Ethoprophos

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:

2018-07-27 to 2018-07-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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:

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

Version / remarks:

1998

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

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:

Ethoprophos

EC Number:

236-152-1

EC Name:

Ethoprophos

Cas Number:

13194-48-4

Molecular formula:

C8H19O2PS2

IUPAC Name:

ethyl bis(propylsulfanyl)phosphinate

Test material form:

liquid

Method

Target gene:

Salmonella typhimurium: His
Escherichia coli: Trp

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: cell membrane permeability (rfa mutation), deficiency in DNA excision repair system (uvrA/uvrB mutation), pKM101 plasmid in TA1535, TA100, E.coli WP2uvrA

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction was prepared from male Sprague-Dawley derived rats, dosed with phenobarbital/5,6- benzoflavone to stimulate mixed-function oxidases in the liver. S9 fraction was obtained from a suitable source and stored at -90 to -70°C.

Test concentrations with justification for top dose:

5, 15, 50, 150, 500, 1500, 5000 µg/plate (top dose according to OECD 471)

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: 2-aminoanthracene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 2

METHOD OF TREATMENT:
- Cell density at seeding: at least 10E9 cells per mL
- Test substance added in agar: plate incorporation in first test and preincubation for second test

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 30 min
- Duration of treatment: 48 h

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition (reduction in mean revertant colony numbers to ≤50% of the concurrent vehicle control count, by a sparse or absent background bacterial lawn, or both)

METHODS FOR MEASUREMENTS OF GENOTOXICITY:
Revertant colonies were counted using an automated colony counter

ACCEPTANCE CRITERIA
For a test to be considered valid, the mean of the vehicle control revertant colony numbers for each strain should lie within or close to the current historical control range for the laboratory unless otherwise justified by the Study Director. The historical range is maintained as a rolling record over a maximum of two years or a minimum of twenty data sets. Also, the positive control compounds must induce an increase in mean revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537, which have relatively low spontaneous reversion rates) that of the concurrent vehicle controls. Mean viable cell counts in the 10-hour bacterial cultures must be at least 10E9/mL

Evaluation criteria:

The mean number and standard deviation of revertant colonies were calculated for all groups. The “fold-increases” relative to the vehicle controls were calculated in order to compare the means for all treatment groups with those obtained for the vehicle control groups.

If exposure to a test item produces a reproducible increase in mean revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) that of the concurrent vehicle controls, with some evidence of a positive concentration-response relationship, it is considered to exhibit mutagenic activity in this test system.

If exposure to a test item does not produce a reproducible increase in mean revertant colony numbers, it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed.

Statistics:

If the results obtained fail to satisfy the criteria for a clear “positive” or “negative” response, even after additional testing, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers. The Dunnett's test is performed.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate in first and >= 1500 µg/plate in second test

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate in first and >= 1500 µg/plate in second test

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate in first and >= 1500 µg/plate in second test

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate in first and >= 1500 µg/plate in second test

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate in first and >= 1500 µg/plate in second test

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

FIRST TEST
Toxicity, observed as a thin background lawn of non-revertant colonies and/or reduction in the number of revertant colonies, was obtained in all strains following exposure to the test substance at 5000 μg/plate in the absence and presence of S9 mix. In strain TA98, in the absence of S9 mix, toxicity was also observed as a reduction in revertant colony numbers at 1500 μg/plate. No precipitate was observed on any of the plates containing the test item. A maximum exposure concentration of 5000 μg/plate was, therefore, selected for use in the second test.
No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to the test substance at any concentration up to and including 5000 μg/plate in either the presence or absence of S9 mix.

SECOND TEST
Toxicity, observed as a thin or absent background lawn of non-revertant colonies and reduction in the number of revertant colonies, was obtained in strains following exposure to the test substance at 1500 μg/plate and above in the absence and presence of S9 mix. The results with strain TA1537 in the presence of S9 mix were obtained from a repeat test. The original test was deemed invalid due to insufficient number of non-toxic concentrations obtained and the results were, therefore, not reported. No precipitate was observed on any of the plates containing the test substance.
No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to the test substance at any concentration up to and including 5000 μg/plate in either the presence or absence of S9 mix.

ACCEPTABILITY
The viability counts confirmed that the viable cell density of the cultures of the individual organisms exceeded 109 cells/mL in all cases, and therefore met the acceptance criteria.
The mean revertant colony counts for the vehicle controls were within the current historical control range for the laboratory. Appropriate positive control chemicals (with S9 mix where required) induced substantial increases in revertant colony numbers with all strains in all reported tests, confirming sensitivity of the cultures and activity of the S9 mix.

Applicant's summary and conclusion

Conclusions:

The test item was shown to have no mutagenic potential in bacterial cells under the test conditions employed.

Executive summary:

In this in vitro assessment of the mutagenic potential of the test substance, histidine-dependent auxotrophic mutants of Salmonella typhimurium, strains TA1535, TA1537, TA98 and TA100, and a tryptophan-dependent mutant of Escherichia coli, strain WP2 uvrA (pKM101), were exposed to the test substance diluted in dimethyl sulfoxide (DMSO). DMSO was also used as a vehicle control. Two independent mutation tests were performed in the presence and absence of liver preparations (S9 mix) from rats treated with phenobarbital and 5,6-benzoflavone. The first test was a standard plate incorporation assay; the second included a pre-incubation stage. Concentrations of the test substance up to 5000 μg/plate were tested. This is the standard limit concentration recommended in the regulatory guidelines that this assay follows. Other concentrations used were a series of ca half-log10 dilutions of the highest concentration.

In the first test, toxicity (observed as thinning of the background lawn of non-revertant colonies, together with a reduction in revertant colony numbers) was seen in all strains following exposure to the test substance at 5000 μg/plate in the absence and presence of S9 mix. In strain TA98, in the absence of S9 mix, toxicity was also observed as a reduction in revertant colony numbers at 1500 μg/plate. In the second test, toxicity (observed as thinning or an absence of the background lawn of non-revertant colonies, together with a reduction in revertant colony numbers) was seen in all strains following exposure to the test substance at 1500 μg/plate and above in both the absence and presence of S9 mix. No precipitate was observed on any of the plates containing the test substance. No evidence of mutagenic activity was seen at any concentration of the test substance in either mutation test. The concurrent positive controls verified the sensitivity of the assay and the metabolizing activity of the liver preparations. The mean revertant colony counts for the vehicle controls were within the current historical control range for the laboratory. It was concluded that the test substance showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.