1,3-Propanediaminium, 2-hydroxy-N1,N1,N1,N3,N3-pentamethyl-N3-[3-[(2-methyl-1-oxo-2-propen-1-yl)amino]propyl]-, chloride (1:2)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (GLP OECD 471 guideline study, Kr.1): No evidence of mutagenic activity in the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and Escherichia coli WP2 uvrA in the presence or the absence of S9-mix.

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 13 October 2015 to 21 April 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: CY50561001
- Expiration date of the batch: 24/04/2016
- Purity test date: 65.4%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature 15-25°C, below 70 RH%
- Solubility and stability of the test substance in the solvent/vehicle: soluble in water. Analytical determination of the test item concentration, stability and homogeneity was not performed because of the character and the short period of study.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was formulated in the selected vehicle to provide a suitably concentrated stock formulation as follows. The necessary amount of test item was weighed into a calibrated volumetric flask (purity conversion was applied in the study; for this purpose, the 65.4% purity value of the provided substance was taken into consideration in each test). A partial volume of solvent was added and the solution was stirred until homogeneity was reached, then the volume was adjusted to the required level. From the stock solution serial dilutions were prepared to obtain the dosing solutions for lower doses. The stock solutions as well as all dilutions (test formulations) were prepared freshly at the beginning of the experiments in the testing laboratory using the determined vehicle (solvent).
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: N/A
- Final preparation of a solid: N/A

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavone-induced rats.

Test concentrations with justification for top dose:

Concentrations were selected on the basis of the Preliminary Compatibility Test and Preliminary Concentration Range Finding Test (Informatory Toxicity Test). In the Initial Mutation Test and Confirmatory Mutation Test, the same concentrations were used:
- Preliminary Compatibility Test :
The solubility of the test item was examined using Distilled water, Dimethyl sulfoxide (DMSO) and N,N-Dimethylformamide (DMF). The test item was soluble at 100 mg/ml concentration (highest achievable concentration) in all the tested vehicles (clear solution was detected in each case).
- Preliminary Concentration Range Finding Test (Informatory Toxicity Test):
Based on the available information and the solubility and compatibility test, 100 mg/mL stock formulation was prepared in Distilled water, which was diluted in 6 steps by factors of 2, 2.5 and approximately √10. The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98, TA100) were determined at the concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate of the test item.
Test Item Concentrations in the Mutagenicity Tests (Initial Mutation Test and Confirmatory Mutation Test):
Based on the results of the preliminary tests, 100 mg/mL stock formulation was prepared in Distilled water, which was diluted by serial dilutions in several steps to obtain the dosing formulations for lower doses. The maximum test concentration was 5000 μg test item/plate. Examined concentrations in the Initial Mutation Test and Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81 and 5μg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Distilled water
- Justification for choice of solvent/vehicle: The solubility of the test item was examined using Distilled water, Dimethyl sulfoxide (DMSO) and N,N-Dimethylformamide (DMF). The test item was soluble at 100 mg/ml concentration in all the tested vehicles (clear solution was detected in each case). Due to the better biocompatibility, Distilled water was selected as vehicle (solvent) for the study.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Distilled water and DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene and 4-nitro-1,2-phenylene-diamine

Details on test system and experimental conditions:

METHOD OF APPLICATION: In the Preliminary Concentration Range Finding Test as well as in the Initial Mutation Test, the plate incorporation method was used. In the Confirmatory Mutation Test, the pre-incubation method was used.
- Cell density at seeding (if applicable): Not applicable

DURATION
- Preincubation period: 20 min at 37ºC
- Exposure duration: 48±1 hours at 37ºC
- Expression time (cells in growth medium): N/A
- Selection time (if incubation with a selection agent): N/A
- Fixation time (start of exposure up to fixation or harvest of cells): N/A
SELECTION AGENT (mutation assays): N/A
SPINDLE INHIBITOR (cytogenetic assays): N/A
STAIN (for cytogenetic assays): N/A

NUMBER OF REPLICATIONS: In the main tests, each sample (including the controls) was tested in triplicate.

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: N/A
NUMBER OF CELLS EVALUATED: N/A
CRITERIA FOR MICRONUCLEUS IDENTIFICATION: N/A
DETERMINATION OF CYTOTOXICITY: N/A

OTHER EXAMINATIONS:
- Determination of polyploidy: N/A
- Determination of endoreplication: N/A
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): N/A

Evaluation criteria:

The colony numbers on the untreated / negative (vehicle/solvent) / positive control and test item treated plates were determined by manual counting. Visual examination of the plates was also performed; precipitation or signs of growth inhibition (if any) were recorded and reported. The mean number of revertants per plate, the standard deviation and the mutation factor* values were calculated for each concentration level of the test item and for the controls using Microsoft ExcelTM software.

Criteria for Validity:
The study was considered valid if:
- the number of revertant colonies of the negative (vehicle/solvent) and positive controls were in the historical control range in all strains of the main tests;
- at least five analyzable concentrations were presented in all strains of the main tests.
Criteria for a Positive Response:
A test item was considered mutagenic if:
- a dose–related increase in the number of revertants occurred and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurred in at least one strain with or without metabolic activation.
An increase was considered biologically relevant if:
- the number of reversions is more than two times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA bacterial strains;
- the number of reversions is more than three times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA1535 and TA1537 bacterial strains.

