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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
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-11-13 to 2010-01-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP and Guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
In accordance with GLP standards published as OECD Principles on Good Laboratory Practice (revised 1997, ENV/MC/CHEM(98)17); and are in accordance with, and implement, the requirements of Directives 2004/9/EC and 2004/10/EC.
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material : calcium sulfate dihydrate
- Substance type: off white powder
- Physical state: solid
- Analytical purity: > 99 %
- Storage condition of test material: Room temperature in the dark

Method

Target gene:
TK +/- locus (thymidine kinase heterozygote system)
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
Prior to freezing stocks of the cells, they were cleansed of homozygous (TK -/-) mutants by culturing in THMG medium for 24 hours.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 microsomal enzyme fraction
Test concentrations with justification for top dose:
Concentrations were as calcium sulfate
Preliminary toxicity test: 0, 5.32, 10.63, 21.27, 42.53, 85.06, 170.13, 340.25, 680.5 and 1361 µg/mL in the presence and absence of S9

Experiment 1: 21.25, 42.5, 85, 170, 340, 453.33, 566.66 and 680 µg/mL in the absence of S9. 85.06, 170.13, 340.25, 680.5, 1020.75 and 1361 µg/mL in the presence of S9.

Experiment 2: 25, 50, 100, 150, 200, 250, 300 and 350 µg/mL in the absence of S9. 85.06, 170.13, 340.25, 680.5, 1020.75 and 1361 µg/mL in the presence of S9.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: R0 medium
Controlsopen allclose all
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
R0 medium
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Migrated to IUCLID6: (EMS) in the absence of metabolic activation at 400 µg/mL in Experiment 1 and 150 µg/mL in Experiment 2.
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
R0 medium
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Migrated to IUCLID6: (CP) 2 µg/mL in the presence of metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: in Experiment 1, cells were treated with the test material for 4 hours both in the presence and absence on metabolic activation. In Experiment 2, cells were treated with the test material for 4 hours in the presence of metabolic activation and 24 hours in the absence of metabolic activation.
- Expression time : 2 days
- Selection time : up to 48 hours

SELECTION AGENT :5 trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: performed in duplicate (denoted as A and B)

NUMBER OF CELLS EVALUATED: 10^4 cells/mL (2000 cells/well) plated in selective medium for evaluation of mutant frequency and 10 cells/mL (2 cells/well) to assess viability (%V) plated in non-selective medium.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

OTHER: MTT (2.5 mg/mL in PBS) was added to each well of the mutation plates to aid in counting viable cells.
Evaluation criteria:
The normal range for mutant frequency per survivor is 50-200 x 10-6 for the TK+/- locus in L5178Y cells at this laboratory. Vehicle controls results should be within this range, minor errors may cause this to be slightly elevated. Experiments where the vehicle control values are markedly greater than 250 x 10-6 mutant frequency per survivor are not normally acceptable and are repeated.
Positive control chemicals should induce at least three to five fold increases in mutant frequency greater than the corresponding vehicle control.
For a test material to demonstrate a mutagenic response it must produce a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value. Any test material dose level that has a mutation frequency value that is greater than the corresponding vehicle control by the Global Evaluation Factor (GEF) of 126 x 10-6 will be considered positive. If a test material produces a modest increase in mutant frequency, only marginally exceeding the GEF and is not reproducible or part of a dose-related response, then it may be considered to have no toxicological significance. However when a test material induces modest reproducible increases in the mutation frequencies that do not exceed the GEF value then scientific judgement will be applied. If the reproducible responses are significantly dose-related and include increases in the absolute numbers of mutant colonies then they may be considered to be toxicologically significant.
Small significant increases designated by the UKEMS statistical package were reviewed using the above criteria (and where necessary disregarded at the Study Director's discretion).
Statistics:
The experimental data was analysed using a dedicated computer program which follows the statistical guidelines recommended by the UKEMS.

Results and discussion

Test results
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
The test material did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
The maximum dose level used was the 10 mM limit dose in the presence of metabolic activation, and was limited by test material induced toxicity in the absence of metabolic activation. The 24-hour exposure without metabolic activation demonstrated that th
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No marked change was noted in the pH when the test material was added to the media.
- Effects of osmolality: osmolality did not increase by more than 50 mOsm when test material was dosed into the media
- Precipitation: In the preliminary toxicity test, a precipitate of the test material was observed at and above 170.13 µg/mL in all three of the exposure groups. In Experiment 1 a precipitate of the test material observed at and above 85 µg/mL in the absence of metabolic activation, and at and above 340.25 µg/mL in the presence of metabolic activation. In Experiment 2, a precipitate of the test material was observed at and above 100 µg/mL in the absence of metabolic activation, and at and above 170.13 µg/mL in the presence of metabolic activation.

