Octasodium 2,2'-[1,4-phenylenebis[imino(6-chloro-1,3,5-triazine-4,2-diyl)imino(1-hydroxy-3,6-disulphonatonaphthalene-2,8-diyl)azo]]bisnaphthalene-1,5-disulphonate

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:

Experimental start date: 09 November 2017. Experimental completion date: 06 December 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro mammalian forward mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Octasodium 2,2'-[1,4-phenylenebis[imino(6-chloro-1,3,5-triazine-4,2-diyl)imino(1-hydroxy-3,6-disulphonatonaphthalene-2,8-diyl)azo]]bisnaphthalene-1,5-disulphonate

EC Number:

275-108-6

EC Name:

Octasodium 2,2'-[1,4-phenylenebis[imino(6-chloro-1,3,5-triazine-4,2-diyl)imino(1-hydroxy-3,6-disulphonatonaphthalene-2,8-diyl)azo]]bisnaphthalene-1,5-disulphonate

Cas Number:

71002-20-5

Molecular formula:

C52H26Cl2N14Na8O26S8

IUPAC Name:

octasodium 3-({4-chloro-6-[(4-{[4-chloro-6-({8-[(1E)-2-(1,5-disulfonatonaphthalen-2-yl)diazen-1-yl]-1-hydroxy-3,6-disulfonatonaphthalen-2-yl}amino)-1,3,5-triazin-2-yl]amino}phenyl)amino]-1,3,5-triazin-2-yl}amino)-5-[(1E)-2-(1,5-disulfonatonaphthalen-2-yl)diazen-1-yl]-4-hydroxynaphthalene-2,7-disulfonate

Test material form:

solid

Specific details on test material used for the study:

Chemical Name: 1,5-Naphthalenedisulfonic acid, 2,2'-[1,4-phenylenebis[imino (6-chloro-1,3,5-triazine-4,2-diyl)imino(8-hydroxy -3,6-disulfo-1,7-naphthalenediyl)-2,1-diazenediyl]]bis-,sodium salt (1:8)
Appearance: Solid brown to black, golden shiny
Expiry / Retest Date: 17 June 2018
Storage Conditions: At room temperature

Method

Target gene:

HPRT (hypoxanthine-guanine phosphoribosyl transferase) gene locus

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

The V79 cell line has been used successfully in in vitro experiments for many years. Especially the high proliferation rate (doubling time 12 - 16 h in stock cultures) and a good cloning efficiency of untreated cells (as a rule more than 50%) both necessary for the appropriate performance of the study, recommend the use of this cell line. The cells have a stable karyotype with a modal chromosome number of 22.

Cell Cultures
Large stocks of the V79 cell line (supplied by Laboratory for Mutagenicity Testing; Techni¬cal University, 64287 Darmstadt, Germany) are stored in liquid nitrogen in the cell bank of Envigo CRS GmbH allowing the repeated use of the same cell culture batch in experiments. Before free¬zing, the level of spontaneous mutants may be reduced by treatment with HAT-medium. Each master cell stock is screened for mycoplasm contamination and checked for karyotype stability and spontaneous mutant frequency. Consequently, the parameters of the experiments remain similar because of the reproducible characteristics of the cells.
Thawed stock cultures were propagated at 37 °C in 75 cm2 plastic flasks. About 2-3 x10^6 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%). The cells were sub-cultured once or twice weekly.
All incubations were done at 37°C with 1.5% carbon dioxide (CO2) in humidified air.

Culture Medium
For seeding of the cell cultures the complete culture medium was 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. For the selection of mutant cells the complete medium was supplemented with 11 µg/mL 6-thioguanine. All cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 (98.5 % air).

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Pre-Test on Toxicity
The pre-experiment was performed in the presence and absence of metabolic activation. Test item concentrations between 20.2 µg/mL and 2581.0 µg/mL were used. The highest concentration was chosen with respect to the current OECD guideline 476 regarding the purity of the test substance (77.5%).
Cytotoxic effects indicated by a relative cloning efficiency below 50% were observed at 645.3 µg/mL and above without metabolic activation, and at 1290.5 µg/mL and above after 4 hours treatment.

