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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial Mutagenicity assay (Ames)

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:
August 08, 2016 to October 22, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: The Dow Chemical Company, Midland, Michigan, Lot no. ZA01202016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: As per the instruction received from the Sponsor on storage of the test item, the test item was stored at:
Storage Temperature : Ambient condition
Other specification : No Specification required
Storage Container : In original container as supplied by theSponsor
Storage Location : Test Item Control Office, JRF
Target gene:
Histidine loci in the tester strains of Salmonella typhimurium i.e., TA1537, TA1535, TA98, TA100 and at tryptophan locus in Escherichia coli WP2uvrA (pKM101).
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S-9 homogenate
Test concentrations with justification for top dose:
In the initial toxicity-mutation assay, the maximum concentration tested was 5000 μg per plate, which is the maximum concentration recommended by test guidelines. The concentrations tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg per plate. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Precipitate was not observed with any of the tester strains in either the presence or absence of S9 activation. Toxicity was not observed up to the tested concentration 5000 μg per plate in the absence of S9 activation as well in the presence of S9 activation. Based on the findings of the initial toxicity-mutation assay, the maximum dose plated in the confirmatory mutation assay was 5000 μg per plate.
In the confirmatory mutation assay, the concentrations tested were 156.25, 312.5, 625, 1250, 2500 and 5000 μg per plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: Acetone was selected as the solvent of choice based on the prestudy evaluation. The test item formed a clear solution in acetone at 100 mg/mL.

Solubility and Precipitation Test:
Prestudy evaluation was conducted to determine the solubility of the test item before commencement of the study. Test item was found insoluble in dimethyl sulfoxide while was soluble in acetone (100 mg/mL).
Precipitation was checked during initial toxicity mutation assay. Precipitation was not observed at the concentration of 5000 μg/plate.
Untreated negative controls:
yes
Remarks:
Distilled Water
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
other: 9-Aminoacridine hydrochloride hydrate (CAS N° 52417-22-8) ; 2-aminoanthracene (613-13-8)
Details on test system and experimental conditions:
METHOD OF APPLICATION: Preincubation assay

Number of Replicates:
There will be two replicates for the initial toxicity-mutation assay and three replicates for the confirmatory mutation assay.

Plating Procedure"
The initial toxicity-mutation, as well as the confirmatory mutation assay will be conducted using the preincubation assay method.
A. Presence of metabolic activation
a) 50 or 100 μL test dose/vehicle/appropriate positive control
b) 100 μL bacterial culture
c) 500 μL S-9 mix

B. Absence of metabolic activation
a) 50 or 100 μL test dose/vehicle/appropriate positive control
b) 100 μL bacterial culture
c) 500 μL of 0.2 M phosphate buffer

These test constituents will be transferred into sterile test tubes and will be kept in an incubator shaker for approximately 20 ± 2 minutes at 37 ± 1 ºC. After this period, 2 mL of soft agar containing histidine-biotin / tryptophan will be added to each of the tubes. These constituents will be overlaid onto VB agar plates. After the soft agar sets, the plates will be incubated at 37 ± 1°C for 48 to 72 hours.

Justification for Selection of the Test System:
This assay measures the ability of the test item to induce reverse mutations at specific histidine loci in the tester strains of Salmonella typhimurium i.e., TA1537, TA1535, TA98, TA100 and at tryptophan locus in Escherichia coli WP2uvrA (pKM101), which are known for their reliability and reproducibility in a short term mutagenicity assay and are also recommended by the OECD, EPA, EC and other guidelines.
Rationale for test conditions:
In the initial toxicity-mutation assay, the maximum concentration tested was 5000 μg per plate, which is the maximum concentration recommended by test guidelines. The concentrations tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg per plate. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Precipitate was not observed with any of the tester strains in either the presence or absence of S9 activation. Toxicity was not observed up to the tested concentration 5000 μg per plate in the absence of S9 activation as well in the presence of S9 activation. Based on the findings of the initial toxicity-mutation assay, the maximum dose plated in the confirmatory mutation assay was 5000 μg per plate.
In the confirmatory mutation assay, the concentrations tested were 156.25, 312.5, 625, 1250, 2500 and 5000 μg per plate.
Evaluation criteria:
Assay Evaluation Criteria:
Once criteria for a valid assay have been met, responses observed in the assay were evaluated. The conditions necessary for determining a positive result were that there should be a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing doses of the test article either in the absence or presence of the metabolic activation system.

