine the in vivo genotoxic effect of zinc sulfate in mouse peripheral blood leukocytes using alkaline single cell gel electrophoresis. Mice were administered orally with doses of 5.70, 8.55, 11.40, 14.25, 17.10, and 19.95 mg/kg bw of zinc sulfate. The samples of whole blood were collected at 24, 48, 72, 96 h and first week post-treatment and the assay was carried out to determine single strand DNA breaks as represented by comet tail-lengths. Zinc sulfate was found to be positive in this assay. The information summarised in this study record was taken from the EU Risk Assessment report for zinc (EU RAR 2004) where it has been thoroughly reviewed. Due to the unavailability of the primary reference, the study was rated with reliability 4 for formal reasons. However, the results of this study will be used in the hazard assessment of the zinc category substances in a weight of evidence approach.

 

 

2.5 Other information

 

Gocke et al. (1981) performed the sex linked recessive lethal test in Drosophila melanogaster to evaluate the mutagenic potential on germ cell of zinc sulfate. Berlin K (wild type) and Base strains were used for the study. The flies were exposed to zinc sulfate at a concentration level of 5 mM (in 5% saccharose) via the feeding method. Negative and positive (trenimon) control groups were run concurrently. Approximately 1200 X-chromosomes were tested in each of 3 successive broods (3-3-4 days). The assay was performed in three independent experiments. In repeat experiments, sometimes only single broods were tested.

Offspring generations were scored for sex-linked recessive lethals. According to the authors, in the first experiment, a statistically significant increase in the proportion of sex-linked recessive lethals was observed in the first brood of zinc sulfate exposure group. However, no such effect was observed in subsequent broods or the repeat experiments. Thus, the finding was considered to be of an incidental nature. Due to the unsuitable study design with the following major restrictions this study will not be used for hazard assessment purposes but as supplementary mechanistic information. The dipteran Drosophila melanogaster is considered to be a no relevant test system for the human health hazard assessment. The test material is only poorly characterised, since information on the purity, manufacturer, and physical appearance are missing. Further, the description of the test material preparation lacks details. Only one dose group was included, which precludes dose-response relationship evaluations. Moreover, information on the flies age and culture conditions are missing. Furthermore, the number of non-fertile males and toxicity data is not reported. Historical control data is not provided. Evaluation criteria are not specified.

 

Carpenter and Ray (1969) conducted a Sex-linked Recessive Lethal (SLRL) Test in Drosophila melanogaster with zinc chloride. Drosophila melanogaster populations were exposed to 96.88 and 312.40 μCi (radioactive) and 0.247 mg (non-radioactive) per mL of food designated as ‘low’, ‘high’, ‘cold’ media. The population exposed to ‘Difco Drosophila Medium M’ served as control. Very significant differences were observed in viabilities of egg samples and rates of appearance of sex-linked recessive lethal mutations in flies from the 'high' medium and those from the "cold" or "control" medium. Also, three visible wing mutations were recovered in males from the 'high' medium. The non-radioactive test material did not cause any sex-linked recessive lethal mutation at the tested dose level of 0.247 mg/mL under the conditions of this test. However, significant mutations were observed in the 'high' radioactive group of the test material. Due to the unsuitable study design and insufficient documentation this study will not be used for hazard assessment purposes but as supplementary mechanistic information.

 

 

3 Conclusion

 

Data on the inorganic zinc category substances have been used, based on the assumption that after intake the biological activities of the zinc compounds are determined by the zinc cation (further details on the read-across are given in the report attached to IUCLID section 13).

No positive Ames result have been observed for any of the zinc category substances in a range of guideline compliant bacterial reverse mutation tests. This is supported by a larger number of studies of lower quality. The negative findings in the bacterial mutation assays have also been confirmed in in vitro mutagenicity tests in mammalian cells (tk assays). The weak positive responses were primarily seen at high test concentrations in which the findings were confounded by elevated levels of cytotoxicity. Positive responses manifested predominantly in formation of small colonies, which is indicative for a clastogenic event.

Clastogenicity (chromosome breakage) can often be caused by oxidative damage, or by indirect mechanisms such as excessive cytotoxicity, disruption of non-DNA targets etc. Such mechanisms would be expected to have a threshold. The clastogenic potential of zinc substances in vitro, as seen in chromosomal aberration, micronucleus and tk mutation (small colony mutants) assays, has been satisfactorily addressed by negative in vivo micronucleus results with zinc oxide.

Potential site of contact genotoxicity has been addressed by a 14-day inhalation (nose-only) comet assay in rats, showing a pronounced inflammatory response after inhalation of 8 mg/m³ zinc oxide (expressed in increased LDH and TP levels) but no elevated levels of %DNA in tail. It is therefore concluded that zinc oxide does not elicit site of contact genotoxicity.

Site-of-contact genotoxicity evaluation in lung epithelial cells and nasal mucosa cells, as well as systemic genotoxicity evaluation in liver and bone marrow has been addressed by a 14-day inhalation (whole-body) comet assay in rats. Pronounced inflammatory response after inhalation of 8 mg/m³ zinc oxide and 18 mg/m³ zinc sulfate monohydrate (expressed in increased gamma-glutamyl-transferase; lactate dehydrogenase; alkaline phosphatase and beta-N-acetyl glucosaminidase levels) but no elevated levels of %DNA in tail, were shown. It is therefore concluded that zinc oxide and zinc sulfate monohydrate do not elicit site of contact or systemic genotoxicity.

