[2-[[4-[(2-chloro-4-nitrophenyl)azo]phenyl]ethylamino]ethyl](2-hydroxypropyl)dimethylammonium acetate

Genetic toxicity: in vitro

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:

December 2015 - April 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for the testing of chemicals No. 490 (adopted July 2015).

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

[2-[[4-[(2-chloro-4-nitrophenyl)azo]phenyl]ethylamino]ethyl](2-hydroxypropyl)dimethylammonium chloride

EC Number:

259-033-6

EC Name:

[2-[[4-[(2-chloro-4-nitrophenyl)azo]phenyl]ethylamino]ethyl](2-hydroxypropyl)dimethylammonium chloride

Cas Number:

54229-13-9

Molecular formula:

C21H29ClN5O3.Cl

IUPAC Name:

[2-[[4-[(2-chloro-4-nitrophenyl)azo]phenyl]ethylamino]ethyl](2- hydroxypropyl)dimethylammonium chloride

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 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 1640 medium supplemented with 10% Foetal Calf Serum (RPMI
complete)
- Properly maintained: yes; permanent stock of mouse lymphoma L5178Y cells are stored in liquid
nitrogen and subcoltures are prepared from the frozen stocks for experimental use.
- Periodically checked for Mycoplasma contamination: yes
- The generation time, plating efficiency and mutation rates (spontaneous and induced) have been
checked in this laboratory.
- Periodically "cleansed" against high spontaneous background: yes

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 3512

Test concentrations with justification for top dose:

A preliminary cytotoxicity assay was performed at the following dose levels: 1000, 500, 250, 125, 62.5, 31.3, 15.6, 7.81 and 3.91 µg/mL.
Based on the results obtained in the preliminary cytotoxicity assay, two independent assays
for mutation at the TK locus were performed using the following dose levels:
Main Assay I (-S9; treatment time 3 hours): 60.2, 43.0, 30.7, 22.0, 15.7 and 11.2 μg/mL
Main Assay I (+S9; treatment time 3 hours): 100, 50.0, 25.0, 12.5, 6.25 and 3.13 μg/mL
Main Assay II (-S9; treatment time 24 hours): 35.0, 25.0, 17.9, 12.8, 9.11 and 6.51 μg/mL

Vehicle / solvent:

Test item solutions were prepared using dimethylsulfoxide (DMSO)

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

methylmethanesulfonate

Details on test system and experimental conditions:

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.
The mutation assays were performed including vehicle and positive controls, in the absence and presence of S9
metabolising system.
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 without metabolic activation, the 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
incubated to allow expression of the mutant phenotype. At the end of the expression period cells were plated for the evaluation of
5-trifluorothymidine resistance and for viability.

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 (126x10^-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 (RobinsonW.D., 1990).

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Survival after treatment:
In the first experiment, in the absence of S9 metabolic activation, no cell survived after treatment at the highest dose level, moderate toxicity reducing relative total growth (RTG) to 14% of the concurrent negative control was noted at 43.0 µg/mL, slight to mild toxicity was observed between 15.7 and 30.7 µg/mL, while no relevant toxicity was noted at the lowest concentration tested. In the presence of S9 metabolism, treatment with the test item at 100 µg/mL yielded moderate toxicity reducing RTG to 24% of the concurrent negative control value, slight toxicity was noted at the next lower concentration, while no relevant toxicity was observed over the remaining concentrations tested.
In the second experiment, in the absence of S9 metabolic activation using a long treatment time, the highest dose level selected (35 µg/mL)
yielded marked toxicity reducing RTG to 8% of the concurrent negative control value. The next two lower dose levels of 25.0 and 17.9 µg/mL yielded moderate toxicity reducing RTG to 15 and 26%, respectively. Dose-related toxicity was seen over the remaining dose levels tested.

Mutation results:
InMain Assay I, statistically significant increases in mutant frequency were observed at the lowest and at two intermediate concentrations, in the absence of S9 metabolism, and at the highest dose level in its presence. A linear trend was indicated, both in the absence and presence of S9 metabolism.
However, the observed increases were lower than the Global Evaluation Factor both in the absence and presence of S9 metabolism, thus they were
considered of no biological relevance. In Main Assay II, no increases in mutant frequency were observed at any concentration tested.

Any other information on results incl. tables

See Final Report

Applicant's summary and conclusion

Conclusions:

It is concluded that Basic Red 18:1 Chloride 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 Basic Red 18:1 Chloride 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. A preliminary solubility trial indicated that the maximum practicable concentration of the test item in the final treatment medium was 1000 µg/mL using dimethylsulfoxide (DMSO) as solvent. On the basis of this result, a cytotoxicity assay was performed. Both in the absence and presence of S9 metabolic activation, the test item was assayed at a maximum dose level of 1000 µg/mL and at a wide range of lower dose levels: 500, 250, 125, 62.5, 31.3, 15.6, 7.81 and 3.91 µg/mL. In the absence of S9 metabolic activation, using the 3 hour treatment time, no cells were recovered after treatment starting from 62.5 µg/mL. At the next two lower dose levels of 31.3 and 15.6 µg/mL, slight toxicity was observed, reducing relative survival (RS) to 57 and 67% of the concurrent negative control value, respectively. No relevant toxicity was noted over the remaining concentrations tested. Using the 24 hour treatment time, no cells survived to treatment starting from 62.5 µg/mL, moderate toxicity was noted at the next lower concentration (RS=16%), while slight toxicity was observed at 15.6 µg/mL, reducing relative survival to 58%. No relevant toxicity was observed over the remaining dose levels tested. Following treatment in the presence of S9 metabolic activation, using the short treatment time (3 hours), no cells survived to treatment at 1000, 500 and 250 µg/mL. Test item treatment at 125 µg/mL yielded severe toxicity reducing RS to 1%, while no relevant toxicity was observed over the remaining dose levels tested. Based on the results obtained in the preliminary trial, two independent assays for mutation at the TK locus were performed using the dose levels described in the following table:

Main Assay I (-S9; treatment time 3 hours): 60.2, 43.0, 30.7, 22.0, 15.7 and 11.2 μg/mL

Main Assay I (+S9; treatment time 3 hours): 100, 50.0, 25.0, 12.5, 6.25 and 3.13 μg/mL

Main Assay II (-S9; treatment time 24 hours): 35.0, 25.0, 17.9, 12.8, 9.11 and 6.51 μg/mL

Adequate levels of cytotoxicity, covering a range from the maximum to slight or no toxicity, were observed in all treatment series. No relevant increases in mutant frequencies were observed following treatment with the test item, in the absence or presence of S9 metabolism. Negative and positive control treatments were included in each mutation experiment in the absence and presence of S9 metabolism. 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. It is concluded that Basic Red 18:1 Chloride 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.