Strains TA98, TA1535, and TA1537:
Data sets were judged positive, if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0-times the mean negative control value.

Strains TA100 and Escherichia coli WP2uvrA (pKM101):
Data sets were judged positive, if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0- times the mean negative control value.

A response that did not meet all three of the above criteria (magnitude, concentration-responsiveness, reproducibility) was determined to be non mutagenic.
Statistics:
No statistical information in report.
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
All doses tested.
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
All doses tested.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
All doses tested.
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
All doses tested.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
All doses tested.
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
All doses tested.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
All doses tested.
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
All doses tested.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
All doses tested.
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
All doses tested.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Negative Controls:
In both the initial toxicity-mutation assay and the confirmatory mutagenicity assay, the values of the vehicle and negative controls (acetone and sterile distilled water) in all strains were within limits of historical range of distilled water. Results of the concurrent vehicle control (acetone) were comparable with the results of concurrently run additional negative control (distilled water).

Positive Controls:
2-Aminoanthracene was used as the positive control in the presence of metabolic activation for all the tester strains during the Initial Toxicity Mutation Assay and Confirmatory Mutation Assay. Historical control data of this laboratory proved the efficiency and suitability of 2-aminoanthracene as a positive control in the presence of metabolic activation.
Positive controls (both in the absence and presence of metabolic activation during both the trials) exhibited a clear increase in the number of revertants when compared with the concurrent negative control and were within the historical ranges.This demonstrated the efficiency of the test system and suitability of the procedures employed in the assay.
An increase in the mean number of revertants was not observed in Salmonella typhimurium tester strain TA100 (Initial Toxicity Mutation Assay and Confirmatory Mutation Assay) treated with 2-aminoanthracene in the absence of metabolic activation, but a clear increase was observed in the presence of metabolic activation. This demonstrated the efficiency of the S9 fraction used in this assay.

Initial Toxicity-mutation Test:
In the initial toxicity-mutation assay, the maximum concentration tested was 5000 μg per plate, which is the maximum concentration recommended by test guidelines. This concentration was achieved using a concentration of 100 mg/mL and a plating aliquot of 50 μL. The concentrations tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg per plate (two plates/concentration). Normal growth was observed up to the tested concentration of 5000 μg/plate in all tester strains. Precipitation was not observed up to the tested concentration of 5000 μg/plate. No positive increase in the number of revertant colonies was observed in any of the tester strains at any of the tested concentrations when compared with the concurrent vehicle or negative control. Hence, 5000 μg 1-Phenyl-3-methyl-4-(p-dodecylphenylazo)-5-pyrazolone/plate was selected as the highest concentration to be tested in the Confirmatory Mutation Assay both in the absence and presence of metabolic activation system (10% v/v S9 mix).

Confirmatory Mutation Assay:
In the Confirmatory Mutation Assay, bacterial cultures were exposed to 1-Phenyl-3-methyl-4-(pdodecylphenylazo)-5-pyrazolone at concentrations of 156.25, 312.5, 625, 1250, 2500 and 5000 μg/plate (three plates/concentration) both in the absence and presence of metabolic activation system (10% v/v S9 mix). Normal growth was observed up to the tested concentration of 5000 μg/plate in all tester strains. Precipitation was not observed up to the tested concentration of 5000 μg/plate. No positive increase in the number of revertant colonies was observed in any of the tester strains at any of the tested concentrations when compared with the concurrent vehicle or negative control.
Conclusions:
CONCLUSION:
All criteria for a valid study were met as described in the protocol. From the results of this study, under the specified experimental conditions, 1-Phenyl-3-methyl-4-(p-dodecylphenylazo)-5-pyrazolone was concluded to be non-mutagenic in the Bacterial Reverse Mutation Assay using Salmonella typhimurium TA1537, TA1535, TA98, TA100 strains and Escherichia coli WP2 uvrA (pKM101) strain.
Executive summary:

The potential of the 1-Phenyl-3-methyl-4-(p-dodecylphenylazo)-5-pyrazolone to induce reverse mutations in Salmonella typhimurium (strains: TA1537, TA1535, TA98 and TA100) and a tryptophan deficient strain, Escherichia coli WP2uvrA (pKM101) in the presence or absence of Aroclor-induced rat liver S9 was evaluated in the bacterial reverse mutation test. The assay was performed in two phases by the preincubation method. The first phase, the initial toxicity-mutation assay, was used to establish the dose-range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. The second phase, the confirmatory mutagenicity assay, was used to evaluate and confirm the mutagenic potential of the test item.

