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]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No mutagenic activity in bacteria was observed with the compound and the test item did not induce gene mutations at the HPRT locus in V79 cells. In addition, the substance is not suspect to be able to induce structural chromosome aberrations in V79 in vitro cells.

Link to relevant study records

Reference

Reference 1

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

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

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.

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.

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.

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

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August from the 03rd to the 24th, 1981

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium, other: TA1535, TA1537, TA98, TA100 and TA1538

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Prof. B.N. Ames, University of California Berkeley, USA.
- Storage: the strains were kept in sterile 0.5 ml ampoules (0.45 ml bacteria culture + 0.05 ml dimethylsulfoxide) at -70 °C and in liquid nitrogen.

REGULAR CHECK
- Periodically checked forgenetic markers: performed.

PREPARATION FOR ANALYSIS
Starting the experiment, bacteria were grown overnight in a shaking waterbath for 16 hours at 37 °C, using nutrient broth No 2 Fa. Oxidid. After centrifugation, bacteria were resuspended to a titer of about 5 x 10^8 - 2 x 10^9 cells for milliliter in 0.16 % nutrient broth and 0.5 % NaCl. The titer is controlled photometrically and determined in an experimental test with histidin rich KCl solution on minimal agar plate.

Metabolic activation:

with and without

Metabolic activation system:

rat liver microsomal activation system

Test concentrations with justification for top dose:

1.58, 5, 15.8, 50, 158, 500, 1580 and 5000 µg/plate

Vehicle / solvent:

- Vehicle used: aqua dest.

Untreated negative controls:

yes

True negative controls:

yes

Remarks:

aseptic control

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

methylmethanesulfonate

other: 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation).
In a test tube the following materials were mixed and poured onto minimal agar plate: 100 µl test solution, or control solvent, or positive control solution; 500 µl S9 mix or KCl 0.15 M; 100 µl bacteria suspension (5 x 10^8 - 2 x 10^9 cells/ml); 2 ml Molten agar, consisting of 0.6 % Bacto agar and 0.6 % NaCl supplemented with 10 % 0.5 mM L-Histdine and 0.5 mM Biotine solution.

ASEPTIC CONTROL: 100 µl of the solution of the test compound or 100 µl of S9 mix were added to 2 ml Molten agar and treated as the other plates.

NUMBER OF REPLICATIONS: each compound concentration including control experiments was tested in triplicate.

INCUBATION: in the dark for 3 days.

TEST CONDITIONS
All experiments were conducted under sterile conditions. In order to avoid any possible light effect on test compound, all experimentation was carried out under yellow light.

COUNT
The His+ revertant colonies were counted with Fisher counter 880.

CYTOTOXICITY
To estimate the toxicity of the test compund, prototrophe bacteri (His+) TA1537 were used. These bacteria were added as an internal standard to plates together with the bacteria strain TA1537, which gives low numbers of revertant colonies and their survival was determined. The ratio of the differences in the numbers of colonies of the RTA and the TA1537 plates for each substance concentration and solvent control gives the relative survival rate.
In addition, the toxicity of the compound may be evidenced by a reduction of the number of spontaneous revertants in the tests with the inserted strains and by an examination of the background law of bacterial growth resulting from traces of histidine added to the top Agar.

LIVER MICROSOMAL FRACTION
-Animal source: male Wistar rats (180 - 250 g), specific pathogen free, from Kleintierfarm Madoerin AG, Fuellinsdorf/BL, Switzerland.
- Item administrated: Aeoclor 1254, dissolved in oleum arachidis.
- Dosage: 500 mg/kg bw
- Administration: single i.p. injection.
- Period: 5 days.
-Homogenate preparation: homogenate were centrifuged at 9000 g for 10 minutes at 0 to 2 °C.
- Composition of 1 ml S9 mix: Na2HPO4: 100 µmol; MgCl2 8 µmol, KCl 22 µmol, NADP+ 4 µmol, glucose-6-phosphate 4 µmol, S9 fraction 0.3 ml.

