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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:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From 2010-01-08 to 2010-05-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent

Method

Target gene:
thymidine kinase (TK) locus
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: The cells were stored in a cryoprotective medium (10% horse serum and 10% dimethylsulfoxide (DMSO)) at -80°C.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: not indicated
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix (liver post-mitochondrial fraction and cofactors of rats induced with Aroclor 1254)
Test concentrations with justification for top dose:
Preliminary test:
-1, 10, 50, 100, 250, 500 µg/mL
Mutagenicity experiments without S9 mix:
- 2.34, 4.69, 9.38, 18.75, 37.5 and 75 µg/mL for the first experiment (3-hour treatment),
- 1.56, 3.13, 6.25, 12.5, 25 and 50 µg/mL for the second experiment (24 hour treatment)
Mutagenicity experiements with S9 mix:
- 4.69, 9.38, 18.75, 37.5, 75 and 150 µg/mL for the first experiment (3 hours),
- 2.34, 4.69, 9.38, 18.75, 37.5 and 75 µg/mL for the second experiment (3 hours)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: Soluble in ethanol
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9 mix

Migrated to IUCLID6: without S9 mix, 25 µg/mL (3h treatment) or 5 µg/mL (24h treatment)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9 mix

Migrated to IUCLID6: with S9 mix, 3 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: first experiment 3h with and without S9 mix; second experiment 3 h with S9 mix and 24h without S9 mix (as results of the first experiment were negative)
- Expression time (cells in growth medium): 48 hours (at 37°C in a humidified atmosphere of 5% CO2/95% air)

SELECTION AGENT (mutation assays): trifluorothymidine

NUMBER OF REPLICATIONS: 2 cultures/dose-level and at least duplicate cultures for the controls

NUMBER OF CELLS EVALUATED: 2 000cells/ well (four 96-well plates/culture = eight plates/dose-level) to select the (trifluorothymidine resistant) mutant cells.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (RTG) ; relative suspension growth (RSG) ; cloning efficiency (CE2)
Evaluation criteria:
IWGT recommendations were followed and a positive result is considered when following criteria are fulfilled :
- at least at one dose-level the mutation frequency minus the mutation frequency of the vehicle control equals or exceeds the global evaluation factor (126 x 10E-6 for the microtiter method)
- a dose-related trend is demonstrated by a statistically significant trend test

Unless considered as clearly positive, the reproducibility of a positive effect should be confirmed.

Noteworthy increases in the mutation frequency observed only at high levels of cytotoxicity (RTG lower than 10%), but with no evidence of mutagenicity at dose-levels with RTG between 10 and 20%, is not considered as positive result.

A test item is determined to be non-mutagenic when there is no culture showing an Adj. RTG value between 10-20% if:
- there is at least one negative data point between 20 and 25% Adj. RTG and no evidence on mutagenicity in a series of data points between 100 to 20% Adj. RTG
- there is no evidence of mutagenicity in a series of data points between 100 to 25% and there is also a negative data point between 10 and 1% Adj. RTG
Statistics:
None

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:
without S9 mix ≥ 50 µg/mL at the 3-hour treatment; ≥ 10 µg/mL at the 24-hour treatment with S9 mix ≥ 100 µg/mL at the 3-hour treatment
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:

In the solubility assay, the test item was soluble in the vehicle (ethanol) at 1000 mg/mL. Using a treatment volume of 0.5% (v/v), the final dose-level of 5000 µg/mL showed a strong precipitate. Further assays were performed lowering down the concentration of the stock preparation to reach the lowest precipitating dose-level in the culture medium. Using a stock preparation at 100 mg/ml and the maximal practicable treatment volume of 0.5% (v/v), the final dose-level of 500 µg/mL showed a moderate precipitate. At this dose-level, the pH was approximately 7.4 (7.7 for the vehicle control) and the osmolality was equal to 382 mOsm/kg H2O (399 mOsm/kg H2O for the vehicle control).
Therefore, the dose-levels selected for treatment of the preliminary test were 1, 10, 50, 100, 250 and 500 µg/mL.

ADDITIONAL INFORMATION ON CYTOTOXICITY (PRELIMINARY TEST):
Following the 3-hour treatment without S9 mix, a marked to severe toxicity was induced at dose-levels ≥ 10 µg/mL as shown by a 62-100% decrease in adjusted relative total growth (Adj. RTG).
Following the 24-hour treatment without S9 mix, a severe toxicity was induced at dose-levels ≥ 10 µg/mL as shown by a 65-100% decrease in Adj. RTG.
Following the 3-hour treatment with S9 mix, a severe toxicity was induced at dose-levels ≥ 100 µg/mL as shown by a 73-100% decrease in Adj. RTG.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

The cloning efficiencies CE2and the mutation frequencies of the vehicle and positive controls were as specified in the acceptance criteria. The study was therefore considered as valid.

