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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-compliant guideline study available as unpublished report, no restrictions, fully adequate for assessment.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Qualifier:
according to
Guideline:
other: ICH Guidance S2(R1): Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use, June 2012
GLP compliance:
yes (incl. certificate)
Remarks:
Toxi-Coop Zrt., Arácsi út 97., 8230 Balatonfüred, Hungary
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Petol PM 410-4N
- Physical state: yellow-brown (apparently yellow), viscous, clear liquid
- Storage condition of test material: Store in tightly closed container, in a dry and well ventilated area, between 20-30°C.

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital and β-naphthoflavone-induced rat liver
Test concentrations with justification for top dose:
- Concentration Range Finding Test: 5000, 1581, 500, 158, 50, 15.8, 5 μg/plate (Salmonella typhimurium TA98 and TA100)
- Initial Mutation Test: 1000, 316, 100, 31.6, 10, 3.16, 1 μg/plate
- Confirmatory Mutation Test: Salmonella typhimurium strains: -S9 Mix: 100; 31.6; 10; 3.16; 1; 0.32, 0.1 μg/plate;+S9 Mix: 1000; 316; 100; 31.6; 10; 3.16, 1 μg/plate. Escherichia coli WP2 uvrA: 5000, 2500, 1000, 316, 100, 31.6, 10 μg/plate.
- Completed Plate Incorporation Test: 5000, 2500, 1000, 316 μg/plate (Escherichia coli WP2 uvrA)
Vehicle / solvent:
Acetone
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-1,2-phenylene-diamine
Remarks:
Salmonella TA98, Without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Salmonella TA100 and TA1535, Without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Salmonella TA1537, Without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
E.coli WP2 uvrA, Without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2AA)
Remarks:
all strains, With metabolic activation
Details on test system and experimental conditions:
METHOD
- Procedure for the Preliminary Range Finding Test, Initial Mutation Test, and Completed Plate Incorporation Test: Bacteria (cultured in Nutrient Broth No.2.) were exposed to the test item both in the presence and absence of an appropriate metabolic activation system. Molten top agar was prepared and kept at 45°C. 2 mL of top agar was aliquoted into individual test tubes (3 tubes per control or concentration level). The equivalent number of minimal glucose agar plates was properly labeled. The test item and other components were prepared fresh and added to the overlay (45°C). The typical content of the tubes: top agar 2000 μL; vehicle or solution of test item or reference controls 50 μL; over night culture of test strain 100 μL phosphate buffer (pH: 7.4) or S9 mix 500 μL. This solution was mixed and poured on the surface of the properly labelled minimal agar plates (3 plates per control or concentration level). For activation studies, instead of phosphate buffer, 0.5 mL of the S9 Mix was added to each overlay tube. The entire test consisted of non-activated and activated test conditions and each of them with the addition of negative and positive controls. The plates were incubated at 37°C for 48 hours.
- Procedure for the Confirmatory Mutation Test: A pre-incubation procedure was performed in the Confirmatory Mutation Test because of the unequivocal negative results of the Initial Mutation Test. Before the overlaying of the test item, the bacterial culture and the S9 mix or phosphate buffer was added into appropriate tubes to provide direct contact between bacteria and the test item (in its vehicle). These tubes were gently mixed and incubated for 20 min at 37°C in a shaking incubator. After the incubation period, 2 mL of molten top agar was added to the tubes, the content mixed and poured onto minimal glucose agar plates as described for the standard plate incorporation method. The entire test consisted of non-activation and activation test conditions and each of them with the addition of negative and positive controls. After preparation the plates were incubated at 37°C for about 48 hours.

NUMBER OF REPLICATIONS: 2
Evaluation criteria:
- A test item is considered mutagenic if a dose–related increase in the number of revertants occurs and/or a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
- An increase is considered biologically relevant if in strain Salmonella typhimurium TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control and in strain TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.
- A test item is considered non-mutagenic if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
- Range-Finding Test (Informatory Toxicity Test): Strong inhibitory effect of the test item was observed in both strains examined (Salmonella typhimurium TA98 and TA100). No bacterial growth indicated the cytotoxic effect in S. typhimurium TA100, at 5000 μg/plate, without metabolic activation (-S9 Mix).