Main Test
without S9 mix: 162.5, 325.0 487.5, 650.0, 975.0* μg/mL
with S9 mix: 78.1, 156.3, 312.5, 625.0, 937.5 μg/mL
Based on the cytotoxicity observed in the pre-experiment, 1300 µg/mL without metabolic activation and 1875.0 µg/mL with metabolic activation were chosen as top concentrations for the main experiment. The individual concentrations were generally spaced by a factor of 2. Narrower spacing was used at higher concentrations to cover the range of cytotoxicity more closely.
To overcome problems with possible deviations in toxicity the main experiment was started with more than four concentrations

Vehicle / solvent:

The test item was dissolved in deionised water. The final concentration of deionised water in the culture medium was 10 % (v/v). The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

deionised water

True negative controls:

no

Positive controls:

yes

Remarks:

300 µg/mL = 2.4 mM

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolica activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

deionised water

True negative controls:

no

Positive controls:

yes

Remarks:

2.3 µg/mL = 8.9 µM

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

Pre-Test on Toxicity
A pre-test was performed in order to determine the toxicity of the test item. In addition the pH and osmolarity were measured. The general culturing and experimental conditions in this pre-test were the same as described below for the mutagenicity experiment.
In this pre-test 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.

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 x 10^7 were seeded in plastic flasks. The cells were grown for 24 hours prior to treatment.

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. Concurrent solvent and positive controls were treated in parallel. 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 x 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 as described below.
Three or four days after first sub-cultivation approximately 2.0 x10^6 cells per experimental point were sub-cultivated in 175 cm² flasks containing 30 mL medium.
Following the expression time of 6 days five 75 cm² cell culture flasks were seeded with about 4 to 5 x 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.

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 (e.g. Poisson-based 95% control limits).
There is no requirement for verification of a clearly positive or negative response. In case the response is neither clearly negative nor clearly positive as described above 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.
In rare cases, even after further investigations, the data set will preclude making a conclusion of positive or negative results, and therefore the test chemical response will be concluded to be equivocal.

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.
A t-Test was not performed since none of the mutation frequencies calculated for the evaluated test item concentrations exceeded the 95% confidence interval.
However, both, biological and statistical significance were considered together

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The test item Bayscript Magenta BB was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster. The treatment period was 4 hours with and without metabolic activation.
Cytotoxic effects indicated by a relative total growth of less than 50% of relative survival as mean of both parallel cultures occurred at 650.0 µg/mL and above without metabolic activation, and at 625.0 µg/mL and above with metabolic activation. The data at 975.0 µg/mL without metabolic activation are rejected as the relative adjusted cloning efficiency I was below 10%.
In the main experiment with and without S9 mix the mean mutant frequency of the solvent control was 19.9 and 21.6 mutants per 10^6 cells. 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 groups treated with the test item ranged from 9.8 up to 23.9 mutants per 106 cells.
No relevant increase in mutant colony numbers/10^6 cells was observed in the main experiment up to the maximum concentration.
The 95% confidence interval was not exceeded at any experimental point. All mutant frequencies remained well within the range of the historical solvent control data.
The linear regression analysis showed no significant dose dependent trend of the mutation frequency.
EMS (300 µg/mL) and DMBA (2.3 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

Any other information on results incl. tables

The main experiment was analyzed at the following concentrations:

S9 mix	concentrations in µg/mL
	Main experiment / exposure period 4 hours
-	162.5	325.0	487.5	650.0	975.0*
+	78.1	156.3	312.5	625.0	937.5

*Not taken into evaluation due to strong toxic effects (rel. adjusted CE I <10%)

Applicant's summary and conclusion

Conclusions:

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, Bayscript Magenta BB is considered to be non-mutagenic in this HPRT assay.

Executive summary:

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

The treatment period was 4 hours with and without metabolic activation.

The maximum test item concentration of the pre-experiment (2581 µg/mL) was chosen with respect to the OECD guideline 476 (2016) regarding the purity of the test item. The concentration range of the main experiment was limited by cytotoxicity observed in the pre-experiment.

No relevant increase in mutant colony numbers/10⁶cells was observed in the main experiment up to the maximum concentration.

Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

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, Bayscript Magenta BB is considered to be non-mutagenic in this HPRT assay.