1,5-Naphthalenedisulfonic acid, 2-[2-[8-[[4-chloro-6-[[4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]amino]-1,3,5-triazin-2-yl]amino]-1-hydroxy-3,6-disulfo-2-naphthalenyl]diazenyl]-, sodium salt (1:5)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 2020 - January 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Test material

Test material information

Constituent 1

Reference substance name:

Structural Analogue 01

IUPAC Name:

Structural Analogue 01

Test material form:

solid: particulate/powder

Details on test material:

see below

Method

Target gene:

The mutation assay method used in this study is based on the identification of L5178Y colonies which have become resistant to a toxic thymidine analogue trifluorothymidine (TFT).This analogue can be metabolised by the enzyme thymidine kinase (TK) into nucleosides, which are used in nucleic acid synthesis resulting in the death of TK-competent cells.TK-deficient cells, which are presumed to arise through mutations in the TK gene, cannot metabolise trifluorothymidine and thus survive and grow in its presence. In the L5178Y mouse lymphoma cells, the gene which codes for the TK enzyme is located on chromosome 11. Cells which are heterozygous at the TK locus (TK+/−) may undergo a single step forward mutation to the TK−/− genotype in which little or no TK activity remains. The cells used, L5178Y TK+/−, are derived from one of the two clones originated from a thymic tumour induced in a DBA/2 mouse by methylcholanthrene. The use of the TK mutation system in L5178Y mouse lymphoma cells has been well characterised and validated (D. Clive et al., 1979) and is accepted by most of the regulatory authorities. The mouse lymphoma assay often produces a bimodal size distribution of TFT resistant colonies designated as small or large. It has been evaluated that point mutations and deletions within the active allele (intragenic event) produce large colonies. Small colonies result in part from lesions that affect not only the active TK allele but also a flanking gene whose expression modulates the growth rate of cells.

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI medium supplemented with Horse serum.
- Properly maintained: yes; Permanent stocks of the L5178Y TK+/− cells are stored in liquid nitrogen, and subcultures are prepared from the frozen stocks for experimental use.
- Periodically checked for Mycoplasma contamination: yes
- The generation time and mutation rates (spontaneous and induced) have been checked in this laboratory.
- Prior to use, cells were cleansed of pre-existing mutants.

Metabolic activation:

with and without

Metabolic activation system:

S9 tissue fraction: Species: Rat; Strain: Sprague Dawley; Tissue: Liver Inducing Agents: Phenobarbital – 5,6-Benzoflavone Producer: MOLTOX, Molecular Toxicology, Inc. Batch Number: 4263

Test concentrations with justification for top dose:

The test item was found to be soluble in RPMI 1640 minimal medium at 20.0 mg/mL. On the basis of this result, a concentration of 2000 µg/mL was selected as the top dose level for the preliminary cytotoxicity assay.

Based on the results obtained in the preliminary trial, the assy for mutation at the TK locus was performed using the following dose levels:
Main Assay 1 (+/-S9, 3 hour treatment): 2000, 1000, 500, 250 and 125 ug/mL.

Negative results were obtained in Main Assay II, thus a third experiment (Main Assay III) in the absence of S9 metabolism was performed, using a longer treatment time (24 hours) and the dose levels described in the following table:
Main Assay 2 (-S9, 24 hour treatment): 2000, 1600, 1280, 1020, 512, 256 and 128

Vehicle / solvent:

Test item solutions were prepared using complete medium (RPMI 1640 Minimal medium).

Controls

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

methylmethanesulfonate

Details on test system and experimental conditions:

Cytotoxicity assay
A preliminary cytotoxicity test was performed in order to select appropriate dose levels for the mutation assays. In this test a wide range of dose levels of the test item was used and the survival of the cells was subsequently determined. Treatments were performed in the absence and presence of S9 metabolic activation for 3 hours and for 24 hours only in the absence of S9 metabolic activation. A single culture was used at each test point. After washing in Phosphate Buffered Saline (PBS), cells were resuspended in 20mL of complete medium (10%). Cell concentrations were adjusted to 8 cells/mL using complete medium (20%) and, for each dose level, 0.2mL was plated into 96 microtitre wells. The plates were incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) for 8 days. Wells containing viable clones were identified by eye using background illumination and then counted.

Mutation assay
The mutation assay was performed including vehicle and positive controls, in the absence and presence of S9 metabolising system.
Preparation of test cell cultures was performed as described in section 4.4. Duplicate cultures were prepared at each test point, with the exception of the positive controls which were prepared in a single culture. In the first experiment, the cells were exposed to the test item for a short treatment time (3 hours). Since negative results were obtained, a second experiment in the absence of S9 metabolism was performed, using a longer treatment time (24 hours).
After washing in Phosphate Buffered Saline (PBS), cells were resuspended in fresh complete medium (10%) and cell densities were determined. The number of cells was adjusted to give 2×105 cells/mL. The cultures were incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) to allow for expression of the mutant phenotype.

