O-(ethoxycarbonyl)-N-(1-methyl-2-oxo-2-phenylethylidene)hydroxylamine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 March 2017 to 31 March 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

O-(ethoxycarbonyl)-N-(1-methyl-2-oxo-2-phenylethylidene)hydroxylamine

EC Number:

265-967-5

EC Name:

O-(ethoxycarbonyl)-N-(1-methyl-2-oxo-2-phenylethylidene)hydroxylamine

Cas Number:

65894-76-0

Molecular formula:

C12H13NO4

IUPAC Name:

O-(ethoxycarbonyl)-N-(1-methyl-2-oxo-2-phenylethylidene)hydroxylamine

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: Off white powder

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING:
- The test material was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer on the day of each experiment.
- No correction for purity was required.
- Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino silicate pellets with a nominal pore diameter of 4 x 10^-4 microns.

Method

Target gene:

- Histidine requirement in the Salmonella typhimurium strains (Histidine operon).
- Tryptophan requirement in the Escherichia coli strain (Tryptophan operon).

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

-All of the strains were stored at approximately -196 °C in a Statebourne liquid nitrogen freezer, model SXR 34.
-In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth (Oxoid Limited; lot number 1865318 05/21) and incubated at 37 °C for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.

MEDIA USED
Top agar was prepared using 0.6 % Bacto agar and 0.5% sodium chloride with 5 mL of 1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan solution added to each 100 mL of top agar. Vogel-Bonner Minimal agar plates were purchased from SGL Ltd.

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

-All of the strains were stored at approximately -196 °C in a Statebourne liquid nitrogen freezer, model SXR 34.
-In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth (Oxoid Limited; lot number 1865318 05/21) and incubated at 37 °C for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.

MEDIA USED
Top agar was prepared using 0.6 % Bacto agar and 0.5% sodium chloride with 5 mL of 1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan solution added to each 100 mL of top agar. Vogel-Bonner Minimal agar plates were purchased from SGL Ltd.

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix

Test concentrations with justification for top dose:

Test for Mutagenicity: Experiment 1 - Plate Incorporation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate
Test for Mutagenicity: Experiment 2 – Pre-Incubation Method: 15, 50, 150, 500, 1500 and 5000 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (dimethyl sulphoxide)
- Justification for choice of solvent/vehicle: The test material was insoluble in sterile distilled water at 50 mg/mL but was fully soluble in dimethyl sulphoxide at the same concentration in solubility checks performed in–house. Dimethyl sulphoxide was, therefore, selected as the vehicle.

Controls

Untreated negative controls:

yes

Remarks:

Top agar and histidine/biotin or tryptophan in the absence of S9-mix; top agar and histidine/biotin or tryptophan in the presence of S9-mix; and the maximum dosing solution of the test item in the absence of S9-mix only (test in singular only).

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene in DMSO with metabolic activation at 1 µg/plate for Salmonella strain TA100, at 2 µg/plate for Salomnella strains TA1535 and TA 1537 and at 10 µg/plate for E. coli strain WP2uvrA.

Details on test system and experimental conditions:

EXPERIMENT 1: PLATE INCORPORATION METHOD
- The test item was tested using the following method. The maximum concentration was 5000 μg/plate (the maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
- Without metaolic activation: 0.1 mL of the appropriate concentration of test material, solvent vehicle or appropriate positive control was added together with 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer to 2 mL of molten, trace amino-acid supplemented media. These were then mixed and overlayed onto a Vogel-Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test.
- With metabolic activation: The same procedure was used for testing with metabolic activation except that following the addition of the test material formulation and bacterial culture, 0.5 mL of S9-mix was added to the molten, trace amino-acid supplemented media instead of phosphate buffer.


