Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From August 03 to September 29, 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:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted on 21st July 1997
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

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 storage: stock cultures 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. These master plates were stored in a refrigerator between 2 to 8 ºC for 3 months

GENETIC CHARACTERIZATION OF TEST STRAINS
After preparation of the master plates, the growth requirements and the genetic identity of strains like histidine requirement, sensitivity to UV radiation and resistance of strains TA98, TA100, TA1535, TA1537 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:
S9 rat and hamster liver fraction
Test concentrations with justification for top dose:
MUTAGENICITY ASSAY: 0.01, 0.03, 0.10, 0.32 and 1 mg a.i/plate were selected (with half-log dose interval), both experiment I and II, with and without metabolic activation
CYTOTOXICITY: 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 mg a.i/plate
Vehicle / solvent:
- Vehicle: distilled water.
- Justification for choice of /vehicle: the solubility test was carried out with distilled water, dimethyl sulphoxide, ethanol and acetone. A quantity of 500 mg of test item was mixed with 10 ml of respective solvents and vortexed. The test item was found to be non soluble in dimethyl sulphoxide, ethanol and acetone. On the contrary, test item formed suspension with distilled water at 50 mg a.i/ml (1.070 g of test item in 10 ml was taken considering purity correction factor).
Controls
Untreated negative 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
Experiment I - plate incorporation method.
- In presence of metabolic activation: 2.0 ml soft agar containing histidine-biotin, 500 µl S9 mix, 100 µl test concentrations /vehicle/appropriate positive control, 100 µl bacterial culture (containing approximately 10^8 viable cells).
- In absence of metabolic activation: 2.0 ml soft agar containing histidine-biotin, 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: after soft agar set, the plates were incubated at 37±1 ºC for 65 hours and 40 minutes.
- Count: the revertant colonies were counted manually and the plates were examined for bacterial background lawn.

Experiment II - preincubation method.
- In presence of metabolic activation: 500 µl S9 mix, 100 µl test concentrations/vehicle/ appropriate positive control, 100 µl bacterial culture (containing approximately 10^8 viable cells).
- In absence of metabolic activation: 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: preparations were kept in an incubator shaker at 180 rpm for 20 minutes at 37±1 ºC. After the incubation period, 2 ml of molten soft agar containing histidine-biotin for Salmonella typhimurium TA98, TA100, TA102, TA1535 and TA1537 tester strains 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 and 12 minutes.
- Count: the revertant colonies were counted manually and the plates were examined for bacterial background lawn.

NUMBER OF REPLICATIONS: triplicate.

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 24 hours and 15 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.

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, distilled water, dimethyl sulphoxide and minimal glucose agar plates were evaluated for contamination.

PRECIPITATION TEST
Stock solution of test item was serially diluted to get the different concentrations of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40 and 50 mg a.i/ml using distilled water. 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 to achieve the concentrations 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4 and 5 mg a.i/plate. Plates were incubated for 2 hours at 37±1 °C.

CYTOTOXICITY
Based on the results of precipitation test, an initial cytotoxicity test was conducted for the selection of test concentrations for the mutation assay. Salmonella typhimurium TA100 tester strain was exposed to concentrations 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 mg a.i/plate of test item in triplicate, both in the presence and absence of reductive metabolic activation along with concurrent vehicle control (distilled water).
Each concentration of test item was mixed with soft agar containing histidine and biotin, S9 mix (for presence of reductive metabolic activation), phosphate buffer saline (for absence of reductive 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 71 hours.

PREPARATION OF S9 HOMOGENATE AND ACTIVATION MIXTURE
Rat liver S9 homogenate
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 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 tester strain. 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 7.33 for plate incorporation to get the concentration of 10 % (v/v).