Criteria for a Negative Response:
The test item was considered to have shown no mutagenic activity in this study 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.

Statistics:

No statistics performed.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

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

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity reported in the confirmatory Mutation Test at 5000 μg/plate concentration without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

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

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity reported in the confirmatory Mutation Test at 5000 μg/plate concentration without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

INITIAL AND CONFIRMATORY MUTATION TESTS:

In the Initial Mutation Test (using the plate incorporation method), the highest revertant rate was observed in Salmonella typhimurium TA1535 bacterial strain at 50 μg/plate concentration with metabolic activation (the mutation factor value was 1.79). However, there was no dose response, the observed mutation factor value was below the biologically relevant threshold limit of 3 and the numbers of revertant colonies were within the historical control range.

In the Confirmatory Mutation Tests (using the pre-incubation method), the highest revertant rate was observed in Salmonella typhimurium TA1537 bacterial strain at 158.1 μg/plate concentration without metabolic activation. The observed mutation factor value was 2.50. However, there was no dose response, the observed mutation factor value was below the biologically relevant threshold limit of 3 and the numbers of revertant colonies were within the historical control range. Furthermore, increased number of revertant colonies compared to the Distilled water control (MF=1.83) was observed for untreated control also in this strain indicating higher than usual variability.

Higher numbers of revertant colonies compared to the vehicle (solvent) control were detected in the main test in some other sporadic cases. However, no dose-dependence was observed in those cases and they were below the biologically relevant threshold value. The numbers of revertant colonies were within the historical control range in each case, so they were considered as reflecting the biological variability of the test.

Sporadically, lower revertant counts compared to the vehicle (solvent) control were observed in the main test at some non-cytotoxic concentrations. However, no background inhibition was recorded and the mean numbers of revertant colonies were in the historical control range in all cases, thus they were considered as biological variability of the test system.

Inhibitory, cytotoxic effect of the test item (slightly reduced background lawn development) was observed in the Confirmatory Mutation Test in Salmonella typhimurium TA100 and TA1537 strain at 5000 μg/plate concentration without metabolic activation

No precipitate was detected in the Initial Mutation Test and Confirmatory Mutation Test.

Conclusions:

The test item Diquat was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay. The experiments were carried out using Salmonella typhimurium TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2 uvrA)in the presence and absence of a metabolic activation system (S9). In the Preliminary Concentration Range Finding Test as well as in the Initial Mutation test the plate incorporation method was used. In the Confirmatory Mutation Test, the pre-incubation method was used.
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 Diquat had no mutagenic activity in the examined bacterial strains under the test conditions of this study.

Executive summary:

The test item Diquat was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay according to OECD Test Guideline No.471.

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavone-induced rats.

The study included a Preliminary Compatibility Test, a Preliminary Concentration Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Method) and a Confirmatory Mutation Test (Pre-Incubation Method).

Based on the results of a solubility test, the test item was formulated in Distilled water. Concentrations of 5000; 2500; 1000; 316; 100, 31.6 and 10 μg/plate were examined in the Preliminary Concentration Range Finding Test. Based on the results of the Range Finding Test, the test item concentrations in the Initial Mutation and Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate (purity conversion was applied in the study).

In the Initial Mutation Test and Confirmatory Mutation Test, none of the observed revertant colony numbers were above the respective biological threshold value. There were no consistent dose-related trends and no indication of any treatment effect. In all test item treated groups, the numbers of revertant colonies were below the biological relevance when compared to the solvent controls.

Inhibitory, cytotoxic effect of the test item (slightly reduced background lawn development) was observed in the Confirmatory Mutation Test in Salmonella typhimurium TA100 and TA1537 strains at 5000 μg/plate concentration without metabolic activation.

No precipitate was detected in the Initial Mutation Test and Confirmatory Mutation Test.

The mean values of revertant colonies of the solvent control plates were within the general historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analyzable concentrations were presented in all strains of the main tests, the examined concentration range was considered to be adequate. The tests were considered to be valid.

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 Diquat had no mutagenic activity in the examined bacterial strains under the test conditions of this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

A GLP-compliant Ames test conducted according to OECD TG 471 (of reliability 1 according to Klimisch cotation criteria) is available and was selected as key study (CIToxLAB, 2016). The registered substance, formulated in distilled water, did not induce gene mutations by base pair changes or frameshifts in the genome of Salmonella typhimurium TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2 uvrA in the presence and absence of a exogenous metabolic activation system (S9 fraction) at any dose levels (up to 5000 µg/plate). The registered substance Diquat is therefore considered not to be mutagenic in this Ames test.

Justification for classification or non-classification

The registered substance had no mutagenic activity in Salmonella typhimurium TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2 uvrA in the presence and absence of a exogenous metabolic activation system and is therefore not classified for genotoxicity according to GHS and CLP regulations.

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