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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:
From October 18, 2016 to November 28, 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)
Deviations:
yes
Remarks:
regarding the cloning efficiency but it did not affect the intergity of the study
GLP compliance:
yes
Type of assay:
other: in vitro gene mutation study in mammalian cells

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
- Name of test material (as cited in study report): Phosphoric acid, mono- and bis (C16-20 branched and linear alkyl) esters
- Batch: CH 200529/001
- Composition: UVCB
- Appearance: Lightly yellow liquid

Method

Target gene:
Thymidine-kinase locus (TK-locus)
(The TK mutational system is able to detect base pair alterations, frame shift mutations and small deletions and clastogenic effect. Cells deficient in thymidine kinase (TK), due to the forward mutation (TK+/- to TK-/-) are resistant to the cytotoxic effects of the pyrimidine analogue trifluorothymidine (TFT)).
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
0 - 100 µg/L
(Since the test substance was not toxic and difficult to dissolve in aqueous solutions, the highest concentration was determined by the solubility in the culture medium)
Vehicle / solvent:
Ethanol
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Details on test system and experimental conditions:
- In the first experiment, the test substance was tested up to concentrations of 100 µg/mL in the absence and presence of S9-mix. The incubation time was 3 hours. No toxicity was observed at this dose level in the absence and presence of S9-mix. The test substance precipitated in the culture medium at this dose level.
- In the second experiment, the test substance was again tested up to concentrations of 100 µg/mL in the absence of S9-mix. The incubation time was 24 hours. No toxicity was observed at this dose level. The test substance precipitated in the culture medium at this dose level.
Per culture 8.0E06 cells (10E06 cells/mL for 3 hour treatment) or 6.0E06 cells (1.25E05 cells/mL for 24 hour treatment) were used.
For expression of the mutant phenotype, cells were cultured for 2 days after the treatment period. During this culture period at least 4.0E06 cells (where possible) were subcultured every day in order to maintain log phase growth. Two days after the end of the treatment with the test substance, the cells were plated for determination of the cloning efficiency (CEday2) and the mutation frequency (MF).
Rationale for test conditions:
Dose range finding test
Solubility of the test substance
Evaluation criteria:
- Determination of the mutant colonies, calculation of the survival or viability, calculation of the mutation frequency (MF) and cloning efficiency (CEday2)
- Acceptability of the assay:
A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120% in order to have an acceptable number of surviving cells analysed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 106 survivors and ≤ 170 per 106 survivors.
c) The suspension growth (SG) over the 2-day expression period for the solvent controls should be between 8 and 32 for the 3 hour treatment, and between 32 and 180 for the 24 hour treatment.
d) The positive control should demonstrate an absolute increase in the total mutation frequency above the spontaneous background MF (an induced MF (IMF) of at least 300E10-6). At least 40% of the IMF should be reflected in the small colony MF. And/or, the positive control should have an increase in the small colony MF of at least 150E10-6 above that seen in the concurrent solvent/control (a small colony IMF of at least 150E10-6).
Statistics:
In addition to the criteria stated below, any increase of the mutation frequency should be evaluated for its biological relevance including comparison of the results with the historical control data range. The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.
A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range. A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study. A test substance is considered negative (not mutagenic) in the mutation assay if: none of the tested concentrations reaches a mutation frequency of MF(controls) + 126.

Results and discussion

Test results
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No severe toxicity was observed up to and including the highest tested dose level of 100 µg/mL.
In the first mutation experiment (3 hour treatment), no significant increase in the mutation frequency at the TK locus was observed after treatment with the test substance either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the test substance treated cultures were comparable to the numbers of small and large colonies of the solvent controls. This result was confirmed in an independent experiment with modification in the duration of treatment. In the second mutation experiment (24 hour treatment), no significant increase in the mutation frequency at the TK locus was observed after treatment with the test substance. The numbers of small and large colonies in the test substance treated cultures were comparable to the numbers of small and large colonies of the solvent controls.
The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database. Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.
Remarks on result:
other: precipitation was observed at 100 µg/mL

Applicant's summary and conclusion

Conclusions:
Under the study conditions, the test substance was not mutagenic in mouse lymphoma L5178Y cells (in vitro gene mutation study in mammalian cells).
Executive summary:

An in vitro study was conducted to determine the genetic toxicity of the test substance according to OECD Guideline 490 (mouse lymphoma assay), in compliance with GLP. Two experiments were performed on mouse lymphoma L5178Y cells at the thymidine-kinase locus (TK-locus). In the first experiment, the test substance was tested up to concentrations of 100 µg/mL in the absence and presence of S9-mix. The incubation time was 3 h. No toxicity was observed at this dose level in the absence and presence of S9-mix. The test substance precipitated in the culture medium at this dose level. In the second experiment, the test substance was again tested up to concentrations of 100 µg/mL in the absence of S9-mix. The incubation time was 24 h. No toxicity was observed at this dose level. The test substance precipitated in the culture medium at this dose level. Per culture 8.0E06 cells (10E06 cells/mL for 3 hour treatment) or 6.0E06 cells (1.25E05 cells/mL for 24 hour treatment) were used. For expression of the mutant phenotype, cells were cultured for 2 d after the treatment period. During this culture period at least 4.0E06 cells (where possible) were subcultured every day in order to maintain log phase growth. Two days after the end of the treatment with the test substance, the cells were plated for determination of the cloning efficiency (CEday2) and the mutation frequency (MF). The determination of the mutant colonies and the calculation of the survival or viability were recorded as well. No severe toxicity was observed up to and including the highest tested dose level of 100 µg/mL. In the first mutation experiment (3-h treatment), no significant increase in the mutation frequency at the TK locus was observed after treatment with the test substance either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the test substance treated cultures were comparable to the numbers of small and large colonies of the solvent controls. This result was confirmed in an independent experiment with modification in the duration of treatment. In the second mutation experiment (24 -h treatment), no significant increase in the mutation frequency at the TK locus was observed after treatment with the test substance. The numbers of small and large colonies in the test substance treated cultures were comparable to the numbers of small and large colonies of the solvent controls. The mutation frequency found in the solvent control (ethanol) cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database. Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. Under the study conditions, the test substance was not mutagenic in mouse lymphoma L5178Y cells (Verspeek-Rip, 2017).