Evaluation criteria:

For a test item to be considered mutagenic in this assay, it is required that:
1. The induced mutant frequency (IMF) is higher than the global evaluation factor (GEF) suggested for the microwell method (126×10^−6) at one or more doses.
2. There is a significant dose-relationship as indicated by the linear trend analysis.
Results which only partially satisfy the above criteria will be dealt with on a case-by-case basis. Similarly, positive responses seen only at high levels of cytotoxicity will require careful interpretation when assessing their biological significance. Any increase in mutant frequency should lie outside the historical control range to have biological relevance.

Statistics:

Statistical analysis was performed according to UKEMS guidelines (Robinson W.D., 1990).

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:

cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Survival after treatment
In the first experiment, using the 3 hour treatment time in the absence of S9 metabolism, moderate toxicity reducing RTG to 44% of the concurrent negative control, was observed at the highest concentration tested. In the presence of S9 metabolic activation the highest concentration (2000 μg/mL) yielded slight toxicity (RTG=69%), while no remarkable toxic effects were observed over the remaining dose levels.
A more pronounced toxic effect was noticed in the second experiment, using a long treatment time in the absence of S9 metabolic activation. Dose related toxic effects were observed from 512 μg/mL onwards reducing RTG to 9% and 1% at the two highest dose levels, 1600 and 2000 μg/mL, respectively. The next lower concentration (1280 μg/mL) yelded 20% RTG.
At low survival levels, the mutation data are prone to a variety of artefacts (selection effects, sampling error, founder effects). Mechanisms other than direct genotoxicity per se can lead to positive results that are related to cytotoxicity and not genotoxicity (e.g. events associated with apoptosis, endonuclease release fromlysosomes, etc.). For this reason it is generally recommended that such data are treated with caution or excluded from consideration.
Accordingly, we have excluded from the statistical analyses, mutation data obtained using the long treatment time at 1600 and 2000 µg/mL.

Mutation results
No statistically significant or biologically relevant increase in mutant frequency values was observed in any experiment, at any concentration tested, in the absence or presence of S9 metabolism, using the short or long treatment time. On the basis of these results and in accordance with the criteria for outcome of the study, the test item was not considered to induce mutation in L5178Y mouse lymphoma cells.

Any other information on results incl. tables

see Final Report

Applicant's summary and conclusion

Conclusions:

It is concluded that the test substance does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

Executive summary:

The test item was examined for mutagenic activity by assaying for the induction of 5 trifluorothymidine resistant mutants in mouse lymphoma L5178Y cells after in vitro treatment, in the absence and presence of S9 metabolic activation, using a fluctuation method. The test item was found to be soluble in RPMI 1640 minimal medium at 20.0 mg/mL. On the basis of this result, a concentration of 2000 µg/mL was selected as the top dose level for the preliminary cytotoxicity assay. Both in the absence and presence of S9 metabolic activation, the test item was assayed at a maximum dose level of 2000 µg/mL and at a wide range of lower dose levels: 1000, 500, 250, 125, 62.5, 31.3, 15.6 and 7.81 µg/mL.

No precipitation of the test item was noted upon addition of the test item to the cultures or by the end of any treatment period. Using the 3 hour treatment time in the absence of S9 metabolic activation, slight or mild toxicity was observed at the three highest dose levels reducing relative survival (RS) up to 53% of the concurrent negative control value. In the presence of S9 metabolism mild toxicity was noticed only at the highest dose level. No remarkable toxicity was observed over the remaining concentrations tested. Using the 24 hour treatment time in the absence of S9 metabolic activation, dose ralated toxicity was observed reducing RS to 37% of the concurrent negative control value at 1000 µg/mL. At the next higher concentration (2000 µg/mL) a severe toxic effect was noticed (RS=1%).

Based on the results obtained in the preliminary trial, two independent assays for mutation at the TK locus were performed using the following dose levels:

 

Main Assay 1: (+/- S9, 3 hour treatment): 2000, 1000, 500, 250 and 125 µg/mL

Main Assay 2: (-S9, 24 hour treatment): 2000, 1600, 1280, 1020, 512, 256 and 128 µg/mL

 

Negative and positive control treatments were included in each mutation experiment in the absence and presence of S9 metabolism. The cloning efficiency at the time of mutant selection and the suspension growth for the solvent control cultures were within the acceptable range in all treatment series. The mutant frequencies in the solvent control cultures fell within the normal range. Marked increases were obtained with the positive control treatments indicating the correct functioning of the assay system. The study was then accepted as valid.

Using the 3 hour treatment in the absence of S9 metabolism, moderate toxicity reducing the Relative Total Growth (RTG) to 44% was observed at 2000 µg/mL, while, in the presence of S9 metabolic activation, this concentration yielded 69% RTG. Using the 24 treatment in the absence of S9 metabolism, dose related toxic effects were observed from 512 μg/mL onwards reducing RTG below 10% at the two highest dose levels. No relevant increases in mutant frequency were observed at any concentration tested, in any experiment.

It is concluded that the test substance does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.