[2-[4-(dihexylamino)-2-methylbenzylidene]benzo[b]thien-3(2H)-ylidene]malononitrile S,S-dioxide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From January 21st to February 16th, 2018

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 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted on 21st July 1997

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Disperse Blue 354

IUPAC Name:

Disperse Blue 354

Test material form:

solid: particulate/powder

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Molecular Toxicology, Inc. PO Box 1189 Boone, NC 28607 USA.
- Storage of the Test System: stock cultures of tester strains in oxoid nutrient broth no. 2 were stored in the test facility as frozen permanents in -80 ± 10 ºC. Laboratory stocks of each strain were maintained on minimal glucose agar as master plates. The master plates were stored in a refrigerator between 2 to 8 ºC for 3 months.

GENETIC CHARACTERIZATION OF TESTER STRAINS
After preparation of the master plates, the growth requirements and the genetic identity of Salmonella typhimurium strains like histidine requirement, sensitivity to UV radiation, resistance of strains TA 98, TA 100 and TA102 to ampicillin, resistance of TA102 for tetracycline and rfa mutation of Salmonella typhimurium strains were checked along with the range of spontaneous revertants.

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9

Test concentrations with justification for top dose:

Main experiments: 0.009, 0.03, 0.09, 0.3 and 0.9 mg/plate, in the presence and absence of metabolic activation system
Cytotoxicity: 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 and 0.9 mg/plate, in the presence and absence of metabolic activation system

Vehicle / solvent:

- Solvent used: dimethyl sulphoxide.
- Justification for choice of solvent: the solubility test was carried out with distilled water and dimethyl sulphoxide. A quantity of 500 mg of test item was mixed with 10 ml of respective solvents and vortexed. The test item formed uniform suspension in dimethyl sulphoxide at 50 mg/ml.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

other: 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: first experiment plate incorporation; second experiment preincubation method.
- Plate Incorporation Method: test item, vehicle, positive control and the tester strains along with S9/phosphate buffer saline were mixed with 2 ml soft agar and poured on to minimal glucose agar plates.
- Preincubation Method: test item, vehicle, positive control and the tester strains along with S9/Phosphate Buffer Saline were mixed and incubated in an incubator shaker for 30 minutes. Post incubation, the test constituents were mixed with 2 ml soft agar and poured on to minimal glucose agar plates.

INCUBATION
- Plate incorporation method: aplates were incubated at 37 ± 1 °C for 66 hrs and 30 mins.
- Pre incubation method: plates were incubated at 37 ± 1 °C for 71 hrs and 20 mins.
- Cell density: approximately 10^8 viable cells, in both experiments.

TEST STRAINS CULTURES - growth
- Initial cytotoxicity: at 37 ± 1 ºC for 15 hrs and 41 minutes.
- Plate incorporation method: at 37 ± 1 °C for 15 hours.
- Pre incubation method: at 37 ± 1 °C for 10 minutes.
- Cell density: the inoculum was adjusted to a density of 18 × 10^8 cells/ml.

NUMBER OF REPLICATIONS: triplicate plates for five treatment levels, vehicle control and positive control were maintained in the experiment

PRECIPITATION TEST
Stock solution of test item was serially diluted to get different concentrations of 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4 and 5 mg/plate using dimethyl sulphoxide. A quantity of 100 µl of different concentrations of test item was separately mixed with 2 ml of molten soft agar, vortexed and spread onto minimal glucose agar plates. Plates were incubated for 2 hrs at 37 ± 1 °C.

CYTOTOXICITY
Salmonella typhimurium TA100 strain was exposed to test item concentrations, in triplicate, both in the presence and absence of metabolic activation along with concurrent vehicle control (dimethyl sulphoxide).
Each concentration of test item was mixed with soft agar containing histidine and biotin, S9 mix (for presence of metabolic activation), phosphate buffer saline (for absence of metabolic activation), Salmonella typhimurium TA100 of cell density approximately 18 ×10^8 cells/ml and overlaid on to prelabeled minimal glucose agar plates. The plates were incubated at 37±1 ºC for 48 hrs and 5 mins.

VIABLE COUNT
The bacterial suspension of each tester strain was diluted up to 10^-7 in phosphate buffer saline and 1000 µl of the diluted suspension from each tester strain was plated onto nutrient agar plates in triplicate. The plates were incubated at 37 ± 1 ºC for 47 hours and 55 mins for plate incorporation method for pre incubation method. After incubation, the number of colonies in each plate were counted manually and expressed as number of Colony Forming Units per mL (CFU/ml) of the bacterial suspension.

COLONY COUNT OF REVERTANT
Revertant colonies for a respective strain, within the test item dilution series were counted manually.

QUALITY CONTROL PLATES
Control plates with S9 mix, PBS, test item, soft agar, dimethyl sulphoxide and minimal glucose agar plates were evaluated for contamination.

METABOLIC ACTIVATION SYSTEM
The S9 homogenate was prepared from male Wistar rats induced with intraperitoneal injection of sodium phenobarbitone and β-Naphthoflavone at 16 mg/ml and 20 mg/ml respectively, for 3 days prior to sacrifice.
The S9 homogenate was prepared and stored in the test facility at -80 ± 10 ºC until use. Each batch of S9 homogenate was assessed for sterility by streaking the supernatant fluid on Nutrient Agar plates and incubated at 37 ± 1 ºC for 48 hours. It was found sterile and was further evaluated for its protein content and for its ability to metabolize the promutagens 2-Aminoanthracene and Benzo(a)pyrene to mutagens using Salmonella typhimurium TA100. The results were found to be acceptable for the tested parameters.
A volume of 1 ml of S9 homogenate was thawed immediately before use and mixed with 9 ml of co-factor solution containing 4 mM Nicotinamide Adenine Dinucleotide Phosphate (NADP) disodium salt, 5 mM Glucose-6-phosphate, 8 mM MgCl2 and 33 mM KCl in Phosphate Buffer Saline (PBS) of pH 7.32 for initial cytotoxicity and plate incorporation and 7.28 for preincubation method to get the concentration of 10 % (v/v).