- Initial Mutation Test (Plate Incorporation Test): All of the obtained revertant colony number increases (in comparison with the vehicle controls) remained in the actual historical control data ranges, and have no biological significance. The revertant colony counts remained in the vehicle control data ranges, no increases or decreases were observed; however were above the corresponding historical control data ranges in S. typhimurium TA98 at the concentrations of 31.6 and 1 μg/plate, with addition of metabolic activation (+S9 Mix). Inhibitory effect of the test item was observed in all examined Salmonella typhimurium strains. No revertant growth was obtained in all Salmonella typhimurium strains at 1000 μg/plate, and in S. typhimurium TA1535 and TA1537 at 316 μg/plate, without metabolic activation (-S9 Mix). The decreased revertant colony numbers (in comparison with the revertant colony numbers of the vehicle controls) were below the corresponding historical control data ranges and additionally reduced or slightly reduced background lawn development was observed at 1000 μg/plate in S. typhimurium TA98, TA100 and TA1537, with addition of metabolic activation (+S9 Mix); at 316 μg/plate in TA98, without metabolic activation (-S9 Mix), in TA100, with and without metabolic activation (±S9 Mix); furthermore at 100 μg/plate in TA100 (-S9 Mix). The lower revertant colony numbers (than the revertant colony numbers of the vehicle control) were below the corresponding historical control data range in S. typhimurium TA98, at 100 μg/plate (-S9 Mix). The lower revertant colony numbers remained in the historical control data range, however slightly reduced background lawn development indicated the inhibitory effect of the test item in S. typhimurium TA1537 at 100 μg/plate (-S9 Mix) and in TA1535 at 316 μg/plate (+S9 Mix). In S. typhimurium TA1535 at 1000 μg/plate (+S9 Mix) pinpoint colonies and reduced background lawn development showed the inhibitory effect of the test item. All other obtained lower revertant colony numbers than the revertant colony numbers of the vehicle control plates remained in the corresponding historical control data ranges and were considered as reflect the biological variability of the applied test system.
- Confirmatory Mutation Test (Pre-Incubation Test): All of the observed revertant colony number increases were of minor intensity, far below the biologically relevant thresholds for being positive. The obtained increases remained in the historical control data ranges and were considered as to reflect the biological variability of the test system. In S. typhimurium TA1537 the obtained revertant colony numbers remained in the vehicle control data range; however were slightly above the historical control data range, without any biological significance, at 1 μg/plate in absence of metabolic activation (-S9 Mix). The inhibitory effect of the test item following the pre-incubation procedure was also observed. No revertant growth and reduced background lawn development was observed in S. typhimurium TA100, TA1535 and TA1537 at 1000 μg/plate (+S9 Mix). The obtained low (in comparison with the revertant colony numbers of the vehicle control) revertant colony numbers were below the corresponding historical control data ranges and reduced or slightly reduced background lawn development was observed at 5000 μg/plate in E. coli WP2 uvrA (±S9 Mix), at 1000 μg/plate in S. typhimurium TA98 (+S9 Mix), at 316 μg/plate in TA100 (+S9 Mix), at 100 μg/plate in TA98, TA100 (-S9 Mix) and at 31.6 μg/plate in TA100 (-S9 Mix). The lower revertant colony numbers remained in the corresponding historical control data ranges, but reduced background lawn development indicated the inhibitory effect of the test item in S. typhimurium TA1537 at 100 μg/plate (-S9 Mix). The revertant colony numbers remained in the vehicle control range, but the affected background lawn development (reduced background lawn development) indicated the inhibitory effect of the test item in S. typhimurium TA1535, at 100 μg/plate, without metabolic activation (-S9 Mix). As sign of the inhibition, the lower revertant colony numbers (than the revertant colony numbers of the vehicle control) were below the corresponding historical control data range in E. coli WP2 uvrA, at 2500 μg/plate (-S9 Mix). All of the other obtained lower revertant colony numbers than the revertant colony numbers of the vehicle control plates remained in the corresponding historical control data ranges.ntrol data ranges3) and were considered as to reflect the biological variability of the test system.