Species / strain:

S. typhimurium, other: TA1535, TA1537, TA98, TA100 and TA1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Up to the highest tested dose with and without metabolic activation system, no relevant differences, i.e. less that 2 fold increase of revertant colony numbers in any Salmonella t.yphimurium strain were obtained in comparison with the corresponding controls.

CONTROLS
The control plates with the solvent (negative control) showed numbers of spontaneous revertant colonies per plate within the normal range of the testing laboratory experience and similar to those described in literature.
The control plates with reference mutagens (positive control) showed a distinct elevation of the revertant colonies by the tester strain. This confirmed the reversion properties of each strain. The positive results of the mutagens 2-aminoanthracene and benzo[a]pyrene indicate that the metabolizing system was functioning.
The aseptic control showed no contamination for either the compound solution or for the S9 mix.

TOXICITY
Neither quantitative nor qualitative evidence of a toxic effect of the compound was observed.

Conclusions:

No mutagenic activity was observed with the compound, under the experimental conditions reported.

Executive summary:

The test was conducted to detect the potential mutagenic activity of the test item, according to the plate incorporation method of Ames. The mutation assay was conducted on five different Salmonella typhimurium strains (TA1535, TA1537, TA98, TA100 and TA1538), with and without metabolic activation system. The compound was tested at 8 concentrations: 1.58, 5, 15.8, 50, 158, 500, 1580 and 5000 µg/plate. Each compound concentration including control experiments was tested in triplicate.

Up to the highest tested dose with and without metabolic activation system, no relevant differences, i.e. less that 2 fold increase of revertant colony numbers in any Salmonella typhimurium strain were obtained in comparison with the corresponding controls. No toxic effect of the compound was observed.

In conclusion, in the described bacterial test, no mutagenic activity was observed with the compound, under the experimental conditions reported.

Conclusion

No mutagenic activity was observed with the compound, under the experimental conditions reported.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From December the 03rd, 2003 to March the 25th, 2004

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Justification for type of information:

Justification for read across is detailed in section 13.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

adopted on 21 June, 1997

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

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, D-64287 Darmstadt).
- Cell cycle length: 12 hours.
- Incubation conditions: 37 °C in humidified atmosphere with 1.5 % CO2.
- Properly maintained: yes.
- Periodically checked for Mycoplasma contamination: yes.
- Periodically checked for karyotype stability: yes.

Metabolic activation:

with and without

Metabolic activation system:

S9 rat liver induced

Test concentrations with justification for top dose:

With and without S9 mix: 156.3, 312.5, 625.0, 1250.0, 2500.0 and 5000.0 µg/ml
- Dose selection: maximum testable concentration was determined as suggested by the followed guideline in case of nontoxicity of the substance.
In addition, the rage finding test did not show significant reduction in cell numbers after 24 hours exposure, up to the highest tested concentration, i.e. 5000 µg/ml.

Vehicle / solvent:

Water

Untreated negative controls:

yes

Remarks:

culture medium

Negative solvent / vehicle controls:

yes

Remarks:

deionised water

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

RANGE FINDING TEST
A pre-test on cell growth inhibition with 4 and 24 hours treatment was performed, in order to assess the toxicity of the test item. General experimental conditions were the same as the main study.
- Concentrations tested: 39.1, 78.1, 156.3, 321.5, 625.0, 1250.0, 2500.0 and 5000 µg/ml.
- Cell culture: exponentially growing, seeding ca. 40.000 cells.
- Observations: at 4 and 24 hours after start of treatment.
- Cell count: with a 400-fold microscopic magnification.

METHOD OF APPLICATION
Cells were seeded into Quadriperm dishes which contained microscopic slides (2 chamber per dish and test group).
- Cell density at seeding: 10.000 - 60.000 cells in each chamber.
- Medium: MEM with 10 % FCS

NUMBER OF REPLICATION
Two independed experiments were performed, each with two parallel cultures set up.

DURATION
- Exposure duration experiment I
With and without S9: 4 hours
- Exposure duration experiment II
With S9: 4 hours
Without S9: 18 and 28 hours
- Fixation time: at 18 hours (experiment I and II) and 28 hours (experiment II).