ACCEPTABILITY OF THE TEST
The mutation test is considered acceptable as it meets the following criteria:
- all tester strains confirmed to their genetic characteristics.
- the positive controls showed increase in revertant colony numbers of at least twice or thrice the concurrent vehicle control levels with the appropriate bacterial strain.

Evaluation criteria:

The conditions necessary for determining a positive result are: there should be a dose related increase over the range tested and/or a reproducible increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing doses of the test item either in the presence or absence of the metabolic activation system.
The test will be judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than 2 times the mean vehicle control value in Salmonella typhimurium strains TA98, TA100 and TA102 or equal to or greater than 3 times the mean vehicle control value in tester strains TA1535 and TA1537.
An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose responsive increase that does not achieve the respective threshold cited above or a non dose responsive increase that is equal to or greater than the respective threshold cited. A response will be evaluated as negative, if it is neither positive nor equivocal.

Results and discussion

Test results

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 102

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:

Plate Incorporation Method: Trial I
All the tester strains treated with test item, at all the tested concentrations, showed very close effects to the vehicle control one, when tested with and without metabolic activation. There was no appreciable increase in number of revertant colonies and no change in bacterial background lawn when compared to that of the vehicle control, among the tester strains. The mean number of revertant colonies/plate and bacterial background lawn in the treatment groups for the tested strains were comparable to that of vehicle control.

Preincubation Method: Trial II
All the tester strains treated with test item, at all the tested concentrations, showed very close effects to the vehicle control one when tested with and without metabolic activation. There was no appreciable increase in number of revertant colonies and no change in bacterial background lawn when compared to that of the vehicle control, among the tester strains. The mean number of revertant colonies/plate and bacterial background lawn in the treatment groups for the tested strains were comparable to that of vehicle control.

CONTROLS
Plate Incorporation Method: Trial I: the specific positive controls tested simultaneously produced approximately 2.2 to 17.0 fold increase in mean number of revertants as compared to the vehicle control.

Preincubation Method: Trial II: the specific positive controls tested simultaneously produced approximately 2.3 to 16.3 fold increase in mean number of revertants as compared to the vehicle control.

PRECIPITATION TEST
The test item showed heavy precipitation at 3, 4 and 5 mg/plate, moderate precipitation at 1 and 2 mg/plate, mild precipitation at 0.8 and 0.9 mg/plate, minimal precipitation at 0.7 mg/plate and no precipitation at 0.6 mg/plate.

CYTOTOXICITY
The tester strain exposed with test item in the presence and absence of metabolic activation system resulted to be not cytotoxic, when compared to vehicle control.
On the basis of cytotoxicity results 0.9 mg/plate was considered as the highest test concentration for mutation assay.

VIABLE COUNT
Each tester strain was serially diluted to 10^-7 and plated on nutrient agar. After 47 hrs 55 mins of incubation for plate incorporation method and for pre incubation method, the numbers of colonies were counted manually and results were expressed as Colony Forming Units (CFU). Each tester strains resulted in acceptable range of 1 to 2×10^9 CFU/ml.

QUALITY CONTROL PLATES
No microbial contamination was observed.

Applicant's summary and conclusion

Conclusions:

Based on the results obtained from the study, it is concluded that the test item is “non-mutagenic” in the Bacterial Reverse Mutation Test.

Executive summary:

The test item was evaluated for mutagenicity in Bacterial Reverse Mutation Test, according to the OECD guideline for testing of chemicals No. 471, “Bacterial Reverse Mutation Test”, adopted on 21st July 1997.

The test item was soluble in dimethyl sulphoxide and showed mild precipitation at 0.9 mg/plate. Based on these results, the initial cytotoxicity test was performed at 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 and 0.9 mg/plate. Initial cytotoxicity test was performed with TA100 both in the presence and absence of metabolic activation system. The tester strain, TA100 treated with test item, in the presence and absence of metabolic activation system, resulted in no cytotoxicity when compared to vehicle control.

On the basis of cytotoxicity results 0.9 mg/plate was considered as the highest test concentration for mutation assay.

The concentrations tested in the mutation assay were selected based on the results of solubility, precipitation and initial cytotoxicity test. The two independent trials (trial 1 and 2) were conducted by plate incorporation method and pre incubation method in the presence and absence of metabolic activation system. In mutation assay the test item was tested at the concentrations of 0.009, 0.03, 0.09, 0.3 and 0.9 mg/plate Vehicle control (dimethyl sulphoxide) and appropriate positive controls (2-nitrofluorene, sodium azide and 9-Aminoacridine, Mitomycin C for trials “without metabolic activation” and 2-Aminoanthracene for trials “with metabolic activation”) were tested simultaneously.

The tester strains used in the mutation assay were Salmonella typhimurium TA98, TA100, TA102, TA1535 and TA1537.

Based on the experimental results obtained, the mean numbers of revertant colonies at the tested concentrations were comparable to those of the vehicle control, in both the trials, in the presence and absence of metabolic activation. There was no appreciable increase in number of revertant colonies at any of the tested concentrations in both the trials.

The number of revertant colonies in the positive controls resulted in 2.2 to 17.0 fold increase under identical conditions.

Conclusion

Based on the results obtained from the study, it is concluded that the test item is “non-mutagenic” in the Bacterial Reverse Mutation Test up to the highest tested concentration of 0.9 mg/plate under the test conditions.