EXPERIMENT 2: PRE-INCUBATION METHOD
- As the result of Experiment 1 was deemed negative, Experiment 2 was performed using the pre-incubation method in the presence and absence of metabolic activation.
- Concentrations: 15, 50, 150, 500, 1500 and 5000 μg/plate (determined by the results of Experiment 1). Six test item concentrations per bacterial strain were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the potential toxic limit of the test item following the change in test methodology from plate incorporation to pre-incubation.
- Without metabolic activation: 0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.1 mL of the test item formulation or solvent vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3 °C for 20 minutes (with shaking) prior to addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating onto Vogel-Bonner plates. Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method. All testing for this experiment was performed in triplicate.
- With metabolic activation: The same procedure was used for testing with metabolic activationexcept that following the addition of the test item formulation and bacterial strain culture, 0.5 mL of S9-mix was added to the tube instead of phosphate buffer, prior to incubation at 37 ± 3 °C for 20 minutes (with shaking) and addition of molten, trace amino-acid supplemented media. All testing for this experiment was performed in triplicate.


REPLICATES: Each concentration of the test material, appropriate positive, vehicle and negative controls, and each bacterial strain, in both experiments, was assayed using triplicate plates.


INCUBATION AND SCORING
- All of the plates (in both experiments) were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). A number of manual counts were performed due to colonies spreading, thus distorting the actual plate count.

Evaluation criteria:

ACCEPTABILITY CRITERIA
The reverse mutation assay may be considered valid if the following criteria are met:
- All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks.
- All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls (negative controls). Acceptable ranges are presented as follows; TA1535: 7 to 40, TA100: 60 to 200, TA1537: 2 to 30, TA98: 8 to 60 and WP2uvrA:10 to 60.
- All tester strain cultures should be in the range of 0.9 to 9 x 10^9 bacteria/ mL.
- Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation.
- There should be a minimum of four non-toxic test material dose levels.
- There should be no evidence of excessive contamination.

EVALUATION CRITERIA
Any, one, or all of the following can be used to determine the overall result of the study:
- A dose-related increase in mutant frequency over the dose range tested.
- A reproducible increase at one or more concentrations.
- Biological relevance against in-house historical control ranges.
- Statistical analysis of data as determined by UKEMS.
- Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response).
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met. Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test material activity. Results of this type will be reported as equivocal.

Statistics:

Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium, other: TA1535, TA1537, TA98 and TA100

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

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A

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

Additional information on results:

-The maximum dose level of the test material in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method) and consequently the same maximum dose level was used in the second mutation test. Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test (pre-incubation method). No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.
-There were no increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation (S9-mix), in Experiment 1 (plate incorporation method). Similarly, no biologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation (S9-mix), in Experiment 2 (pre incubation method). A small, statistically significant increase in TA100 revertant colony frequency was observed in the second mutation test at 5000 µg/plate in the presence of S9-mix. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at 5000 µg/plate were within the in-house historical untreated/vehicle control range for this specific tester strain and the increase was only 1.2 times the concurrent vehicle control.
- The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

Applicant's summary and conclusion

Conclusions:

Under the conditions of the study the test material was considered to be non-mutagenic, both in the presence and absence of metabolic activation.

Executive summary:

The genetic toxicity of the test material was investigated in accordance with the standardised guidelines OECD 471, EU Method B13/14, USA EPA 870.5100 and Japanese testing guidelines, under GLP in a bacterial reverse mutation assay.

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test material using both the Ames plate incorporation and pre-incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10 % liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5000 µg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test material formulations. The dose range was amended following the results of Experiment 1 and was 15 to 5000 µg/plate. Six test material concentrations were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the potential toxic limit of the test material following the change in test methodology.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The maximum dose level of the test material in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method) and consequently the same maximum dose level was used in the second mutation test. Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test (pre-incubation method). No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

There were no increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation (S9-mix), in Experiment 1 (plate incorporation method). Similarly, no biologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation (S9-mix), in Experiment 2 (pre incubation method). A small, statistically significant increase in TA100 revertant colony frequency was observed in the second mutation test at 5000 µg/plate in the presence of S9-mix. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at 5000 µg/plate were within the in-house historical untreated/vehicle control range for this specific tester strain and the increase was only 1.2 times the concurrent vehicle control.

Under the conditions of the study the test material was considered to be non-mutagenic, both in the presence and absence of metabolic activation.