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED
- Colcemid was added at 0.2 µg/ml concentration to the cultures. 2.5 hours later, cells were treated in chambers with hypotonic solution (0.4 % KCl) for 20 min at 37 °C. After incubation, cells were fixed with a mixture of methanol and glacial acetic acid (3:1).
- Cells were stained with Giemsa (D-64293 Darmstadt).

NUMBER OF CELLS EVALUATED
100 metaphase plates per culture were scored.

DETERMINATION OF CYTOTOXICITY
Percentage of cells in mitosis was determined, in order to calculate the mitotic index. In addition, number of polyploid cells in 500 metaphase cells per culture was calculated.

Rationale for test conditions:

Maximum testable concentration according to guideline in case of nontoxicity.
In addition, the rage finding test did not show significant reduction in cell numbers after 24 hours exposure, up to the highest tested concentration, i.e. 5000 µg/ml.

Evaluation criteria:

Test item has to be considered as clastogenic if:
- The number of induced structural chromosome aberrations in all evaluated dose groups is outside the range of the historical control data of the performing laboratory.
- Significant increase of the number of chromosome aberrations occurred.

Statistics:

Fisher's exact test (p < 0.05).

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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

CHROMOSOME ABERRATION RESULTS
In both experiments with and without S9, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed.
There were although two exceptions in the presence of S9, where two significant increases were observed: (a) in experiment I (2.5 %) at preparation interval 18 hours with 5000 µg/ml and (b) in experiment II (2.0 %) at preparation interval 28 hours with 2500 µg/ml. Despite the increases of aberrant cells were statistically significant compared to the solvent control (0.0 % aberrant cells, both), the responses are within the historical control data range. Therefore, the statistical significances have to be regarded as being biologically irrelevant.
The aberration rates of the cells after treatment with the test item (0.5 - 2.5 % aberrant cells, exclusive gaps) were close to the range of the solvent control values (0.0 - 2.0 % aberrant cells, exclusive gaps) and within the range of historical control data.

CYTOTOXIC EFFECTS RESULTS
Neither reduced mitotic indices nor reduced cell numbers could be observed up to the highest evaluated concentrations, except in experiment II with S9, where the cell numbers were clearly reduced after 4 hrs treatment with 5000 µg/ml (47% of control) at the 28 hours interval. Besides, in experiment II after 18 hrs and 28 hours continuous treatment without S9 the two highest applied concentrations (2500 and 5000 µg/ml, respectively) were not evaluable for cytogenetic damage, due to toxic effects indicated by clearly reduced mitotic indices and/or low metaphase quality.
In both experiments, no biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (1.1 - 3.3 %) as compared to the rates of the solvent controls (1.4- 3.6 %).
The positive controls with CPA and EMS showed significant increases in chromosome aberrations.

TEST-SPECIFIC CONFOUNDING FACTORS
In the range finding study, neither precipitation nor influence of the test item on the pH value or osmolarity was observed (275 mOsm and pH 7.1 versus 340 mOsm and pH 7.3 for solvent control and test item respectively, at 5000 µg/ml).

RANGE-FINDING
No clear toxic effects with values below 50 % of control were observed after treatment, up the the highest concentration of 5000 µg/ml.

HISTORICAL CONTROL DATA
- Positive historical control data (exclusive gap)
CPA: 17.2 % aberrant cells (mean ± SD range from 11.5 - 22.9 %).
EMS: 21.7 % aberrant cells (mean ± SD range from 10.7 - 32.8 %).
- Negative (water) historical control data (exclusive gap)
Without S9: 1.1 % aberrant cells (mean ± SD range from 0.5 - 1.7 %).
With S9: 1.1 % aberrant cells (mean ± SD range from 0.4 - 1.7).

Experiment	Preparation interval	Test item concentration [µg/ml]	Polyploid cells in %	Cell numbers in % of control	Mitotic indices in % of control	Abberrant cells in %
						Incl. gaps	Excl. gaps	With exchanges
Exposure period: 4 hours without S9
I	18 hours	Negative control	1.9	n.t.	100	1.0	1.0	0.5
		Solvent control	3.6	100	100	2.0	2.0	0.0
		Positive control	1.6	n.t.	117	10.5	10.0	4.5
		1250.0	2.7	93	102	0.5	0.5	0.0
		2500.0	2.6	114	107	1.0	1.0	0.0
		5000.0	1.6	104	105	1.5	1.0	0.0
Exposure period: 18 hours without S9
II	18 hours	Negative control	2.5	n.t.	100	1.0	1.0	0.0
		Solvent control	2.4	100	100	0.5	0.5	0.0
		Positive control	2.4	n.t.	90	11.5	11.0	5.0
		312.5	2.6	107	93	3.5	1.5	0.0
		625.0	1.4	114	86	1.5	1.5	0.0
		1250.0	1.4	113	82	2.5	1.5	0.0
Exposure period: 28 hours without S9
II	18 hours	Negative control	2.6	n.t.	100	3.0	3.0	0.5
		Solvent control	2.9	100	100	2.0	1.5	0.0
		Positive control	2.3	n.t.	77	15.5	15.0	6.0
		1250.0	1.2	84	62	3.0	2.5	0.0
Exposure period: 4 hours with S9
I	18 hours	Negative control	1.7	n.t.	100	0.5	0.0	0.0
		Solvent control	1.4	100	100	0.0	0.0	0.0
		Positive control	1.1	n.t.	99	0.0	9.5	3.5
		1250.0	2.5	103	108	1.5	1.0	0.0
		2500.0	1.6	108	103	1.5	1.0	0.5
		5000.0	1.1	97	123	2.5	2.5	0.0
II	18 hours	Negative control	1.9	n.t.	100	0.5	0.5	0.0
		Solvent control	2.0	100	100	0.0	0.0	0.0
		Positive control	2.0	n.t.	111	13.5	12.5	0.0
		1250.0	3.3	74	87	1.0	1.0	0.0
		2500.0	2.3	59	94	2.5	2.0	0.5
		5000.0	1.8	47	90	3.0	1.0	0.0

n.t. = not tested

in bold character are marked statistical significant values higher than the corresponding control.

Conclusions:

The test item did not induce structural chromosome aberrations in V79 in vitro cells, both with and without metabolic activation.

Executive summary:

The test was performed according to OECD 473 (1997). In each experimental group two parallel cultures were set up. Per culture, 100 metaphase plates were scored for structural chromosome aberrations; the highest testable concentration determined in the pre-test and was 5000 µg/ml.

In both experiments with and without S9, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed.

No relevant increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls.

No clear cytotoxicity indicated by reduced mitotic indices and cell numbers were observed, except in the presence of S9 mix in experiment II at the highest applied concentration.

Appropriate mutagens were used as positive controls. They induced statistically significant increases in aberrant cells.

Conclusion

The test item did not induce structural chromosome aberrations in V79 in vitro cells, both with and without metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The test article is not suspect to be able to induce micronuclei in the bone marrow cells of the mouse.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From the 02nd February to the 23th March, 1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

Justification for read across is detailed in section 13.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted on 26th May, 1983

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

mammalian germ cell cytogenetic assay

Species:

mouse

Strain:

Swiss

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Wiga GmbH, Sulzfeld, FRG
- Age at study initiation: ca. 8 weeks
- Weight at study initiation: males 27 - 32 g; females 20 - 25 g
- Fasting period before study: withheld the night before dosing until 4 hours after administration.
- Diet: standard laboratory animal diet (RMH-B); certificate of analysis performed.
- Water: tap water; certificate of analysis performed.
- Accomodation: in groups of 5 per sex in polycarbonate cages.
- Acclimation: at least 6 days under laboratory conditions.
- Identification: by unique cage number and a mark on the tail.

ENVIRONMENTAL CONDITIONS
- Temperature: 21 ± 3 °C
- Humidity: 40 - 70 %
- Air changes: 7.5 ACH
- Photoperiod: 12 hours dark

Route of administration:

oral: gavage

Vehicle:

- Vehicle: Milli-RO water.
- Concentration of test material in vehicle: 4000 mg/kg b.w., single dose.
- Amount of vehicle: 10 ml/kg b.w.
- Sampling time: 24, 48 and 72 hours.

Details on exposure:

Single oral dose of 4000 mg/kg b.w.

Duration of treatment / exposure:

Bone marrow was sampled at 24, 48 and 72 hours after dosing for both treated and control animal groups. Groups A and B were dosed with 4000 and 2000 mg/kg b.w. substance respectively, and did not show any signs of reaction to the treatment.
Concentration higher than 4000 could not be assessed due to aggregation of test article in suspension.

Dose / conc.:

4 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 males and 5 females.

Control animals:

yes, concurrent vehicle

Positive control(s):

Yes, cyclophosphamide.
- Route of administration: oral gavage.
- Dose: single dose at 50 mg/kg b.w. dissolved in 0.9 % NaCl in Milli-RO water.
- Sampling time: 48 hours after treatment.

Tissues and cell types examined:

Bone marrow and erythrocytes.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION
A pilot study was performed on 3 males and 3 females per each dose group (five).

DETAILS OF SLIDE PREPARATION
Both femurs were flushed with 2 ml of foetal calf serum. The cell suspension was collected and centrifuged at 1000 rmp for 5 minutes. A drop of the cell suspension was placed on a slide which was previously cleaned (24 hours immersed in a 1:1 mixture of 96 % ethanol/ether). The preparations were then air-dried and thereafter fixed for 5 min in 100 % methanol and air-dried overnight. Two slides were prepared per animal.
- Staining: stained for 3 minutes in undiluted May-Grunwald solution followed by 2 minutes in May Grunwald solution diluted 1:1 with Sorensen buffer pH 6.8. Thereafter slides were rinsed and stained for 25 minutes in 5 % (v/v) Giemsa solution in Sorensen buffer pH 6.8. The preparations were rinsed for 1 minute in running tap-water and blotted dry between filter paper.

METHOD OF ANALYSIS
Slides were scored at a magnification of 1000 x. The number of micronuclei was counted in 1000 polychromatic erythrocytes. The ratio polychromatic to normochromatic erythrocytes was determined by counting and differentiating the first 1000 erythrocytes at the same time. Averages and standard deviations were calculated.

Evaluation criteria:

Test substance is considered negative if:
- None of the tested concentrations or sampling times showed a statistically significant (p < 0.05) increase in the incidence of micronuclei neither in the combined data for both sexes nor in the data for male or female groups alone.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: no.
- Statistical evaluation: incidence of micronuclei in the control animals was found to be in the range of historical data of the performing laboratory. Positive control showed a statistical significant increase in the number of micronuclei.

Mean number of micronuclei

Sex	Group	Treatment	Dose [mg/kg b.w.]	Sampling time [h]	Micronuclei no. per 1000 polychromatic erythrocytes		Ratio polychromatic / normochromatic erythrocytes
					Mean	SD	Mean	SD
M	A	Vehicle	-	24	0.0	0.0	1.50	0.27
	B	Vehicle	-	48	0.0	0.0	1.27	0.44
	C	Vehicle	-	72	0.0	0.0	1.31	0.17
	D	Test item	4000	24	0.0	0.0	1.46	0.89
	E	Test item	4000	48	0.6	0.9	1.06	0.16
	F	Test item	4000	72	0.0	0.0	1.64	0.45
	G	Positive control	50	48	8.6	2.1	0.44	0.12
F	A	Vehicle	-	24	0.0	0.0	2.23	0.86
	B	Vehicle	-	48	0.4	0.9	1.94	1.45
	C	Vehicle	-	72	0.0	0.0	1.38	0.40
	D	Test item	4000	24	0.2	0.4	1.33	0.77
	E	Test item	4000	48	0.2	0.4	1.39	0.41
	F	Test item	4000	72	0.0	0.0	1.26	0.21
	G	Positive control	50	48	8.0	0.7	0.40	0.11

Conclusions:

The test substance can be considered as not mutagenic in the Mouse Micronucleus Test, under the experimental conditions of the test.