- Completed Plate Incorporation Test: The originally planned concentration range (for the Initial Mutation Test) was not appropriate in the case of Escherichia coli WP2 uvrA where the highest examined concentration level should have been in the cytotoxic concentration range or 5000 μg/plate. Therefore a short completion of this experimental part was necessary. This experiment was carried out using Escherichia coli WP2 uvrA in absence and also in the presence of metabolic activation (±S9 Mix) with appropriate positive and negative controls. The revertant colony numbers were higher than the revertant colony numbers of the vehicle control (without any biological significance), however the higher colony counts remained in the historical control data range at the concentration of 316 μg/plate (+S9 Mix). Unequivocal inhibitory effect of the test item was noticed at the highest two concentration levels of 5000 and 2500 μg/plate, without and with addition of exogenous metabolic activation (±S9 Mix). The revertant colony numbers were lower than the revertant colony numbers of the vehicle control and were below the historical control data ranges at these concentration levels (±S9 Mix). Additionally slightly reduced background lawn development was observed, without metabolic activation. Lower revertant colony numbers (lower than the revertant colony numbers of the vehicle control) within the historical control data range were obtained at 1000 μg/plate (-S9 Mix).
- Controls: The spontaneous revertant colony numbers of the acetone vehicle control plates showed mostly the characteristic mean numbers agreed with the actual historical control data ranges. In the Initial Mutation Test in Escherichia coli WP2 uvrA in presence of metabolic activation (+S9 Mix) the spontaneous revertant colony numbers of the acetone vehicle control plates were slightly above the actual laboratory’s historical control data range. The higher counts were evaluated as acceptable without any influence on the final conclusion of the study. The reference mutagen treatments (positive controls) showed the expected, biological relevant increases in induced revertant colonies in all experimental phases, in all tester strains. The revertant colony numbers of the untreated, DMSO and ultrapure water control plates in the different experimental phases were slightly higher or lower than the vehicle control plates, but these higher or lower revertant counts of these controls remained in the corresponding historical control data ranges.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The test substance is not mutagenic in the bacterial reverse mutation test (Ames test) with or without metabolic activation.
Executive summary:

Petol PM 410-4N was examined for mutagenic activity in the bacterial reverse mutation test (GLP compliant and according to OECD guideline 471) using the histidine-requiring Salmonella typhimuriumstrains TA1535, TA1537, TA98, TA100 and the tryptophan-requiring Escherichia coli strain WP2uvrA, in the absence and presence of S9-mix. In the initial mutation test (plate incorperation test) the following concentrations were tested:1000, 316, 100, 31.6, 10, 3.16 and 1 μg/plate based on the preliminary range finding test.Because of the observed cytotoxic effect of the test item (in the Salmonella typhimurium strains) the above concentrations were modified in the Confirmatory Mutation Test (Pre Incubation Test). The following concentrations were examined in the Salmonella typhimurium strains: -S9 Mix: 100; 31.6; 10; 3.16; 1; 0.32 and 0.1 μg/plate, +S9 Mix: 1000; 316; 100; 31.6; 10; 3.16 and 1 μg/plate. No inhibition was obtained in the Initial Mutation Test in the case of Escherichia coli WP2 uvrA. Therefore, the following modified concentration range was examined in the Confirmatory Mutation Test for the E. Coli strain: 5000; 2500; 1000; 316; 100; 31.6 and 10 μg/plate. In the Initial Mutation Test in Escherichia coli WP2 uvrA the examined concentration range did not fulfill the guideline criterion as the highest examined concentration level was 1000 μg/plate and was not unequivocally a cytotoxic concentration, therefore a short completion of this experimental part was necessary (Completed Plate Incorporation Test). In the Completed Plate Incorporation Test, the concentrations were: 5000; 2500; 1000 and 316 μg/plate.No substantial increases were observed in revertant colony numbers of any of the five test strains following treatment with Petol PM 410-4N at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments.The positive controls showed the expected, biological relevant increases in induced revertant colonies in all experimental phases, in all tester strains. The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. In conclusion, the test item Petol PM 410-4N has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.