Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2011

Materials and methods

Test guideline
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

Test material

Constituent 1
Chemical structure
Reference substance name:
Zinc oxide
EC Number:
215-222-5
EC Name:
Zinc oxide
Cas Number:
1314-13-2
Molecular formula:
ZnO
IUPAC Name:
oxozinc
Test material form:
solid: nanoform
Details on test material:
- Name of test material (as cited in study report): Z-COTE HP1
- Molecular weight (if other than submission substance): 81.38 g/mol
- Physical state: solid
- Composition of test material, percentage of components: nanoscaled ZnO (content W/W 98%) coated on surface with triethoxycaprylylsilane CAS # 2943-75-1 (content W/W 2%)
- Lot/batch No.: NPL Ref #: ZB250#65
- Expiration date of the lot/batch: June 2014
- Stability under test conditions: stable in vehicle for at least 14 days
- Storage condition of test material: room temperature, dry place, in the dark

Method

Target gene:
thymidine kinase (TK)
Species / strain
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)
Controls
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 : 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

Results and discussion

Test results
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:
"Test system'

Any other information on results incl. tables

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

Applicant's summary and conclusion

Conclusions:
Interpretation of results:
ambiguous relevant increase in mutant frequency always linked to cytotoxicity
Executive summary:

Mouse lymphoma L5178Y/ TK±cells in suspension culture were treated for 4 hours with different concentrations of the test item 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. The gene mutation potential of the test item Z-COTE HP1 (coated nanoscaled ZnO) was determined in comparison to the reference items Z-COTE (non-coated nanoscaled ZnO) and microscaled ZnO.

Z-COTE HP1 induced relevant increases of MF with and without S9 -mix with a clear concentration-dependency in the presence of S9 -mix.

However, significantly increased MF was always linked to acute 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.