Based on the entire database of genetic toxicity studies it is concluded that in summary, soluble zinc substances do not elicit any mutagenic activity either in bacterial or mammalian test systems. However, they induce some genotoxic effects in vitro, mainly manifest as DNA strand or chromosome breaks. A weight-of-evidence approach was applied, considering guideline compliant negative in vivo clastogenicity and site of contact comet assay studies. It is concluded that effective protective processes exist in vivo to prevent genetic toxicity with relevance for humans from the soluble zinc salts category.

 

Table: Weight of evidence analysis of all existing genetic toxicity data for non-nano zinc category substances.

		RL1		RL2	RL3	RL4
In vitro
Bacterial reverse mutation test		NEGATIVE: Zinc bis(dihydrogen phosphate), Taublova, 2010, KL1   NEGATIVE: Zinc nitrate, Taublova, 2010, KL1		NEGATIVE: ZnO Li et al., 2012, KL2	NEGATIVE: Zinc sulphate: Marzin 1985, KL3	NEGATIVE: ZnO, Ames, Litton Bionetics 1976, KL4     NEGATIVE: Zinc monoglycerolate, Jones 1994, KL4
In vitro gene mutation test		AMBIGUOUS: ZnO, Ziemann (2011), TK assay, KL1		NEGATIVE: ZnCl2 TK, Amacher and Paillet, 1980, KL2		POSITIVE: Zinc monoglycerolate, Adams 1994, KL4
In vitro chromosome aberration test		NEGATIVE: ZnO, Ziemann (2011), CA, KL1		AMBIGUOUS: ZnO, Someya et al, 2008, KL2   NEGATIVE: ZnCl2, CA test, Someya et al, 2008, KL2	NEGATIVE: Nano and micro ZnO, Chromosomal aberration, Li C-H et al.,2012, KL3     POSITIVE: ZnO, Hikiba et al, 2005, KL3     NEGATIVE: Zinc sulphate, Cytogenetic assay, Litton Bionetics Inc., 1974, KL3	POSITIVE: Zinc monoglycerolate, Akhurst et al, 1994, KL4   AMBIGUOUS: ZnCl2, Deknudt & Deminatti, 1978, KL4
In vitro micronucleus test					NEGATIVE: ZnCl2, MN, Kononenko 2017 KL3
In vivo
In vivo chromosome aberration test					NEGATIVE: Zinc sulphate, DLA rat, Litton Bionetics, 1974, KL3   NEGATIVE: Zinc sulphate CA rat, Litton Bionetics 1974, KL3   POSITIVE: ZnCl2 CA mouse, Gupta et al, 1991, KL3
In vivo micronucleus test		NEGATIVE: ZnO, Creutzenberg 2013, MN, KL1				NEGATIVE: Zinc monoglycerolate, Windebank 1995, KL4
In vivo DNA damage				NEGATIVE: ZnO, Comet assay, Creutzenberg 2013, KL2   ZnO/Zinc sulphate, Comet assay rat, Hornarvar 2022, KL2		POSITIVE: Zinc sulphate, Comet rat, Banu 2001, KL4

 

Studies disregarded for hazard assessment purposes:

In vitro

In vivo

POSITIVE: Zinc oxide, Suzuki 1987, UDS KL4 AMBIGUOUS: Zinc oxide, Suzuki 1987, SCE KL4 AMBIGUOUS: Zinc sulfate, Litton Bionetics 1974, host mediated assay KL3 NEGATIVE: Zinc chloride, Kada et al. 1980, REC assay KL4 AMBIGOUS: Zinc chloride, Rossmann et al. 1984, induction of λ prophage in E. coli KL3 NEGATIVE: Zinc chloride, DiPaolo and Casto 1979, cell transformation assay KL3 POSITIVE: Zinc sulfate, Singh 1983, reverse mutation S cerev KL3 NEGATIVE: Zinc sulfate, Siebert 1970, mitotic recombination KL3 NEGATIVE: Zinc oxide, Crebelli 1985, bacterial reverse mutation KL3 NEGATIVE: Zinc sulfate, Gocke et al. 1981, bacterial reverse mutation KL3 POSITIVE: Zinc oxide, Suzuki 1987, cell transformation KL3 POSITIVE: Zinc oxide, Roszak et al. 2016, MN KL3 NEGATIVE: Zinc oxide, zinc chloride, Kononenko 2017, comet assay KL3

POSITIVE: Zinc oxide, Voroshilin et al. 1978, CA KL3 AMBIGUOUS: Zinc chloride, Deknudt & Gerber 1979, CA KL3 NEGATIVE: Zinc sulfate, Gocke et al. 1981, MN KL3 NEGATIVE: Zinc oxide, Li et al. 2012, MN KL3 NEGATIVE: Zinc chloride, Carpenter and Ray 1969, Drosophila SLRL KL3 NEGATIVE: Zinc sulfate, Gocke et al. 1981, Drosophila SLRL KL3

Justification for classification or non-classification

Based on the weight of the evidence from the existing in vitro and in vivo genotoxicity assays as presented and discussed above, it is concluded that the zinc category substances do not have biologically relevant genotoxic activity. Consequently, no classification for germ cell mutagenicity is applicable.

This conclusion is in line with those achieved by other regulatory reviews of the genotoxicity of zinc compounds (WHO, 2001; SCF, 2003; EU RAR, 2004, MAK, 2009). Hence, no classification and labelling for mutagenicity is required.