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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item resulted to be non-mutagenic in the Bacterial Reverse Mutation Test, up to the highest tested concentration of 1 mg/plate under the tested conditions.

Link to relevant study records
Reference
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 20th, 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
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
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 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.
- Cells growth: a fresh culture of bacteria was grown up to late exponential or early stationary phase of growth.

GENETIC CHARACTERIZATION OF TEST STRAINS
- Checked for histidine requirement: performed.
- Checked for sensitivity to UV radiation: performed.
- Checked for resistance to ampicillin: performed for strains TA 98, TA 100 and TA102.
- Checked for resistance to tetracycline: performed for TA102.
- Checked for rfa mutation: 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: 0.01, 0.03, 0.10, 0.32 and 1 mg/plate
Cytotoxicity assay: 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1 mg/plate
Vehicle / solvent:
- Vehicle used: dimethyl sulphoxide.
- Selection of vehicle: 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.
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
Experiment 1 - plate incorporation method: bacterial suspensions were mixed with soft agar and plated immediately onto minimal glucose agar medium viz., his- for Salmonella typhimurium.
Experiment 2 - preincubation method: the test constituents were mixed with the bacteria inside a tube, incubated in an incubator shaker, mixed with soft agar and plated immediately onto minimal glucose agar medium his- for Salmonella typhimurium.

INOCULUM: the inoculum was adjusted to a density of 18×10^8 cells/ml.

INCUBATION
Experiment 1 - plate incorporation method: plates were incubated at 37 ± 1 °C for 48 hrs and 5 mins.
Experiment 2 - preincubation method: plates were incubated at 37 ± 1 °C for 48 hrs.

NUMBER OF REPLICATIONS: triplicate, both in the presence and absence of metabolic activation along with concurrent vehicle control (dimethyl sulphoxide).

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

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 46 hours and 40 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.
Viable counts for the different bacterial strains on nutrient agar plates were counted manually.

CYTOTOXICITY
- Strain: Salmonella typhimurium TA100
- Cell density: approximately 18 ×10^8 cells/ml.
- Incubation: the plates were incubated at 37 ± 1 ºC for 48 hrs and 11 mins.

PRECIPITATION TEST
Stock solution of test item was serially diluted to get different concentrations of 0.2, 0.3, 0.4, 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.

S9 HOMOGENATE AND ACTIVATION MIXTURE
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 pH 7.33 for initial cytotoxicity and plate incorporation and pH 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.
Species / strain:
S. typhimurium, other: TA98, TA100, TA102, TA1535, and TA1537
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: experiment 1
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: experiment 2
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.

CONTROLS
Plate Incorporation method: experiment 1
The specific positive controls tested simultaneously produced approximately 2.2 to 18.1 fold increase in mean number of revertants as compared to the vehicle control.

Preincubation method: experiment 2
The specific positive controls tested simultaneously produced approximately 2.2 to 18.2 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 46 hrs 40 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.

CYTOTOXICITY
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.

PRECIPITATION TEST
The test item resulted in heavy precipitation at 2, 3, 4 and 5 mg/plate, mild precipitation at 0.9 and 1 mg/plate, minimal precipitation at 0.7, 0.8 mg/plate and no precipitation at 0.2, 0.3, 0.4, 0.5, 0.6 mg/plate.

SOLUBILITY TEST
The test item was soluble in dimethyl sulphoxide at a concentration of 50 mg/ml.
Conclusions:
The test item resulted to be non-mutagenic in the Bacterial Reverse Mutation Test, up to the highest tested concentration of 1 mg/plate under the test conditions
Executive summary:

The test item was evaluated for mutagenicity in Bacterial Reverse Mutation Test as per 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 at a concentration of 50 mg/ml and showed mild precipitation at 1 mg/plate.

On the basis of test item solubility and precipitation tests, the initial cytotoxicity test was performed at 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1 mg/plate. Initial cytotoxicity test was performed with Salmonella typhimurium TA100 both in the presence and absence of metabolic activation system.

The tester strain, Salmonella typhimurium 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 1 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 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 0.01, 0.03, 0.10, 0.32 and 1 mg/plate vehicle control (dimethyl sulphoxide) and appropriate positive controls 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 2.2 to 18.2 fold increase under identical conditions.

Conclusion

Based on the results obtained from the study, it is concluded that the test item resulted to be non-mutagenic in the Bacterial Reverse Mutation Test, up to the highest tested concentration of 1 mg/plate under the test conditions.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

The test item was evaluated for mutagenicity in Bacterial Reverse Mutation Test as per the OECD guideline 471. The test item was soluble in dimethyl sulphoxide at a concentration of 50 mg/ml and showed mild precipitation at 1 mg/plate.

On the basis of test item solubility and precipitation tests, the initial cytotoxicity test was performed at 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1 mg/plate. Initial cytotoxicity test was performed withSalmonella typhimuriumTA100 both in the presence and absence of metabolic activation system.

The tester strain, Salmonella typhimurium 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 1 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 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 0.01, 0.03, 0.10, 0.32 and 1 mg/plate vehicle control (dimethyl sulphoxide) and appropriate positive controls 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 2.2 to 18.2 fold increase under identical conditions.

Justification for classification or non-classification

According to the CLP Regulation (EC ) No 1272/2008, for the purpose of the classification for germ cell mutagenicity, substances are allocated in one of two categories in consideration of the fact that they are:

- substances known to induce heritable mutations or to be regarded as if they induce heritable mutations in the germ cells of humans or substances known to induce heritable mutations in the germ cells of humans or

- substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans.

The available information suggest that test substance did not show any reasons of concern from the genotoxicity point of view.

 

In conclusion, the substance does not meet the criteria to be classified for genetic toxicity according to the CLP Regulation (EC) No 1272/2008.