Reaction mass of sodium 3-[[4-[(4-ethoxy-m-tolyl)azo]-1-naphthyl]azo]benzenesulphonate and sodium 3-[[4-[(4-ethoxyphenyl)azo]-1-naphthyl]azo]benzenesulphonate

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 March 30 to May 19, 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 (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Acid Orange 127

IUPAC Name:

Acid Orange 127

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.
- Stock cultures nutrien broth: oxoid nutrient broth no. 2.
- Storage of stock cultures: frozen permanents in -80 ± 10 ºC. 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 ºC for 3 months.

CULTURES CHECK
The growth requirements and the genetic identity of Salmonella typhimurium strains like histidine requirement, sensitivity to UV radiation, resistance of strains TA98, TA100 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.

PREPARATION OF INOCULUM
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 ºC for 15 hours and 5 minutes for initial cytotoxicity, 15 hours and 14 minutes for plate incorporation method for pre incubation method. The inoculum was adjusted to a density of 18 × 10^8 cells/ml.

Metabolic activation:

with and without

Metabolic activation system:

rat and hamster liver S9 homogenate

Test concentrations with justification for top dose:

Main tests: 0.005, 0.016, 0.05, 0.16 and 0.5 mg/plate
Cytotoxicity test: 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4 and 5 mg/plate

Vehicle / solvent:

- Solvent used: DMSO

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 - experiment I
Plate incorporation method was carried out with the test item, vehicle, positive control and the tester strains along with S9 or phosphate buffer saline were mixed with 2 ml soft agar and poured on to minimal glucose agar plates.
After the soft agar sets, the plates were incubated at 37 ± 1 ºC for 48 hours and 3 minutes.

Preincubation method - experiment II
The test item and the tester strains along with S9 or phosphate buffer saline were transferred into sterile test tubes and were kept in an incubator shaker at 180 rpm for 20 minutes at 37 ± 1 ºC. After the incubation period, 2 ml of soft agar containing histidine-biotin for Salmonella typhimurium was added to each of the tubes. The tube contents were mixed and overlaid onto minimal glucose agar plates. After the soft agar sets, the plates were incubated at 37 ± 1 ºC for 65 hours. The revertant colonies were counted manually and the plates were examined for bacterial background lawn.

INCUBATION
Plate incorporation method: the plates were incubated at 37 ± 1 ºC for 48 hours and 3 minutes.
Preincubation method - experiment II: the plates were incubated at 37 ± 1 ºC for 65 hours.

REPLICATES: triplicate plates for five treatment levels, vehicle control and positive control were maintained in the experiment.

VIABLE TEST 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 25 minutes for plate incorporation method for pre incubation method, respectively. 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.

PRECIPITATION TEST
Stock solution of test item was serially diluted and plated to get the different concentrations of 6, 7, 8, 9, 10, 20, 30, 40 and 50 mg/ml 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 hours at 37 ± 1 °C.

CYTOTOXICITY TEST
Based on the results of precipitation test, an initial cytotoxicity test was conducted. Salmonella typhimurium TA100 strain was exposed to concentrations 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4 and 5 mg/plate of test item 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 70 hours.

METABOLIC ACTIVATION SYSTEM
Experiment I
Sodium phenobarbitone and β-naphthoflavone induced rat liver S9 homogenate was used as the 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 and pH 7.30 plate incorporation to get the concentration of 10 % (v/v).

Experiment II
The S9 homogenate of hamster liver was procured from Molecular Toxicology.
For 75.5 ml S9 homogenate, Flavin mononucleotide (FMN) 72.68 mg in 5.3 ml of water (filter sterilized and kept on ice pack) to a 250 ml conical flask, co-factors were added that have been allowed to thaw at room temperature. 319.4 mg of D-glucose-6-phosphate and 107.2 mg of Nicotine adenine dinucleotide (NADH) were added they were once dissolved; filter sterilization was done through 0.45 µm filter. Aseptically 1 mg of Glucose-6-phosphate dehydrogenase was added to the co-factor mixture in Phosphate Buffer Saline (PBS) pH 7.31 for initial cytotoxicity and 7.30 for preincubation method followed by 22.7 ml of liver S9 fraction. Just prior to performing the experiment the FMN solution was added. The final mixture was kept on ice for the duration of experiment. The post-mitochondrial fraction used at concentration was in the range of 30 % v/v in the S9-mix.

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.

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

Positive controls validity:

valid

Additional information on results:

Plate Incorporation Method
All the tester strains treated with test item 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.

Preincubation Method
All the tester strains treated with test item 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.

VIABLE COUNT
Each tester strain was serially diluted to 10^-7 and plated on nutrient agar. Post 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.

POSITIVE CONTROLS
Plate Incorporation Method: The specific positive controls tested simultaneously produced approximately 2.3 to 17.5 fold increase in mean number of revertants as compared to the vehicle control.
Preincubation Method: the specific positive controls tested simultaneously produced approximately 2.2 to 17.2 fold increase in mean number of revertants as compared to the vehicle control.

CYTOTOXICITY TEST
Test item colour interference was observed after incubation for lawn evaluation at the concentrations of 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4 and 5 mg/plate, therefore these concentrations were not evaluated for mutation test
On the basis of cytotoxicity results 0.5 mg/plate was considered as the highest test concentration for mutation assay.

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

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

Applicant's summary and conclusion

Conclusions:

The test item is “non-mutagenic” in the Bacterial Reverse Mutation Test, up to the highest tested concentration of 0.5 mg/plate and under the test conditions.

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 found soluble in dimethyl sulphoxide at a concentration of 50 mg/ml and resulted in minimal precipitation at 5 mg/plate; thus, 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 Salmonella typhimurium TA100 tester strain both in the presence and absence of metabolic activation system.

After incubation, the test item colouration interferred with the lawn evaluation at the concentrations of 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4 and 5 mg/plate, therefore these concentrations were not evaluated for mutation test.

On the basis of cytotoxicity results 0.5 mg/plate was considered as the highest test concentration for mutation assay by comparing with the vehicle control.

The two independent trials (trial 1 and 2) were conducted by plate incorporation method (standard exogenous metabolic activation system) and pre incubation method (reductive metabolic activation system), in the presence and absence of metabolic activation system. In mutation assay the substance was tested at the concentrations of 0.005, 0.016, 0.05, 0.16 and 0.5 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, 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 17.5 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 0.5 mg/plate and under the test conditions.