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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Specific details on test material used for the study:
Batch no: TROD7BB06

Method

Target gene:
thymidine kinase (TK)
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
Mouse lymphoma L5178Y TK+/-cells (ATCC-CRL-9518) purchased from ATCC (American Type Culture Collection-Rockeville, MD 20852 – USA) have been used successfully in “in vitro” experiments for many years. These cells are characterized by their high proliferation rate (10 h – 12 h doubling time of the stock cultures) and their cloning efficiency, usually more than 50 %. They possess a nearly diploid karyotype (40 ± 2 chromosomes). They are heterozygous at the thymidine kinase (TK) locus which allows to detect mutation events at the TK-locus.
Cells from the cell bank stored at- 80°C are systematically checked to be free from mycoplasma contamination (LEMI operating procedure MB05/02).
To prevent background arising from spontaneous mutation, cells lacking TK have to be eliminated by culturing them in a culture medium (Dulbecco’s modified Eagle’s medium (DMEM) GlutaMAXTM – I) supplemented with 10% (v/v) of inactivated horse serum containing HMTG (Cole et al10): 15 μg/mL hypoxanthine, 0.3 μg/mL methotrexate, 9 μg/mL thymidine, 22.5 μg/mL glycine.
After 24 hours incubation at 37° C in a humidified atmosphere containing 5% (v/v) CO2, the culture is centrifuged (200 x G, 10 min) in order to eliminate methotrexate, and the cell pellet is suspended in medium, without methotrexate, containing HTG (hypoxanthine, thymidine and glycine) and incubated at 37° C in a humidified atmosphere containing 5% (v/v) CO2 for 1 day to 3 days.
Cleaned cells are stored at -80°C. Each cell batch is checked free from mycoplasma contamination. Thawed cultures are maintained in complete culture medium (CCM).
Metabolic activation:
with and without
Metabolic activation system:
S9-mix 2.5% v/v
Test concentrations with justification for top dose:
320- 800- 1400- 2000- 3500 and 5000 μg/mL
Vehicle / solvent:
water
Controls
Negative solvent / vehicle controls:
yes
Remarks:
Culture medium DMEN, GlutaMAX
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Details on test system and experimental conditions:
Mouse lymphoma L5178Y TK+/-cells (ATCC-CRL-9518) purchased from ATCC (American Type Culture Collection-Rockeville, MD 20852 – USA) have been used successfully in “in vitro” experiments for many years. These cells are characterized by their high proliferation rate (10 h – 12 h doubling time of the stock cultures) and their cloning efficiency, usually more than 50 %. They possess a nearly diploid karyotype (40 ± 2 chromosomes). They are heterozygous at the thymidine kinase (TK) locus which allows to detect mutation events at the TK-locus.
Cells from the cell bank stored at- 80°C are systematically checked to be free from mycoplasma contamination (LEMI operating procedure MB05/02).
Evaluation criteria:
An approach for defining positive and negative responses is recommended to assure that the increased MF is biologically relevant. In place of statistical analysis generally used for other tests, it relies on the use of a predefined induced mutant frequency (i.e. increase in MF above concurrent control), designated the Global Evaluation Factor (GEF), which is based on the analysis of the distribution of the negative control MF data from participating laboratories. For the microwell version of the MLA the GEF is 126 x 10-6.
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined, the increase in MF above the concurrent background exceeds the GEF and the increase is concentration related. The test chemical is then considered able to induce mutation in this test system.
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly negative if, in all experimental conditions examined there is no concentration related response or, if there is an increase in MF, it does not exceed the GEF. The test chemical is then considered unable to induce mutations in this test system.
There is no requirement for verification of a clearly positive or negative response.
In cases when the response is neither clearly negative nor clearly positive as described above and/or in order to assist in establishing the biological relevance of a result the data should be evaluated by expert judgement and/or further investigations. Performing a repeat experiment possibly using modified experimental conditions [e.g. concentration spacing to increase the probability of attaining data points within the 10-20 % RTG range, using other metabolic activation conditions (i.e. S9 concentration or S9 origin) and duration of treatment] could be useful.

