Disodium 3,3'-[[6-[bis(2-hydroxyethyl)amino]-1,3,5-triazine-2,4-diyl]bis[imino(3-methoxy-4,1-phenylene)azo]]bis[benzenesulphonate]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Remarks:

source study on the analogous substance

Adequacy of study:

weight of evidence

Study period:

From November 16, to December 13, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

adopted 29 July 2016

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Test material

Test material information

Constituent 1

Reference substance name:

Similar Substance 02

IUPAC Name:

Similar Substance 02

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Supplier: Laboratory for Mutagenicity Testing; Techni-cal University, 64287 Darmstadt, Germany.
- Storage: cells were stored in liquid nitrogen in the cell bank oftesting facility, allowing the repeated use of the same cell culture batch in experiments.
- Stock cultures: thawed stock cultures were propagated at 37 °C in 75 cm2 plastic flasks.
- Seeding: about 2-3×10^6 cells were seeded into each flask with 15 ml of MEM (minimal essential medium) containing Hank’s salts supplemented with 10 % foetal bovine serum (FBS), neomycin (5 μg/ml) and amphotericin B (1 %).
- Subculturing: the cells were sub-cultured once or twice weekly.
- Incubation: at 37 °C with 1.5 % carbon dioxide (CO2) in humidified air.

PERIODICAL CHECKS
- Periodically checked for Mycoplasma contamination: each master cell stock is screened for mycoplasm contamination.
- Periodically checked for karyotype stability: each master cell stock is checked for karyotype stability.
- Periodically 'cleansed' against high spontaneous background: each master cell stock is checked for spontaneous mutant frequency.

Metabolic activation:

with and without

Metabolic activation system:

rat liver microsomal fraction S9 mix

Test concentrations with justification for top dose:

- Main test: without S9 mix 72.6; 145.2, 290.4, 580.8 and 1161.5 μg/ml; with S9 mix 145.2, 290.4, 580.8, 1161.5 and 2323.0 μg/ml.
- Toxicity data, pre-experiment: 18.1, 36.3, 72.6; 145.2, 290.4, 580.8, 1161.5 and 2323.0 μg/ml
- Selection for main test: the concentrations used in the main experiment were selected based on precipitation observed in the pre-experiment.
- Spacing factor: the individual concentrations were spaced by a factor of 2.

Vehicle / solvent:

- Vehocle: on the day of the experiment (immediately before treatment), the test item was dissolved in deionised water.
- Concentratin: the final concentration of deionised water in the culture medium was 10 %.
- Justification for choice of solvent/vehicle: the solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.
- Formulation preparation: all formulations were prepared freshly before treatment and used within two hours of preparation.

Controls

Negative solvent / vehicle controls:

yes

Remarks:

deionised water

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

TREATMENT
After 24 hours the medium was replaced with serum-free medium containing the test item, either without S9 mix or with 50 μl/ml S9 mix. 4 hours after treatment, this medium was replaced with complete medium following two washing steps with PBS.
Immediately after the end of treatment the cells were trypsinised as described above and sub-cultivated. At least 2.0×10^6 cells per experimental point (concentration series plus controls) were subcultured in 175 cm² flasks containing 30 ml medium.
Two additional 25 cm² flasks were seeded per experimental point with approx. 500 cells each to determine the relative survival (cloning efficiency I) as measure of test item induced cytotoxicity. The cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2.
The colonies used to determine the cloning efficiency I were fixed and stained 6 to 8 days after treatment.
Three or four days after first sub-cultivation approximately 2.0×10^6 cells per experimental point were sub-cultivated in 175 cm² flasks containing 30 ml medium.
Following the expression time of 7 days five 75 cm² cell culture flasks were seeded with about 4 to 5×10^5 cells each in medium containing 6-TG. Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability (cloning efficiency II).
The cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 for about 8 days. The colonies were stained with 10 % methylene blue in 0.01 % KOH solution.
The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.

SEEDING
Two to four days after sub-cultivation stock cultures were trypsinized at 37 °C for approximately 5 to 10 minutes. Then the enzymatic digestion was stopped by adding complete culture medium with 10 % FBS and a single cell suspension was prepared. The trypsin concentration for all sub-culturing steps was 0.2 % in saline.
Prior to the trypsin treatment the cells were rinsed with PBS. Approximately 0.7 to 1.2×10^7 were seeded in plastic flasks. The cells were grown for 24 hours prior to treatment.

CULTURE MEDIUM
- Seeding: MEM (minimal essential medium) containing Hank’s salts, neomycin (5 μg/ml), 10 % FBS, and amphotericin B (1 %). During treatment no FBS was added to the medium.
- Mutant selection: for the selection of mutant cells the complete medium was supplemented with 11 μg/ml 6-thioguanine.
- Incubation: all cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 (98.5 % air).

PRE TEST ON TOXICITY
The general culturing and experimental conditions in this pre-test were the same as described below for the mutagenicity experiment.
Approximately 1.5 million cells were seeded in 25 cm² flasks 24 hours prior to treatment. After approximately 24 hours the test item was added and the treatment proceeds for 4 hours (duplicate cultures per concentration level). Immediately after treatment the test item was removed by rinsing with PBS. Subsequently, the cells were trypsinized and suspended in complete culture medium. After an appropriate dilution the cell density was determined with a cell counter. Toxicity of the test item is evident as a reduction of the cell density compared to a corresponding solvent control. A cell density of approximately 1.5 million cells in 25 cm² flasks is about the same as approximately 10 million cells seeded in 175 cm² bottles 24 hours prior to treatment with the main experiment.