COMPARISON WITH HISTORICAL CONTROL DATA: Historical control data were included. All controls performed within the study were found to be valid.

RANGE-FINDING/SCREENING STUDIES: Please refer to Table 1 in the attached Appendix 1 Tabulated data for full results of the range-finding study. At 4 and 24-hours in the absence of metabolic activation there were marked dose-related reductions in the Relative Suspension Growth (%RSG) of treated cells. A slight reduction in %RSG occurred at the 10 mM limit dose in with S9. The toxicity curve was very steep in both the 4-hour and 24-hour exposure groups in the absence of metabolic activation. A precipitate was observed at concentrations ≥ 170.13 µg/mL in all three groups. In the mutagenicity experiments the maximum dose for the 4 and 24-hour exposure groups without S9 was limited by toxicity. The maximum dose level for the 4-hour exposure group in the presence of metabolic activation was the 10 mM limit dose.
Remarks on result:
other: strain/cell type: Only TK +/- cells were used, cultures were cleansed of were cleansed of homozygous TK -/- cells prior to testing.
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

For tabulated data on results, please refer to Appendix 1 Tabulated data for Flanders 2010.

Controls

The positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional. Vehicle control mutant frequency values were within the acceptable range of 50 to 200 x 10-6viable cells. Both positive controls induced acceptable levels of toxicity.

Toxicity

In Experiment 1, evidence of toxicity following exposure to the test material in the absence of metabolic activation was noted. The modest reduction observed in the presence of metabolic activation in the preliminary toxicity test was not reproduced. No significant reductions in viability (%V) in either the absence or presence of metabolic activation occurred indicating no residual toxicity. Near optimum levels of toxicity were achieved in the absence of metabolic activation. Optimum levels of toxicity were not achieved in the absence of metabolic activation due to the sharp onset of toxicity (despite a very narrow dose interval). However, a dose level that exceeded the usual acceptable upper limit of toxicity was plated for viability andresistance.

In Experiment 2, a marked dose-related reduction occurred in % and RTG values in cultures dosed with the test material in the absence of metabolic activation and no evidence of any reductions in the presence of metabolic activation. There were no significant reductions in viability (%V) with or without metabolic activation, indicating no residual toxicity. Optimum levels of test material‑induced toxicity were achieved in the absence of metabolic activation. The 24-hour exposure without metabolic activation demonstrated that the extended time point had a marked effect on the toxicity of the test material.

The author concluded that with no evidence of any toxicologically significant increases in mutant frequency at any of the dose levels, including the dose level that exceeded the usual upper limit of acceptable toxicity or in the 24-hour exposure group of Experiment 2 where optimum levels of toxicity were achieved, the test material had been adequately tested.

Experiment 1

The test material did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10-6 per viable cell in the absence of metabolic activation. In the presence of metabolic activation, a very modest dose related (linear trend) statistically significant response was observed. However, statistically significant increases in mutant frequency were not observed at any of the of the individual dose levels, thewas not exceeded at any of the individual dose levels, the mutant frequency values observed at 1020 and 1361 µg/mL only marginally exceeded the upper acceptable range for vehicle controls, and the response was not reproduced in Experiment 2. Therefore, the response was considered to be of no toxicological significance. A precipitate of the test material was observed at ≥85 µg/mL in the absence of metabolic activation, and ≥340.25 µg/mL in the presence of metabolic activation, this was considered not to affect the purpose and integrity of the study.

Experiment 2

The test material did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10-6per viable cell in either the absence or presence of metabolic activation. The test material precipitated at ≥100 µg/mL without S9, and at ≥170.13 µg/mL with S9

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative in the presence and absence of metabolic activation

The test material did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non mutagenic under the conditions of the test.
Executive summary:

The study includes all data required for the guideline OECD Guideline 476 (In vitro Mammalian Cell Gene Mutation Test) and is performed to GLP. No analysis was conducted to determine the homogeneity, concentration or stability of the test material formulation. This is an exception with regard to GLP and had been reflected in the GLP compliance statement of the study. This exception is considered not to affect the purpose or integrity of the study.