Hamster liver S9 homogenate
The S9 homogenate of hamster liver was procured from Molecular Toxicology.
For 75.5 ml S9 homogenate, flavin mononucleotide (FMN) 72.48 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 7.32 for initial cytotoxicity and 7.33 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.
A response is evaluated as negative, as 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
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Plate Incorporation Method - experiment I
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.
The specific positive controls tested simultaneously produced approximately 2.2 to 14.7 fold increase in mean number of revertants as compared to the vehicle control.

Preincubation Method - experiment II
All the tester strains treated with test item showed very close resemblance to the vehicle control when tested with and without metabolic activation. There showed 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 2.3 to 12.5 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. 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.

QUALITY COUNT PLATES
Control plates with S9 mix, phosphate buffer saline, soft agar, 1 mg a.i/plate of test item, distilled water, dimethyl sulphoxide and minimal glucose agar plates incubated at 37±1 °C for 65 hours and 40 minutes for plate incorporation method and for pre incubation method were checked for contamination. No microbial contamination was observed.

SOLUBILITY TEST
The test item formed suspension in distilled water at a concentration of 50 mg a.i/ml.

PRECIPITATION TEST
The test item resulted in heavy precipitation at 3, 4 and 5 mg a.i/plate, moderate precipitation at 2 mg a.i/plate, mild precipitation at 0.9 and 1 mg a.i/plate, minimal precipitation at 0.7 and 0.8 mg a.i/plate, slight precipitation at 0.6 mg a.i/plate and no precipitation at 0.4 and 0.5 mg a.i/plate was observed at the tested concentration.

INITIAL CYTOTOXICITY TEST
Based on the results of the solubility and precipitation tests, the concentrations of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1 mg a.i/plate were selected for the initial cytotoxicity test.
On the basis of cytotoxicity results 1 mg a.i/plate was considered as the highest test concentration for mutation assay.

Any other information on results incl. tables

SUMMARY OF COLONY COUNTS OF REVERTANTS OF EXPERIMENT I - Plate Incorporation Method

Test Concentration  (mg a.i/plate) No. of revertants (mean of 3 plates)
with S9 without S9
Salmonella typhimurium Salmonella typhimurium
TA 98 TA 100 TA 102 TA 1535 TA 1537 TA 98 TA 100 TA 102 TA 1535 TA 1537
100 µl of distilled water  Mean 25.3 107.7 266.3 20.7 9.3 24.3 97.7 261.3 18.3 8.0
±SD 1.5 9.1 7.4 2.5 2.1 3.2 6.5 9.5 2.1 1.0
Lawn Intensity 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+
Test substance 0.01 Mean 24.7 103 264.3 20.0 7.7 22.0 96.3 258.3 18.7 6.0
±SD 2.1 4.6 3.5 2.6 1.5 2.0 1.5 5.7 2.1 1.0
Fold Increase 1.0 1.0 1.0 1.0 0.8 0.9 1.0 1.0 1.0 0.8
Lawn Intensity 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+
Test substance 0.03 Mean 22.0 102.7 263.0 19.0 8.3 21.7 96.7 258.0 16.3 7.0
±SD 1.0 3.2 8.5 2.0 0.6 1.2 3.2 5.6 1.5 1.0
Fold Increase 0.9 1.0 1.0 0.9 0.9 0.9 1.0 1.0 0.9 0.9
Lawn Intensity 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+
Test substance 0.1 Mean 24.3 106.7 263.3 22.0 8.7 21.0 95.7 255.3 18.3 7.0
±SD 1.5 3.5 11.7 2.6 1.5 1.0 3.2 5.9 1.5 1.0
Fold Increase 1.0 1 .0 1.0 1.1 0.9 0.9 1.0 1.0 1.0 0.9
Lawn Intensity 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+
Test substance 0.32 Mean 25.7 107.0 252.7 19.7 11.0 25.0 104.0 252.7 18.7 10.0
±SD 2.1 4.6 4.7 2.5 1.0 2.6 3.0 5.1 2.5 1.0
Fold Increase 1.0 1.0 0.9 1.0 1.2 1.0 1.1 1.0 1.0 .1.3
Lawn Intensity 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+
Test substance 1 Mean 32.3 118.0 245.7 18.3 13.7 27.0 108.3 241.3 18.0 11.0
±SD 2.3 4.0 4.5 2.3 1.5 2.6 3.5 4.2 2.0 1.0
Fold Increase 1.3 1.1 .0.9 0.9 1.5 1.1 1.1 0.9 1.0 1.4
Lawn Intensity 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+
100 µl of respective Positive Control Mean 371.7 393.7 593.7 146.3 115.0 355.3 386.7 591.7 135.3 107.0
±SD 10 11.2 9.7 10.2 9.5 15.5 9.5 15 5.0 5.6
Fold Increase 14.7 3.7 2.2 7.1 12.3 14.6 4.0 2.3 7.4 13.4
Lawn Intensity 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+