Results and discussion

Test results
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cytotoxic effects observed at largest test concentration (5000 µg/ml) without metabolic activity. All other doses with and without metabolic activity are compatible with the acceptability criteria.
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
1) In the absence of metabolic activation – 4 hours treatment

a) An increase in the mutant frequency is observed.
Moreover, the GEF (Global Evaluating Factor) was calculated in these experimental conditions, since the GEF was recommended by the OECD 490 to help in evaluating the test results (Moore et al12 13 14). The GEF is applied as follows: if the negative control mutant frequency (MF) in a microwell experiment is 100 x 10−6, then one of the treatment groups must have a MF of at least [100+126 (the microwell GEF) = 226] x 10-6 in order to meet the GEF criterion for a positive call.
The above criteria, is met for three concentrations tested 1400 - 2000 and 3500 μg/ml, in the absence of metabolic activation for the short exposure time. The measured MF for these three concentrations is 330.2 - 577.5 and 2000.6 x 10-6.

b) Analysis of the size of colonies shows an increase in induced small colonies from 2 to 1251, and an increase in induced large colony for any concentration tested from 12 to 443

2) In the presence of metabolic activation – 3 hours treatment

a) In the presence of 2.5 % S9-mix, an increase in the mutant frequency is observed.
The above criteria (described in §11.3.1. a), is met for two concentrations tested 3500 and 5000 μg/ml, in the presence of metabolic activation. The measured MF for these two concentration is 270.0 and 318.4 x 10-6 falls above GEF criterion of [126+102.5] 228.5x 10-6

b) Analysis of the size of colonies shows an increase in induced small colonies (Number of induced mutants: from 32 to 166) and in induced large colony (Number of induced mutants: from 16 to 54 for any concentration tested

Applicant's summary and conclusion

Conclusions:
In the framework of OECD 490 under the described experimental conditions, solutions of Acetic acid, oxo-, sodium salt, reaction products with ethanolamine and phenol, sodium hydroxide and ferric chloride (MEAHA Fe) BATCH: TROD7BB06 provided by TRADE Corporation International SA induce a mutagenic effect in L5178Y TK+/-Mouse lymphoma cells in the absence and in the presence of metabolic activation (2.5% S9-mix).
Executive summary:

Two aqueous solutions of Acetic acid, oxo-, sodium salt, reaction products with ethanolamine and phenol, sodium hydroxide and ferric chloride (MEAHA Fe) BATCH: TROD7BB06 were tested for their capacity to induce mutagenic activity in L5178Y Mouse Lymphoma cells. No long-term treatment has been conducted, only short-term treatment without or with metabolic activation was carried out according to the acceptability criteria of OECD 490.

320- 800- 1400- 2000- 3500 and 5000 μg/mL of Acetic acid, oxo-, sodium salt, reaction products with ethanolamine and phenol, sodium hydroxide and ferric chloride (MEAHA Fe) BATCH: TROD7BB06 were evaluated in contact with the cells in the absence of a metabolic activation system.

320- 800- 1400- 2000- 3500 and 5000 μg/mL of Acetic acid, oxo-, sodium salt, reaction products with ethanolamine and phenol, sodium hydroxide and ferric chloride (MEAHA Fe) BATCH: TROD7BB06 were evaluated in contact with the cells in the presence of metabolic activation. (S9-mix 2.5 % (v/v)).

For short-term treatment without or with metabolic activation studies, negative and positive controls were carried out in parallel. Positive controls induced a significant increase in the number of colonies compared to negative controls. These results validate the assays.

In the absence and in presence of the metabolic activation system (S9-mix 2.5 % (v/v)) a concentration-related increase in the mutant frequency was measured in presence of Acetic acid, oxo-, sodium salt, reaction products with ethanolamine and phenol, sodium hydroxide and ferric chloride (MEAHA Fe) BATCH: TROD7BB06 .

Conclusion:

In the framework of OECD 490 under the described experimental conditions, solutions obtained from Acetic acid, oxo-, sodium salt, reaction products with ethanolamine and phenol, sodium hydroxide and ferric chloride (MEAHA Fe) BATCH: TROD7BB06 provided by TRADE Corporation International SA induce a mutagenic effect in L5178Y TK+/-Mouse lymphoma cells in the absence and in the presence of metabolic activation (2.5% S9-mix).