Sodium 2-[[4-(cyclohexylamino)-9,10-dihydro-9,10-dioxo-1-anthryl]amino]-5-ethoxybenzenesulphonate

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 December 19th 2017 to January 9th 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted on 21st July 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Acid Blue 280

IUPAC Name:

Acid Blue 280

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: stock cultures of tester strains in oxoid nutrient broth no. 2 were stored in the test facility as frozen permanents in -80 ± 10 °C. Laboratory stocks of each strain were maintained on minimal glucose agar as master plates. These master plates were stored in a refrigerator between 2 to 8 °C for 3 months .
- Culturing: each of the tester strains from the master plates were grown in oxoid nutrient broth no. 2. A fresh culture of bacteria was grown up to late exponential or early stationary phase of growth. The inoculum was incubated in a water bath at 37 ±1 °C for 15 hrs and 7 minutes for initial cytotoxicity, 15 hrs and 14 minutes for plate incorporation method for pre incubation method. The inoculum was adjusted to a density of 18×10^8 cells/ml.

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 homogenate

Test concentrations with justification for top dose:

MAIN EXPERIMENTS Plate Incorporation Method and Preincubation Method: 0.05, 0.16, 0.5, 1.6 and 5 mg/plate, with and without S9
PRELIMINARY CYTOTOXICITY TEST: 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4 and 5 mg/plate of test item, with and without S9

Vehicle / solvent:

- Solvent: the test item formed uniform suspension in dimethyl sulphoxide at 50 mg/ml.

Controls

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: plate incorporation method and pre incubation method.

Plate incorporation
- Plate composition: 2.0 ml soft agar containing histidine-biotin, 500 µl S9 mix or 500 µl Phosphate Buffer Saline (PBS), 100 µl test concentrations /vehicle/ appropriate positive control, 100 µl bacterial culture (containing approximately 10^8 viable cells).
- PIncubation: at 37 ± 1 °C
- Incubation period: 48 hrs and 15 mins.

Preincubation Method
- Plate composition: 500 µl S9 mix or 500 µl Phosphate Buffer Saline (PBS), 100 µl test concentrations /vehicle/ appropriate positive control, 100 µl bacterial culture (containing approximately 10^8 viable cells).
- Incubation: at 37 ± 1 °C
- Incubation period: 30 minutes.
- Post incubation: the test constituents were mixed with 2 ml soft agar and poured on to minimal glucose agar plates.
- Second incubation: plates were incubated at 37 ± 1°C
- Second incubation period: 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 °C for 47 hours and 12 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.

PRELIMINARY CYTOTOXICITY TEST
- Test strains: Salmonella typhimurium TA100.
- Replicates: in triplicate.
- Plate composition: 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 cells and overlaid on to prelabeled minimal glucose agar plates.
- Cell density: approximately 18 × 10^8 cells/ml.
- Incubation consitions: the plates were incubated at 37 ± 1 °C for 48 hrs and 15 mins.
- Incubation period: 48 hrs and 15 mins.

PRECIPITATION TEST
Stock solution of test item was serially diluted to get different concentrations of 0.5, 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.

PREPARATION OF S9 HOMOGENATE AND ACTIVATION MIXTURE
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 °C 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 °C 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 7.33 for initial cytotoxicity and plate incorporation and 7.28 for preincubation method to get the concentration of 10 % (v/v).

CRITERIA FOR 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 98, TA 100, TA 102, TA 1535, and TA 1537

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
All the tester strains treated all the concentrations showed very close resemblance to the vehicle control 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.
The specific positive controls tested simultaneously produced approximately 2.1 to 16.7 fold increase in mean number of revertants as compared to the vehicle control.

Preincubation method
All the tester strains treated at all the concentrations showed very close resemblance to the vehicle control 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.
The specific positive controls tested simultaneously produced approximately 2.1 to 18.3 fold increase in mean number of revertants as compared to the vehicle control.

VIABLE COUNT
Each tester strain was serially diluted to 10^-7 and plated on nutrient agar. After 47 hrs 12 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.

PRELIMINARY CYTOTOXICITY TEST
The tester strain exposed 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 5 mg/plate was considered as the highest test concentration for mutation assay.

PRECIPITATION TEST
The test item resulted in minimal precipitation at 4 and 5 mg/plate and no precipitation up to 0.5 mg/plate.

SOLUBILITY TEST
The test item formed suspension in dimethyl sulphoxide at a concentration of 50 mg/ml.

Applicant's summary and conclusion

Conclusions:

It can be concluded that the test item is “non-mutagenic” in the Bacterial Reverse Mutation Test, up to the highest tested concentration of 5 mg/plate and under the tested conditions

Executive summary:

The test item was evaluated for mutagenicity in Bacterial Reverse Mutation Test, according to OECD guideline 471 (1997).

On the basis of test item solubility and precipitation tests, the initial cytotoxicity test was performed at 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4 and 5 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, didn't show cytotoxicity when compared to vehicle control. On the basis of cytotoxicity results, 5 mg/plate was considered as the highest test concentration for mutation assay.

The test 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 and pre incubation methods in the presence and absence of metabolic activation system. In mutation assay, the test item was tested at the concentrations of 0.05, 0.16, 0.5, 1.6 and 5 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, TA 102, 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 2.1 to 18.3 fold increase under identical conditions.

Conclusion

Based on the results obtained, it can be concluded that the test item is “non-mutagenic” in the Bacterial Reverse Mutation Test, up to the highest tested concentration of 5 mg/plate and under the test conditions.