Acetone was selected as the solvent of choice based on the prestudy evaluation. The test item formed a clear solution in acetone at 100 mg/mL.

In the initial toxicity-mutation assay, the maximum concentration tested was 5000 μg per plate, which is the maximum concentration recommended by test guidelines. The concentrations tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg per plate. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Precipitate was not observed with any of the tester strains in either the presence or absence of S9 activation. Toxicity was not observed up to the tested concentration 5000 μg per plate in the absence of S9 activation as well in the presence of S9 activation. Based on the findings of the initial toxicity-mutation assay, the maximum dose plated in the confirmatory mutation assay was 5000 μg per plate.

In the confirmatory mutation assay, the concentrations tested were 156.25, 312.5, 625, 1250, 2500 and 5000 μg per plate. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Precipitate was not observed with any of the tester strains in either the presence or absence of S9 activation. Toxicity was not observed up to the tested concentration 5000 μg per plate in the absence of S9 activation, as well in the presence of S9 activation.

The negative controls and positive controls in the initial toxicity-mutation and confirmatory mutation assays were within the acceptable historical ranges and fulfilled the requirements for a valid assay.

All criteria for a valid study were met as described in the protocol. From the results of this study, under the specified experimental conditions, 1-Phenyl-3-methyl-4-(p-dodecylphenylazo)-5-pyrazolone is concluded to be non-mutagenic in the Bacterial Reverse Mutation Assay using Salmonella typhimurium TA1537, TA1535, TA98, TA100 strains and Escherichia coli WP2uvrA (pKM101) strain.

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

Additional information

The potential of the 1-Phenyl-3-methyl-4-(p-dodecylphenylazo)-5-pyrazolone to induce reverse mutations in Salmonella typhimurium (strains: TA1537, TA1535, TA98 and TA100) and a tryptophan deficient strain, Escherichia coli WP2uvrA (pKM101) in the presence or absence of Aroclor-induced rat liver S9 was evaluated in the bacterial reverse mutation test. The assay was performed in two phases by the preincubation method. The first phase, the initial toxicity-mutation assay, was used to establish the dose-range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. The second phase, the confirmatory mutagenicity assay, was used to evaluate and confirm the mutagenic potential of the test item.

Acetone was selected as the solvent of choice based on the prestudy evaluation. The test item formed a clear solution in acetone at 100 mg/mL.

In the initial toxicity-mutation assay, the maximum concentration tested was 5000 μg per plate, which is the maximum concentration recommended by test guidelines. The concentrations tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg per plate. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Precipitate was not observed with any of the tester strains in either the presence or absence of S9 activation. Toxicity was not observed up to the tested concentration 5000 μg per plate in the absence of S9 activation as well in the presence of S9 activation. Based on the findings of the initial toxicity-mutation assay, the maximum dose plated in the confirmatory mutation assay was 5000 μg per plate.

In the confirmatory mutation assay, the concentrations tested were 156.25, 312.5, 625, 1250, 2500 and 5000 μg per plate. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Precipitate was not observed with any of the tester strains in either the presence or absence of S9 activation. Toxicity was not observed up to the tested concentration 5000 μg per plate in the absence of S9 activation, as well in the presence of S9 activation.

The negative controls and positive controls in the initial toxicity-mutation and confirmatory mutation assays were within the acceptable historical ranges and fulfilled the requirements for a valid assay.

All criteria for a valid study were met as described in the protocol. From the results of this study, under the specified experimental conditions, 1-Phenyl-3-methyl-4-(p-dodecylphenylazo)-5-pyrazolone is concluded to be non-mutagenic in the Bacterial Reverse Mutation Assay using Salmonella typhimurium TA1537, TA1535, TA98, TA100 strains and Escherichia coli WP2uvrA (pKM101) strain.

Justification for classification or non-classification

Criteria for classification as a mutagen are not met