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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 Jan - 08 Feb 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes
Type of assay:
other: mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
TK locus
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: JHSF (Japan Health Science Foundation)

MEDIA USED
- Type and identity of media: RPMI 1640 medium supplemented with horse serum, sodium pyruvate and penicillin/streptomycin
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically 'cleansed' against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254 for 5 days
Test concentrations with justification for top dose:
Cytotoxicity test, short-term treatment (4 h), with and without metabolic activation: 5000, 2500, 1250, 625, 312, 156, 78 and 39 µg/mL
Cytotoxicity test, long-term treatment (24 h), without metabolic activation: 1000, 500, 250, 125, 62.5, 31.25, 15.63 and 7.8 µg/mL

In each assay 4 doses were selected for the gene mutation test:
Short-term treatment (4 h), without metabolic activation: 625, 312, 156 and 78 µg/m:
Short-term treatment (4 h), with metabolic activation: 2500, 1250, 625 and 312 µg/mL
Long-term treatment (24 h), without metabolic activation: 500, 250, 125 and 62.5 µg/mL

Considering that the test item is a multi-constituent substance, the maximum concentration tested was 5000 µg/mL.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: culture medium
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
culture medium
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION:
Short-term treatment (4 h), with and without metabolic activation: 5000, 2500, 1250, 625, 312, 156, 78 and 39 µg/mL
Long-term treatment (24 h), without metabolic activation: 1000, 500, 250, 125, 62.5, 31.25, 15.63 and 7.8 µg/mL

EXPERIMENTAL PROCEDURE AND DETAILS:
Short-term treatment (4 h), -/+S9:
- A series of tubes containing 6x10E+6 cells in suspension/tube in medium was used. The different concentrations of the test item were added directly into the tubes (2 cultures per concentration).
- At the end of the incubation period of 4 h, the tubes were centrifuged and the medium was aspirated. 2 washes were performed and 10 ml of medium were deposited in each culture. A count of the cells was performed on each tube, then treated cells were transferred into flasks and incubated 24 h (± 1 h) at 37 °C (± 0.5 °C), 5% CO2.
- After the incubation period, cell counting was performed and cell cultures were adjusted if necessary to 2x10E+5 cells/mL and incubated for 24 h (± 1 h) at 37 °C (± 0.5 °C), 5% CO2.
- After the incubation period, cell counting was performed again, i.e. 48 h after the end of the treatment. These counts allowed assessing the cytotoxicity expressed as a percentage of relative cell growth (RSG). The actual gene mutation test was performed using 4 selected concentrations of the test item as determined by cytotoxicity.
- The cells were seeded in 96-well plates at 2 cells/200µL in non-selective culture medium (2 plates/concentration) to determine viability. Plates were incubated at 37 °C (± 0.5 °C), 5% CO2 for 10 to 12 days. Empty wells were recorded to calculate the survival rate (cloning efficiency, CE) to determine the viability.
- In parallel, 96-well plates were seeded at 2x10E+3 cells/200µL in selective culture medium to determine the mutation frequency (4 plates/concentration). Plates were incubated at 37 °C (± 0.5 °C), 5% CO2 for 10 to 12 days. Empty wells were recorded to calculate the mutation frequency.

Long-term treatment (24 h), -S9:
- A series of flasks containing 1.5x10E+6 cells/flask in culture medium was used. The different concentrations of the test item were added directly to the flasks (2 cultures per concentration) and then incubated for 24 h (± 1 h) at 37 °C (± 0.5 °C), 5% CO2.
- After the incubation period, the cultures were transferred into sterile tubes, centrifuged and the medium was aspirated. 2 washes were performed and 10 ml of medium were deposited in each culture. A count of the cells was performed on each tube. The treated cells were placed in new culture flasks at 2x10E+5 cells/mL and incubated for 24 h (± 1 h) at 37 °C (± 0.5 °C), 5% CO2.
- All further steps are identical to the procedure for the short-term treatment (4 h).

SELECTION AGENT (mutation assays): trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: duplicates

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (cytotoxicity corresponds to relative survival compared to the respective negative control values)

OTHER EXAMINATIONS:
Cloning efficiency was determined by seeding exposed cells in one microtiter plate with a density of 2 cells/well in medium without TFT.
Small and large colonies were differentiated as small colonies are capable to indicate chromosomal mutations.
Evaluation criteria:
A test item is considered potentially mutagenic if all of the following conditions are met:

- The mutation rate induced is significantly higher than that of the negative control,
- The observed effect is dose-dependent,
- The observed effect is reproducible,
- The rate of mutation is greater than the value of 126 plus the mutation frequency of the negative control (Moore et al., 2006).

The evaluation results are based on statistical methods, but the statistical significance should not be the only deciding factor.

If the criteria given above are not met, the test item is considered not mutagenic in this test.

Moore et al. (2006). Env. Mol. Mut. 2006, 47: 1-5
Statistics:
The calculations and summary of the results were performed using the Excel software. The mutant frequency for each series studied was subjected to a Chi-2 test.

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
-S9: starting at 1250 µg/mL (4 h) and 1000 µg/mL (24 h); +S9: starting at 5000 µg/mL (4 h)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
VALIDATION AND COMPARISON WITH HISTORICAL CONTROL DATA:
The negative control shows a cloning efficiency (CE) between 65 and 120%, a mutation frequency (MF) between 5x10E-5 and 17x10E-5) cells and a suspension growth (SG) between 8 and 32 (4 h-treatment) and between 32 and 180 (24 h-treatment).

The positive control shows a mutation frequency (MF) higher than 3x10E-4 with at least 40% in small colonies.

Except for the positive control for the long-term treatment (24 h), without S9, these values are slightly outside the IC (inhibition concentration) 95% of the historical control data. However, they are all consistent with the validity criteria and no deviation to the study plan or incidents have been observed during the study. The control values will, therefore, be added to the historical control data of the laboratory. The results validate the test.

Any other information on results incl. tables

Tables and further details on results are included in a separeate pdf document attached to this robust study summary.

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative