5-(diisopropylamino)-2-[[4-(dimethylamino)phenyl]azo]-3-methyl-1,3,4-thiadiazolium methyl sulphate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

HPRT method

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

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:

2015

Deviations:

yes

Remarks:

detailed below

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

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:

5-(diisopropylamino)-2-[[4-(dimethylamino)phenyl]azo]-3-methyl-1,3,4-thiadiazolium trichlorozincate(1-)

EC Number:

298-265-2

EC Name:

5-(diisopropylamino)-2-[[4-(dimethylamino)phenyl]azo]-3-methyl-1,3,4-thiadiazolium trichlorozincate(1-)

Cas Number:

93783-70-1

Molecular formula:

C17H27N6S.Cl3Zn

IUPAC Name:

5-(diisopropylamino)-2-{[4-(dimethylamino)phenyl]diazenyl}-3-methyl-1,3,4-thiadiazol-3-ium trichlorozincate(1-)

Test material form:

solid: particulate/powder

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: frozen permananet cell culture from European Collection of cells cultures; cells kept at -196 °C under liquid nitrogen. After activation, cells were grown in Dulbecco's modified Eagles's medium with 4 mM L-glutamine and 10 % FBS in humidified incubator (5 % CO2, 37 ± 1 °C).
Cells underwent maximum 5 passages after thawing the original culture delivered
from cell collection before using for mutagenicity testing.
Cleansing of cultures was performed 5 days before treatment with complete medium supplemented with HAT supplement due to elimination of mutants. Cleansing was not performed in experiment without metabolic activation, because of bad growth of cells in HAT medium.

MEDIA USED
- DMEM (Dulbecco's minimal essential medium): minimal medium, part of complete growth medium
- HAT supplement: liquid mixture of sodium hypoxanthine (5 mM), aminopterin (20 µM) and thymidine (0.8 mM). HAT-supplemented medium is suitable for post-fusion selection against unfused or self-fused HGPRT–myeloma cells. Hypoxanthine and thymidine supply preformed purines and pyrimidines for DNA synthesis by hybridomas via the salvage pathway that utilizes HGPRT- contributed by the fused spleen cell. Aminopterin, a folic acid antagonist, inhibits the de novo nucleoside biosynthesis pathway. For HAT medium is diluted 50×. It is used as cleansing medium for reduction of mutants at the start of experiment
- FBS: fetal bovine serum, part of complete growth medium
- trypsin-EDTA (0.5%) solution: for release of cells from the bottom of dishes
- Atb - penicillin (10000 U/ml) streptomycin (10000 µg/ml): for prevention of contamination, part of complete growth medium
Complete growth medium DMEM : FBS : Atb = 500 : 55 : 5.5, prepared in laboratory.

- Periodically checked for mycoplasma contamination: yes

Additional strain / cell type characteristics:

other: HPRT deficient

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of rat liver homogenate and a mixture of cofactors

Test concentrations with justification for top dose:

Test substance is soluble in water till 25.22 g/l. Test substance was dissolved in assay medium (DMEM). The highest recommended concentration is 2 mg/ml.
On the basis of cytotoxicity test results, the mutagenicity test with metabolic activation was arranged with range of concentrations 0.02-0.2 mg/ml. Concentrations for the mutagenicity test without metabolic activation were 0.01-0.04 mg/ml.

Vehicle / solvent:

- Solvent used: DMEM

Controls

Untreated negative controls:

yes

Remarks:

DMEN

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

Rinsing of cells after treatment: PBS prepared in laboratory
Selective agent: 6-thioguanine 98 % diluted in 0.5 % Na2CO3, 5 µg/ml (final concentration) for selection of mutants
Dye for staining of colonies: methylene blue, 0.1 % solution

Cytotoxicity
The first cytotoxicity experiment was performed with as well as without metabolic activation. The concentrations for cytotoxicity were in range 0.01-2.0 mg/ml.
The first experiment failed in determination of doses for the mutagenicity tests, because doses with low, medium and high cytotoxicity were not possible to determine. Toxicity test had to be repeated for two more times.
On the basis of cytotoxicity test results, the mutagenicity test with metabolic activation was arranged with range of concentrations 0.02-0.2 mg/ml. Concentrations for the mutagenicity test without metabolic activation were 0.01-0.04 mg/ml.

Treatment: at least 2E+06 cells will be treated for each concentration/control for 3 hours. After treatment, every dish will be trypsinised and 300 cells will be seeded to 3 Petri dishes. Relative viability will be counted by comparison of viability in single concentration to viability in solvent control.

Mutation assay procedure
Each concentration was tested in two simultaneously performed independent runs (duplicates).
Experimental design
Concentrations for mutagenicity experiments were chosen on the basis of cytotoxicity testing. No precipitation was observed in any concentration in experiment with as well as without metabolic activation.
Test substance was dosed to dishes with cells in volume of 100 μl.
Cells were treated for 3 hours (with as well as without metabolic activation; day 1). After treatment, approximately 2E+06 cells were transferred to suitable number of dishes to seed enough cells. At the same time, cells were seeded for detection of number of cells (PE estimation).
On the 3rd, 6th and 8th day, approximately 2E+06 cells from every culture were transferred and 10th day, extractions of mutants was performed with using selective medium together with PE estimation again.

Experimental design (3 hours treatment)
Day Activity
1 treatment, passage of 1E+06 cells, plating of an aliquot (300 cells) for estimating of viability
3 passage of approx. 2E+06 cells
6 passage of approx. 2E+06 cells
8 passage of approx. 2E+06 cells
10 extraction of mutant cells, plating of an aliquot (300 cells) for estimating of viability

Fresh solutions of test substance were prepared for every experiment; solutions were prepared on a weight/volume in volumetric vials.
Neither assay of test substance stability, nor assays of its concentration and homogeneity in vehicle was undertaken.

Determination of survival
After treatment period, the cultures were trypsinised and an aliquot (0.3 ml of 1000/ml cell suspension) was diluted and plated to 6 cm Petri dishes to estimate the viability of the cells.
A number of cells were then replaced in order to maintain the treated cell populations; the number of cells taken forward was adjusted according to the expected viability of the cultures, to give two millions of viable cells. Cells were grown in 10 cm Petri dishes.

Subculturing
On day 3, 6 and 8, cell populations were subcultured in order to maintain them in exponential growth. The number of cells taken forward was adjusted according to the expected viability, to give two millions viable cells seeded in 10 cm Petri dishes.

Incubation, staining and scoring
Survival and plating efficiency plates were incubated for at least six days (37±1 ºC, 5 % CO2, moistened) prior to scoring. Mutant plates were incubated for an appropriate period to ensure adequate colony size (about 10 days). After incubation, the plates were stained with methylene blue and colonies were scored.

Determination of mutant frequency
At expression time, each culture was trypsinised, resuspended in complete medium and counted by microscopy. Then, an adequate number of cells were subcultured to maintain the treated populations of cells. This step is not performed on day 10.
After dilution, an estimated 220000 cells were plated in each of ten 100 mm tissue culture Petri dishes (together 2200000 cells). After about 1 hour, 6-thioguanine was added to each the Petri dish to final concentration of 5 µg/ml. Only HPRT mutant colonies are able to grow in the presence of 6-thioguanine; these plates were subsequently scored for the presence of mutants.
After dilution, an estimated 300 cells were plated in each of three 60 mm tissue culture Petri dishes. These plates were used to estimate plating efficiency.

Evaluation criteria:

Each experiment will be evaluated separately using modified two-fold increase rule according to Claxton L.D. et al, Mutat. Res.,189, 83-91, 1987.
The mutagenic potential is indicated by increasing number of mutants in treated groups in comparison to negative solvent control (modified two-fold increase rule and any of the results outside the distribution of the historical negative control data) and/or by dependence of increasing number of mutants on dose (dose-response relationship).
There is no requirement for verification of a clearly positive or negative response.
In cases when the response is neither clearly negative nor clearly positive than a repeated experiment possibly using modified experimental conditions (e.g. concentration spacing, other metabolic activation conditions [i.e. S9 concentration or S9 origin]) could be performed.

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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Solubility
Precipitation occurred in the first toxicity test starting from 1.0 mg/ml. In the end, due to cytotoxicity of test substance, lower concentrations were used for mutagenicity testing in which no precipitation occurred.

Cytotoxicity experiments
In the first cytotoxicity test (concentration range 0.01 - 2 mg/ml) with as well as without metabolic activation, cytotoxicity was seen at concentration of 0.01 (without metabolic activation) and 0.1 (with metabolic activation). A second experiment was necessary to be performed for determination of doses with high, medium and low cytotoxicity.

Concentrations between 0.0005 - 0.01 mg/ml were used for second cytotoxicity experiment without metabolic activation. Concentrations 0.0025 - 0.05 mg/ml were used for the second cytotoxicity experiment with metabolic activation.
In this experiment doses appropriate for mutagenicity testing were not found because of no or small toxicity.
The third experiment had to be done for clarification. Concentrations 0.005, 0.01, 0.025, 0.05, 0.1, 0.25 and 0.5 were used without metabolic activation; concentrations 0.005, 0.01, 0.025, 0.05, 0.1, 0.25, 0.50 and 0.75 mg/ml were used with metabolic activation.
Based on all the cytotoxicity tests, the following concentrations were determined for mutagenicity testing:
- without metabolic activation: 0.01; 0.015; 0.02; 0.025, 0.030 and 0.040 mg/ml
- with metabolic activation: 0.02, 0.05, 0.1 and 0.2 mg/ml.

Cytotoxicity in mutagenicity experiments
Experiment without metabolic activation was started with 6 concentrations, with the idea to choose the 4 best concentrations (OECD 476).
Cytotoxicity immediately after treatment and after the first plating for cultivation to allow expression of the mutant phenotype was recorded. During further passages the influenced cells either were growing slower than control or some cells died. Overall, decrease of cells in all of the three passages was seen.
Concentration of 0.02 mg/ml was similarly toxic as the surrounding concentrations 0.015 and 0.025 mg/ml; so it was not kept untill the end of experiment.

Experiment with metabolic activation was started at concentrations of 0.02, 0.05, 0.1 and 0.2 mg/ml; concentration of 0.4 mg/ml was added during the mutagenicity because no toxicity was observed immediately after treatment in the lower concentrations.
Concentrations of 0.2 and 0.4 mg/ml were completely cytotoxic after plating for other growing so they were not continued. Toxicity of concentration 0.1 mg/ml was in accordance with OECD 476 but less than 2 millions of cells were transferred for other growing.
Additional experiment was performed with concentrations 0.075 and 0.1 mg/ml to get another suitable concentration for evaluation. In the end, four concentrations were available for 0.02, 0.05, 0.075, 0.1 mg/ml.

pH determination
conc. mg/ml measured pH
DMEM 7.62
0.02 7.64
0.05 7.65
0.1 7.65
0.2 7.65

Addition of test substance to cultivation medium changed pH of treatment solutions by 0.03. No pH adjustment was needed.

Mycoplasma determination
Three withdrawals of medium were performed: experiment without metabolic activation, experiment with metabolic activation and additional experiment with metabolic activation, after minimum of 14 days of growing of cells.
Result of test sample was negative so all media after cultivation of cells were free of mycoplasma.

Plating efficiency
For the assessment of number of plated cells, PE was determined always after treatment.
In all concentrations used in all experiments more than 2 millions cells were influenced in single replicates. In both experiments with metabolic activation and concentration of 0.1 mg/ml less than 2 millions of cells were seeded for further cultivation. In the additional experiment in both replicates together, condition of 2 millions cells was fulfilled.

Mutagenicity
Mutation frequency of cells treated with test substance in both experiments was low in all concentrations; Mt/Msc ratios generally never exceeded 3 fold limits (in fact all of them were below 2 fold vs solvent control). At the same time, no dose dependence was observed in any experiment.

Assay acceptance criteria compliance
Mutation frequencies of negative control (DMEM) were 1.07-2.12 mutants per 1E+05 plated cells. Historical control range (97.5 % confidence interval) is 0.78-2.28 mutants per 1E+05cells (123 entries).
Mutation frequency of positive controls was sufficiently high:
EMS 50 µl: 18.97; historical control range (95 % confidence interval) is 10.07 – 18.22 mutants per 1E+05cells (40 entries);
EMS 100 µl: 44.14; historical control range (95 % confidence interval) is 19.23 – 37.41 mutants per 1E+05cells (41 entries);
DMBA: 30.69-38.06 what is an evidence of good function of the test system. Historical control range (95 % confidence interval) is 19.23 – 37.41 mutants per 1E+05cells (54 entries).

Any other information on results incl. tables

Mutagenicity without metabolic activation, 3h treatment

Conc. mg/ml	viability (number of colonies)			avg	PE %	Mutants (number of colonies)										∑M	NPC	MF/10^5cells	Mt/Msc
NC (1)	335	402	394	377	102.8	2	2	2	6	4	5	4	4	6	1	36	2,764,667	1.30	1.09
NC (2)	337	364	368	356	97.2	1	1	6	1	6	5	3	2	1	2	28	2,613,111	1.07	0.90
0.01 (1)	298	311	328	312	85.2	2	4	3	3	1	4	3	3	2	3	28	2,290,444	1.22	1.03
0.01 (2)	304	312	387	334	91.2	3	2	2	4	3	4	3	1	5	4	31	2,451,778	1.26	1.06
0.015 (1)	379	378	376	378	103.0	3	4	2	6	2	2	4	1	4	4	32	2,769,556	1.16	0.97
0.015(2)	320	311	303	311	84.9	6	5	4	5	1	3	1	4	2	2	33	2,283,111	1.45	1.21
0.025 (1)	310	266	301	292	79.7	4	6	2	0	5	5	3	5	2	4	36	2,143,778	1.68	1.41
0.025 (2)	381	386	375	381	103.8	1	4	3	5	2	4	2	2	3	5	31	2,791,556	1.11	0.93
0.03 (1)	259	302	303	288	78.5	3	5	4	1	4	4	2	5	4	5	37	2,112,000	1.75	1.47
0.03 (2)	302	380	389	357	97.4	4	3	4	4	2	5	3	4	6	4	39	2,618,000	1.49	1.25
0.04 (1)	346	324	351	340	92.8	4	5	4	2	4	2	3	3	2	4	33	2,495,778	1.32	1.11
0.04 (2)	346	342	358	349	95.1	3	3	2	7	4	2	2	4	4	9	40	2,556,889	1.56	1.31
EMS50	320	328	286	311	84.9	40	46	45	40	51	41	38	46	39	47	433	2,283,111	18.97	15.94
EMS100	336	326	301	321	87.5	107	104	104	114	120	90	105	105	100	90	1 039	2,354,000	44.14	37.09

∑M = sum of mutants in all 5 dishes

NPC = no. of planted cells

MF/1E+05 cells = mutation frequency

M_t/M_sc = no. of mutants in test conc. vs no. of mutants in solvent control

Mutagenicity with metabolic activation, 3h treatment

Conc. mg/ml	Viability (number of colonies)			avg	PE %	Mutants (number of colonies)										∑M	NPC	MF/1E+05 cells	Mt/Msc
NC (1)	371	385	385	380	112.5	9	1	8	8	7	7	4	7	1	7	59	2,789,111	2.12	1.12
NC (2)	283	295	310	296	87.5	1	4	2	5	3	2	5	4	2	7	35	2,170,667	1.61	0.85
0.02 (1)	399	402	363	388	114.7	4	6	5	5	5	6	6	4	7	5	53	2,845,333	1.86	0.98
0.02 (2)	257	291	277	275	81.3	2	3	2	2	5	2	6	3	2	4	31	2,016,667	1.54	0.81
0.05 (1)	362	331	367	353	104.5	1	2	2	5	3	2	5	4	1	2	27	2,591,111	1.04	0.55
0.05 (2)	395	382	360	379	112.1	5	6	6	4	4	4	4	2	3	4	42	2,779,333	1.51	0.80
0.1 (1)	452	442	409	434	128.4	6	3	5	3	4	1	2	6	4	3	37	3,185,111	1.16	0.61
0.1 (2)	376	345	358	360	106.4	3	9	5	6	5	4	1	4	2	9	48	2,637,556	1.82	0.96
DMBA (1)	338	327	340	335	99.1	77	85	98	93	112	95	85	101	82	107	935	2,456,667	38.06	20.08
DMBA (2)	376	345	358	360	106.4	80	93	115	110	100	91	102	83	89	96	959	2,637,556	36.36	19.18

Mutagenicity with metabolic activation, 3h treatment

Conc. mg/ml	Viability (number of colonies)			avg	PE %	Mutants (number of colonies)										∑M	NPC	MF/1E+05 cells	Mt/Msc
NC (1)	340	302	295	312	97.6	1	2	7	4	2	6	3	3	6	1	35	2,290,444	1.53	1.02
NC (2)	327	315	341	328	102.4	4	3	4	3	3	3	3	2	5	5	35	2,402,889	1.46	0.98
0.075 (1)	273	288	282	281	87.8	7	8	13	2	8	4	4	4	4	2	56	2,060,667	2.72	1.43
0.075 (2)	258	275	276	270	84.3	2	2	3	8	1	5	4	4	5	4	38	1,977,556	1.92	1.01
0.1 (1)	400	465	470	445	139.1	5	8	2	3	3	2	5	5	3	4	40	3,263,333	1.23	0.65
0.1 (2)	255	272	300	276	86.1	4	2	8	8	5	6	6	9	6	5	59	2,021,556	2.92	1.54
DMBA (1)	412	326	343	360	112.6	82	81	82	72	89	81	75	81	84	84	811	2,642,444	30.69	16.19
DMBA (2)	342	375	324	347	108.4	70	82	80	87	85	80	74	92	68	64	782	2,544,667	30.73	16.21

Applicant's summary and conclusion

Conclusions:

Test substance Basic Blue 159 trichlorozincate was not mutagenic for V79 cells with as well as without metabolic activation.

Executive summary:

The test item was assayed for mutagenicity in in vitro mammalian cell gene mutation test, according to OECD guideline 476. Chinese hamster V79 lung fibroblasts were used for testing; test substance was dissolved in DMEM. Cytotoxicity tests without as well as with metabolic activation were performed to provide the optimal dose range for the mutagenicity study. Concentrations used for cytotoxicity testing were in the range of 0.0005 – 2.0 mg/mL.

On the basis of cytotoxicity testing results, six concentrations were chosen for the mutagenicity experiment without metabolic activation, i.e. 0.01, 0.015, 0.020, 0.025, 0.03 and 0.04 mg/mL. Four concentrations 0.02, 0.05, 0.1 and 0.2 mg/mL were used for the experiment with metabolic activation. The concentration of 0.2 mg/mL was completely toxic and in the concentration 0.1 mg/mL insufficient number of cells was transferred for further cultivation. An additional experiment was performed with the concentration of 0.075 mg/mL and the concentration 0.1 mg/mL was repeated to get four analysable concentrations according to guidelines.

The metabolic activation was performed by S9 fraction of rat liver homogenate and mixture of cofactors. Concurrent positive controls verified the sensitivity of the assay and the metabolising activity of the liver preparations. Average mutant colony counts for the vehicle controls were within the current historical control range for the laboratory.

Under the experimental conditions, test substance was non-mutagenic for V79 cells with and without metabolic activation.