Hexasodium 2,2'-[vinylenebis[(3-sulphonato-4,1-phenylene)imino[6-[(2-cyanoethyl)[2-(2-hydroxyethoxy)ethyl]amino]-1,3,5-triazine-4,2-diyl]imino]]bis[benzene-1,4-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:

From November the 02nd to December the 05th, 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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 29 July 2016

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:

Optical Brightener 380

IUPAC Name:

Optical Brightener 380

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Laboratory for Mutagenicity Testing; Techni¬cal University, 64287 Darmstadt, Germany.
- Storage: stored in liquid nitrogen in the cell bank of testing facility allowing the repeated use of the same cell culture batch in experiments.
- Preparation of incubation: before free¬zing, the level of spontaneous mutants may be reduced by treatment with HAT-medium. Thawed stock cultures were propagated at 37 °C in 75 cm2 plastic flasks. About 2-3 x 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 %).
- Sub-culturing: the cells were sub-cultured once or twice weekly.
- Incubation consitions: all incubations were done at 37 °C with 1.5 % carbon dioxide (CO2) in humidified air.

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

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9

Test concentrations with justification for top dose:

MAIN EXPERIMENT: 96.6, 139.2, 278.4, 556.8, 1113.5 and 2227.0 µg/ml, with and without metabolic activation.
JUSTIFFICATION: test item concentrations between 17.4 and 2227.0 µg/ml were tested in preliminary investigation, with and without metabolic activation. The highest concentration was chosen with respect to the current OECD guideline 476, regarding the purity of the test substance.

Vehicle / solvent:

- Solvent used: deionised water.
- Justification for choice of solvent/vehicle: the solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

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).

METHOD OF APPLICATION
On the day of the experiment (immediately before treatment), the test item was dissolved in deionised water. The final concentration of deionised water in the culture medium was 10 % (v/v). All formulations were prepared freshly before treatment and used within two hours of preparation.
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: solvent control osmolarity 2.73 mOsm and pH 7.63; test item osmolarity 296 mOsm and pH 7.28.

DURATION
- Exposure duration: 4 hours
- Expression time: the phenotypic expression is achieved by allowing exponential growth of the cells for 7 - 9 days.

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. 4 hours after treatment, the 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.
Three or four days after first sub-cultivation approximately 2.0 x 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 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.

MUTANT FREQUENCY DETERMINATION
The mutant frequency is determined by seeding known numbers of cells in medium containing the selective agent to detect mutant cells, and in medium without selective agent to determine the surviving cells. After a suitable period the colonies are counted. Mutant frequencies are calculated from the number of mutant colonies corrected for cell survival.

DETERMINATION OF CYTOTOXICITY - PRE TEST
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 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.

MAMMALIAN MICROSOMAL FRACTION S9 MIX
Phenobarbital/β-naphthoflavone induced rat liver S9 was used as metabolic activation system. 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.
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 contained 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).
The protein concentration of the S9 preparation was 32.7 mg/ml in the pre-experiment and in the main experiment.

ACCEPTABILITY OF THE ASSAY
The gene mutation assay is considered acceptable if it meets the following criteria:
- 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. If they fall outside those limits, they are acceptable as long as these data are not extreme outliers and there is evidence that the test system is ‘under control’ and technical or human failure can be excluded.
- Concurrent positive controls should induce responses that are compatible with those generated in the historical positive control data base and produce a statistical significant increase compared with the concurrent negative control.
- Two experimental conditions (i.e. with and without metabolic activation) were tested unless one resulted in positive results.
- 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.
- 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 (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.

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

Remarks:

water

Positive controls validity:

valid

Additional information on results:

In the main experiment with and without S9 mix the range of the solvent controls was from 18.1 up to 20.6 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 was from 10.3 up to 24.5 mutants per 10^6 cells.
No relevant increase in mutant colony numbers/106 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 dependend trend of the mutation frequency at any of the experimental groups.

CONTROLS
EMS (300 µg/ml) and DMBA (2.3 µg/ml were used as positive controls and showed a distinct increase in induced mutant colonies.

CYTOTOXICITY - PRE TEST
No relevant cytotoxic effect indicated by an adjusted cloning efficiency I below 50% was observed neither in absence nor presence of metabolic activation.
No precipitation or phase separation occurred up to the highest concentration following 4 hours treatment with and without metabolic activation.
There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item.