Positive controls: With S9 for Salmonella typhimurium TA98, TA100, TA102, TA1535 and TA1537 4 µg/plate of 2-Aminoanthracene. Without S9 for TA98 2 µg/plate of 2-Nitrofluorene; for TA100 and TA1535 1 µg/plate of Sodium azide; for TA102 0.5 µg/plate of Mitomycin C; for TA1537 50 µg/plate of 9-Aminoacridine

Values of Revertants are in Mean ± SD

SUMMARY OF COLONY COUNTS OF REVERTANTS OF EXPERIMENT II - Preincubation Method

Test Concentration  (mg a.i/plate) No. of Revertants (Mean of 3 Plates)
With S9 Without S9
Salmonella typhimurium Salmonella typhimurium
TA 98 TA 100 TA 102 TA 1535 TA 1537 TA 98 TA 100 TA 102 TA 1535 TA 1537
100 µL of Distilled water  Mean 30.7 100.3 265.7 20.3 10.3 28.3 101.0 257.3 18.3 9.7
±SD 2.5 4.0 7.6 3.5 1.5 4.0 4.4 4.2 2.5 1.5
Lawn Intensity 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+
Test substance 0.01 Mean 28.3 99.7 280.0 21.3 8.7 26.7 94.3 268.3 20.7 6.7
±SD 2.1 3.8 8.0 1.5 2.5 1.5 3.1 1.5 1.5 2.1
Fold Increase 0.9 1 1.1 1 0.8 0.9 0.9 1 1.1 0.7
Lawn Intensity 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+
Test substance 0.03 Mean 28.0 94.0 277.3 22.0 9.0 24.0 93.3 271.3 19.3 7.7
±SD 3.0 2.0 2.5 2.0 2.0 2.0 2.1 7.5 1.5 2.1
Fold Increase 0.9 0.9 1.0 1.1 0.9 0.8 0.9 1.1 1.1 0.8
Lawn Intensity 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+
Test substance 0.1 Mean 27.7 101.0 258.7 22.3 8.7 26.7 95.7 261.3 18.3 7.0
±SD 1.5 2.0 6.5 1.2 1.5 0.6 3.2 6.7 2.1 2.6
Fold Increase 0.9 1.0 1.0 1.1 0.8 0.9 0.9 1.0 1.0 0.7
Lawn Intensity 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+
Test substance 0.32 Mean 27.7 102.0 262.7 21.3 8.7 28.0 98.0 261.7 21.0 8.3
±SD 2.1 6.0 8.3 2.1 1.2 3.6 3.6 9.0 3.0 1.2
Fold Increase 0.9 1.0 1.0 1.0 0.8 1.0 1.0 1.0 1.1 0.9
Lawn Intensity 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+
Test substance 1 Mean 35.7 115.7 246.3 26.0 14.3 28.0 109.0 245.7 22.3 10.7
±SD 2.5 7.4 4.0 1.0 1.5 2.0 2.6 3.1 1.5 1.5
Fold Increase 1.2 1.2 0.9 1.3 1.4 1.0 1.1 1.0 1.2 1.1
Lawn Intensity 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+
100 µL of respective Positive Control Mean 380.3 381.7 599.7 150.3 118.0 353.7 381.7 593.3 142.7 111.7
±SD 8.5 10.1 12.0 3.5 8.2 12.5 8.0 12.0 12.7 8.7
Fold Increase 12.4 3.8 2.3 7.4 11.4 12.5 3.8 2.3 7.8 11.6
Lawn Intensity 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+