Executive summary:

The substance was tested in the Micronucleus Test in mice, according to the method and procedures outlined into the OECD guideline 474. Three groups, each comprising 5 males and 5 females, received a single oral dose of 4000 mg/kg body weight. Bone marrow was sampled at 24, 48 and 72 hours after dosing. Corresponding vehicle treated groups served as negative controls. Bone marrow from a positive control group, treated with a single oral dose of cyclophosphamide (CP) at 50 mg/kg body weight, was harvested at 48 hours after dosing only. The test substance was found to respond negatively in the Micronucleus Test, whereas the positive control substance (CP) produced a statistically significant increase in the incidence of micronuclei in polychromatic erythrocytes.

Conclusion

The test substance can be considered as not mutagenic in the Mouse Micronucleus Test, under the experimental conditions of the test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Investigation on the genetic toxicity has been performed with the integrated evaluation of the following studies: in vitro Ames tests and in vitro gene mutation on mammalian cells conducted on Optical Brightener 380; in addition, the available information of the structural analogous Similar Substance 01 have been taken into consideration in order to evaluate also the chromosomal aberration potential. The read across approach can be considered reliable and appropriate to investigate the property (details for the approach are included into the IUCLID section 13).

IN VITRO GENE MUTATION IN BACTERIA

A test was conducted to detect the potential mutagenic activity of the Optical Brightener 380, according to the plate incorporation method of Ames. The mutation assay was conducted on Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA1538, with and without metabolic activation system. The compound was tested at 8 concentrations: 1.58, 5, 15.8, 50, 158, 500, 1580 and 5000 µg/plate. Each compound concentration including control experiments was tested in triplicate.

Up to the highest tested dose with and without metabolic activation system, no relevant differences, i.e. less that 2 fold increase of revertant colony numbers in any Salmonella typhimurium strain were obtained in comparison with the corresponding controls. No toxic effect of the compound was observed.

A second test, conducted using Salmonella typhimurium strains (TA1535, TA1537, TA98, TA100 and TA1538) and Escherichia coli (WP2 uvrA), is available. The potential mutagenic activity of the test item was assayed with and without metabolic activation system. The compound was tested at concentrations of 10, 50, 100, 500, 1000 and 5000 µg/plate. Each compound concentration including control experiments was tested in duplicate. No significant difference in the number of the colonies between the test compound dosages and the control with any of the bacteria used here.

In conclusion, in the described bacterial tests, no mutagenic activity was observed with the compound, under the experimental conditions reported.

IN VITRO GENE MUTATION IN MAMMALIAN CELLS

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.

IN VITRO and IN VIVO CHROMOSOMAL ABERRATION ASSAY

Similar Substance 01 was tested in in vitro chromosomal aberration assay, according to the OECD 473 (1997). In each experimental group two parallel cultures were set up. Per culture, 100 metaphase plates were scored for structural chromosome aberrations; the highest testable concentration determined in the pre-test and was 5000 µg/ml. In both experiments, with and without S9, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. No relevant increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls. No clear cytotoxicity indicated by reduced mitotic indices and cell numbers were observed, except in the presence of S9 mix in experiment II at the highest applied concentration.

Similar Substance 01 was also tested in in vivo Micronucleus Test in mice, according to the method and procedures outlined into the OECD guideline 474. Three groups, each comprising 5 males and 5 females, received a single oral dose of 4000 mg/kg body weight. Bone marrow was sampled at 24, 48 and 72 hours after dosing. Corresponding vehicle treated groups served as negative controls. Bone marrow from a positive control group, treated with a single oral dose of cyclophosphamide (CP) at 50 mg/kg body weight, was harvested at 48 hours after dosing only. The test substance was found to respond negatively in the Micronucleus Test, whereas the positive control substance (CP) produced a statistically significant increase in the incidence of micronuclei in polychromatic erythrocytes.

Justification for classification or non-classification

According to the CLP Regulation (EC) No 1272/2008, for the purpose of the classification for germ cell mutagenicity, substances are allocated in one of two categories in consideration of the fact that they are:

- substances known to induce heritable mutations or to be regarded as if they induce heritable mutations in the germ cells of humans or substances known to induce heritable mutations in the germ cells of humans or

- substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans.

The available information suggest that test substance did not show any reasons of concern from the genotoxicity point of view.

In conclusion, the substance does not meet the criteria to be classified for genetic toxicity, according to the CLP Regulation (EC) No 1272/2008.