OSMOLARITY AND pH
The osmolarity and the pH-value were determined in culture medium of the solvent control and at the maximum concentration of the pre-experiment without metabolic activation.

MAMMALIAN MICROSOMAL FRACTION S9 MIX
- Metabolic activation system: phenobarbital/β-naphthoflavone induced rat liver S9 was used as metabolic activation system. T
- S9 capability for activation: each batch of S9 was routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test.
- S9 use: an appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/ml in the cultures.
- S9 mix composition: MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium-ortho-phosphate-buffer (100 mM, pH 7.4).
- Protein concentration: 32.7 mg/ml in the pre-experiment and the main experiment.

ACCEPTABILITY OF THE ASSAY
The gene mutation assay is considered acceptable if it meets the following criteria:
a) the mean values of the numbers of mutant colonies per 10^6 cells found in the solvent controls of both parallel cultures remain within the 95 % confidence interval of the laboratory historical control data range.
b) the positive control substances should produce a significant increase in mutant colony frequencies and remain within the historical control range of positive controls.
c) Two experimental conditions (i.e. with and without metabolic activation) were tested unless one resulted in positive results.
d) An adequate number of cells and concentrations (at least four test item concentrations) are analysable even for the cultures treated at concentrations that cause 90 % cytotoxicity during treatment.
e) The criteria for the selection of the top concentration are fulfilled.

Evaluation criteria:

A test item is classified as clearly mutagenic if, in any of the experimental conditions examined, all of the following criteria are met:
a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) the increase is dose-related when evaluated with an appropriate trend test,
c) any of the results are outside the distribution of the historical negative control data (e.g. Poisson-based 95 % control limits).
A test item is classified as clearly non-mutagenic if, in all experimental conditions examined, all of the following criteria are met:
a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) there is no concentration-related increase when evaluated with an appropriate trend test,
c) all results are inside the distribution of the historical negative control data (based 95 % control limits).
In cases when the response is neither clearly negative nor clearly positive as described above, or in order to judge the biological relevance of a result, the data should be evaluated by expert judgement or further investigations.

Statistics:

A linear regression (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible dose dependent increase of mutant frequencies. The numbers of mutant colonies generated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Precipitation visible at the end of treatment was noted at 1161.5 μg/ml without metabolic activation.
No relevant cytotoxic effect indicated by an adjusted cloning efficiency I below 50 % was observed neither in absence nor presence of metabolic activation.
No biologically relevant increase in mutant colony numbers was observed in the main experiment up to the maximum concentrations scored for gene mutations. The range of the mean mutant frequencies of the groups treated with the test item was from 5.4 up to 27.6 mutants per 10^6 cells. The linear regression analysis showed no significant dose dependend trend of the mutation frequency at any of the experimental groups.

CONTROLS
The mean mutant frequency obtained in the solvent controls was 12.3 without S9 mix and 22.4 with S9 mix. The values were well within the 95 % confidence interval of our laboratory’s historical negative control data and, thus, fulfilled the requirements of the current OECD Guideline 476.
EMS (300 μg/ml) and DMBA (2.3 μg/ml) were used as positive controls and showed a distinct increase in induced mutant colonies.

PRE TEST ON TOXICITY
In the pre-experiment no toxic effects were observed after 4 hours in the presence and absence of metabolic activation.
The test medium was checked for precipitation or phase separation at the end of each treatment period (4 hours) before the test item was removed. Precipitation occurred at 2323.0 μg/mL without metabolic activation.
There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item.

OSMOLARITY AND pH
Osmolarity: solvent 291 mOsm; test item 299 mOsm.
pH: solvent 7.40; test item 7.39.

Applicant's summary and conclusion

Conclusions:

The substance is considered to be non-mutagenic in the HPRT assay perfromed.

Executive summary:

The study was performed to investigate the potential of test item to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster.

The main experiment was performed with a treatment period of 4 hours with and without metabolic activation. The maximum test item concentration of the pre-experiment (2323 μg/ml) was chosen with respect to the OECD guideline 476 regarding the purity of the test item. The following concentrations used in the main experiment were selected based on precipitation observed in the pre-experiment: 72.6, 145.2, 290.4, 580.8, 1161.5 and 2323.0 μg/ml.

Precipitation visible at the end of treatment was noted at 1161.5 μg/ml without metabolic activation. No relevant cytotoxic effect indicated by an adjusted cloning efficiency I below 50 % was observed neither in absence nor presence of metabolic activation.

No biologically relevant increase in mutant colony numbers was observed in the main experiment up to the maximum concentrations scored for gene mutations.

The mean mutant frequency obtained in the solvent controls was 12.3 without S9 mix and 22.4 with S9 mix. The values were well within the 95 % confidence interval of our laboratory’s historical negative control data and, thus, fulfilled the requirements of the current OECD Guideline 476. The range of the mean mutant frequencies of the groups treated with the test item was from 5.4 up to 27.6 mutants per 10⁶ cells.

The linear regression analysis showed no significant dose dependent trend of the mutation frequency at any of the experimental groups.

EMS and DMBA were used as positive controls and showed a distinct increase in induced mutant colonies.

Conclusion

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.

Therefore, the substance is considered to be non-mutagenic in this HPRT assay.