Positive controls: With S9 for Salmonella typhimurium TA98, TA100, TA102, TA1535 and TA1537 4 µg/plate of 2-Aminoanthracene. Without S9 for TA98 2 µg/plate of 2-Nitrofluorene; for TA100 and TA1535 1 µg/plate of Sodium azide; for TA1020.5µg/plate of Mitomycin C; for TA1537 50 µg/plate of 9-Aminoacridine

Values of Revertants are in Mean±SD

CYTOTOXICITY TEST

Test Item Concentration (mg a.i/plate) No. of revertants/plate
with S9 without S9
R1 R2 R3 Average ±SD Bacterial Lawn Intensity R1 R2 R3 Average ±SD Bacterial Lawn Intensity
Vehicle Control  91 98 105 98 7 4+ 94 101 90 95 5.6 4+
0.1 97 94 104 98 5.1 4+ 91 98 101 97 5.1 4+
0.2 95 101 98 98 3 4+ 94 100 93 96 3.8 4+
0.3 96 93 102 97 4.6 4+ 92 101 96 96 4.5 4+
0.4 91 102 103 99 6.7 4+ 92 93 95 93 1.5 4+
0.5 105 98 96 100 4.7 4+ 97 95 91 94 3.1 4+
0.6 103 101 99 101 2 4+ 97 93 98 96 2.6 4+
0.7 102 97 102 100 2.9 4+ 97 91 102 97 5.5 4+
0.8 101 95 92 96 4.6 4+ 92 96 98 95 3.1 4+
0.9 89 97 93 93 4 4+ 87 90 92 90 2.5 4+
1 86 84 89 86 2.5 4+ 85 81 86 84 2.6 4+

Values of Revertants are in Mean±SD

Lawn intensity:

4+= Thick lawn: Distinguished by a healthy (Normal) background lawn comparable tovehiclecontrol plates.    

Applicant's summary and conclusion

Conclusions:
Based on the results of the study, it is concluded that test item is “non-mutagenic” in the bacterial reverse mutation test up to the highest tested concentration of 1 mg a.i/plate under test conditions.
Executive summary:

The test item was evaluated for mutagenicity in bacterial reverse mutation test, according to OECD guideline 471 (1997).

The test item was found soluble in distilled water at a concentration of 50 mg a.i/ml and resulted in heavy precipitation at 3, 4 and 5 mg a.i/plate; thus, on the basis of test item solubility and precipitation tests results, the initial cytotoxicity test was performed at 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 mg a.i/plate. Initial cytotoxicity test was performed with Salmonella typhimurium TA100 tester strain both in the presence and absence of metabolic activation system.

On the basis of cytotoxicity results 1 mg a.i/plate was considered as the highest test concentration for mutation assay by comparing with the vehicle control.

Two independent trials (trial 1 and 2) were conducted by plate incorporation method (standard metabolic activation system) and pre incubation method (reductive metabolic activation system), in the presence and absence of metabolic activation system at concentrations of 0.01, 0.03, 0.10, 0.32 and 1 mg a.i/plate.

Vehicle control (distilled water) and the 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.

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 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 14.7 fold increase under identical conditions.

Conclusion

Based on test results, it is concluded that test item is “non-mutagenic” in the bacterial reverse mutation test up to the highest tested concentration of 1 mg a.i/plate under test conditions.