Any other information on results incl. tables

Sumary of results

	Conc. µg/ml	P / PS	S9 mix	Relative colony efficiency I %	Relative cell density %	Rel. Adjusted colony efficiency I %	Mutant colonies / 10^6cells	95 % confidence interval
Column	1	2	3	4	5	6	7	8
Main experiment / 4 hrs treatment				mean values of culture I and II
Solvent control with water		-	-	100.0	100.0	100.0	18.1	1.7 - 30.2
Positive control (EMS)	300	-	-	86.4	115.0	97.6	212.5	1.7 - 30.2
Test item	69.6	-	-	91.8	96.8	83.9	#
Test item	139.2	-	-	94.9	104.6	95.0	24.5	1.7 - 30.2
Test item	278.4	-	-	87.7	93.3	79.1	10.3	1.7 - 30.2
Test item	556.8	-	-	91.3	88.7	78.6	14.5	1.7 - 30.2
Test item	1113.5	-	-	84.1	106.5	88.0	11.2	1.7 - 30.2
Test item	2227.0	-	-	70.5	109.2	75.6	11.4	1.7 - 30.2
Solvent control with water			+	100.0	100.0	100.0	20.6	2.0 - 29.4
Positive control (DMBA)	2.3	-	+	99.7	99.3	99.1	174.9	2.0 - 29.4
Test item	69.6	-	+	99.7	91.8	91.4	#
Test item	139.2	-	+	100.3	81.5	81.8	10.4	2.0 - 29.4
Test item	278.4	-	+	96.9	89.8	87.1	21.0	2.0 - 29.4
Test item	556.8	-	+	98.8	67.9	67.0	17.6	2.0 - 29.4
Test item	1113.5	-	+	99.3	82.6	81.8	21.7	2.0 - 29.4
Test item	2227.0	-	+	99.9	81.9	81.6	22.5	2.0 - 29.4

P / PS = Precipitation / Phase separation

# culture was not continued as a minimum of only four analysable concentrations is required

Toxicity data, pre-experiment

Test group	Conc. µg/ml	S9 mix	Duration of treatment (h)	cells seeded	number of colonies per flask found			CE absolute %	CE relative %	P / PS
				I/II	I	II	mean
Column	1	2	3	4	5	6	7	8	9	10
Solvent control with deion water		-	4	1520000	947400	985400	966400.0	63.6	100.0	~
Test item	17.4	-	4	1520000	1179800	1064800	1122300.0	73.8	116.1	~
Test item	34.8	-	4	1520000	1061600	1025400	1043500.0	68.7	108.0	~
Test item	69.6	-	4	1520000	1021800	1049000	1035400.0	68.1	107.1	~
Test item	139.2	-	4	1520000	1020200	1009200	1014700.0	66.8	105.0	~
Test item	278.4	-	4	1520000	1079800	1048800	1064300.0	70.0	110.1	~
Test item	556.8	-	4	1520000	1090200	926200	1008200.0	66.3	104.3	~
Test item	1113.5	-	4	1520000	1026600	1043600	1035100.0	68.1	107.1	~
Test item	2227.0	-	4	1520000	1084600	875000	979800.0	64.5	101.4	~
Solvent control with deion water		+	4	1520000	1077000	988000	1032500.0	67.9	100.0	~
Test item	17.4	+	4	1520000	1026400	992800	1009600.0	66.4	97.8	~
Test item	34.8	+	4	1520000	1031000	1033600	1032300.0	67.9	100.0	~
Test item	69.6	+	4	1520000	1026800	982000	1004400.0	66.1	97.3	~
Test item	139.2	+	4	1520000	1045000	1070200	1057600.0	69.6	102.4	~
Test item	278.4	+	4	1520000	970400	1021800	996100.0	65.5	96.5	~
Test item	556.8	+	4	1520000	1046200	973900	1010050.0	66.5	97.8	~
Test item	1113.5	+	4	1520000	1034000	1083200	1058600.0	69.6	102.5	~
Test item	2227.0	+	4	1520000	1015400	1056600	1036000.0	68.2	100.3	~

CE = Cloning efficiency

P / PS = Precipitation / Phase separation

Applicant's summary and conclusion

Conclusions:

The test item is considered to be non-mutagenic in this HPRT assay.

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 treatment period was 4 hours with and without metabolic activation.

The maximum test item concentration of the pre-experiment and the main experiment (2227 µg/ml) was chosen with respect to the OECD guideline 476 (2016) regarding the purity of the test item.

No relevant increase in mutant colony numbers/10⁶ cells was observed in the main experiment up to the maximum concentration. No cytotoxic effects indicated by an adjusted cloning efficiency I below 50 % was observed neither in absence nor presence of metabolic activation.

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, test item is considered to be non-mutagenic in this HPRT assay.