 Since the test item was toxic in the preliminary test, the choice of the highest dose-level for the main test was based on the level of toxicity, according to the criteria specified in the international guidelines (decrease in Adj. RTG).

 

Experiments without S9 mix

Using a treatment volume of 100 µL/20 mL (0.5% (v/v)), the selected dose-levels were as follows:

.           2.34, 4.69, 9.38, 18.75, 37.5 and 75 µg/mL for the first experiment (3-hour treatment),

.           1.56, 3.13, 6.25, 12.5, 25 and 50 µg/mL for the second experiment (24‑hour treatment).

  

Cytotoxicity

Following the 3-hour treatment, a marked to severe toxicity was induced at dose-levels ≥ 37.5 µg/mL, as shown by a 61-100% decrease in Adj. RTG.

Following the 24-hour treatment, a marked to severe toxicity was induced at dose‑levels ≥ 6.25 µg/mL, as shown by a 80-100% decrease in Adj. RTG.

Mutagenicity

Following the 3-hour or the 24-hour treatments, no noteworthy increase in the mutation frequency was noted in comparison to the vehicle control.

Experiments with S9 mix

Using a treatment volume of 100 µL/20 mL, the selected dose-levels were as follows:

.           4.69, 9.38, 18.75, 37.5, 75 and 150 µg/mL for the first experiment,

.           2.34, 4.69, 9.38, 18.75, 37.5 and 75 µg/mL for the second experiment.

 In the first experiment, a slight to strong precipitate was noted in the culture medium at the end of the 3-hour treatment at dose-levels ≥ 4.69 µg/mL.

Cytotoxicity

In the first experiment, a severe toxicity was induced at dose-levels ≥ 75 µg/mL, as shown by a 88-100% decrease in Adj. RTG.

In the second experiment, a moderate to severe toxicity was induced at dose-levels ≥ 37.5 µg/mL, as shown by a 49-100% decrease in Adj. RTG.

Mutagenicity

In either experiment, no noteworthy increase in the mutation frequency was noted in comparison to the vehicle control.


Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with and without S9 mix

Under these experimental conditions, no noteworthy increase in the mutation frequency was observed, both with and without S9 mix. The test substance did not show any mutagenic activity in the in vitro mammalian cell gene mutation test with L5178Y TK+/- mouse lymphoma cells.
Executive summary:

The potential for the test substance to induce mutations at the TK locus, was investigated in L5178Y mouse lymphoma cells. The test substance was tested in two independent experiments, both with and without metabolic activation. Approximately 0.5 x 106(3-hour treatment) or 0.15 x 106(24-hour treatment) cells/mL in 20 mL culture medium with 5% horse serum were exposed to the test or control items, in the presence or absence of S9 mix (final concentration of S9 fraction 2%), at 37°C. Since the test item was toxic in the preliminary test, the choice of the highest dose-level for the main experiments was based on the level of toxicity (decrease in Adj. RTG), according to the criteria specified in the international guidelines.

 

In the experiments without metabolic activation, the selected dose-levels were as follows:

.  2.34, 4.69, 9.38, 18.75, 37.5 and 75 µg/mL for the first experiment (3-hour treatment),

.  1.56, 3.13, 6.25, 12.5, 25 and 50 µg/mL for the second experiment (24‑hour treatment).

Cytotoxicity was observed. Following the 3-hour treatment, a marked to severe toxicity was induced at dose-levels ≥ 37.5 µg/mL, as shown by a 61-100% decrease in Adj. RTG. Following the 24-hour treatment, a marked to severe toxicity was induced at dose‑levels ≥ 6.25 µg/mL, as shown by a 80-100% decrease in Adj. RTG.

No noteworthy increase in the mutation frequency was noted in comparison to the vehicle control following the 3-hour or the 24-hour treatments.

In the experiments with metabolic activation,the selected dose-levels were as follows:

.  4.69, 9.38, 18.75, 37.5, 75 and 150 µg/mL for the first experiment,

. 2.34, 4.69, 9.38, 18.75, 37.5 and 75 µg/mL for the second experiment.

 In the first experiment, a slight to strong precipitate was noted in the culture medium at the end of the 3-hour treatment at dose-levels ≥ 4.69 µg/mL.

Cytotoxicity was observed. In the first experiment, a severe toxicity was induced at dose-levels ≥ 75 µg/mL, as shown by a 88-100% decrease in Adj. RTG. In the second experiment, a moderate to severe toxicity was induced at dose-levels ≥ 37.5 µg/mL, as shown by a 49-100% decrease in Adj. RTG.

In either experiment, no noteworthy increase in the mutation frequency was noted in comparison to the vehicle control.