Desmedipham

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1990-11-05 to 1991-03-21

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

The test was conducted according to OECD TG 471. This guideline was revised in 1997 with new recommendations. Two were not meet in this test: Only strains with GC base pair at the primary reversion site were included in the assay which may not detect certain oxidising mutagens, cross-linking agents and hydrazines. 2- Aminoanthracene was used as the only indicator of the efficacy of the S9-mix. Each batch of the S9 should also be characterised with a mutagen that requires metabolic activation by microsomal enzymes, e.g., benzo(a)pyrene, dimethylbenzanthracene.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

yes

Remarks:

See "Rationale for reliability incl. deficiencies" for more information

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Desmedipham

EC Number:

237-198-5

EC Name:

Desmedipham

Cas Number:

13684-56-5

Molecular formula:

C16 H16 N2 O4

IUPAC Name:

ethyl 3´-phenylcarbamoyloxycarbanilate

Test material form:

solid: particulate/powder

Details on test material:

The test material is Desmedipham but the purity has not been specified.

Specific details on test material used for the study:

White powder, stored in the dark at ambient temperature.

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Livers of rats induced by the polychlorinated biphenyl mixture.
- method of preparation of S9 mix: Ice-cold 0.05 M phosphate buffer, pH 7.4, was added to the following pre-weighed reagents to give final concentrations in the S9 mix of:

NADP di-Na salt 4 mM
Glucose-6-phosphate di-Na salt 25 mM
MgCl2.6H2O 8 mM
KCl 33 mM

This solution was immediately filter-sterilised by passage through a 0.45 urn Millipore filter and mixed with the liver 9,000 g supernatant fluid in the following proportion:

co-factor solution 9 parts liver preparation 1 part
This combination of co-factors and liver preparation was called the S9 mix.

- quality controls of S9 (enzymatic activity, metabolic capability, total protein concentration)

Test concentrations with justification for top dose:

33, 100, 333, 1000, 3333 and 10000 mg/plate. The dose levels used selected on the basis of the results of the toxicity test.

Vehicle / solvent:

DMSO:
Desmedipham technical and the positive control substances, except sodium azide, were dissolved in dimethylsulphoxide. Sodium azide was dissolved in sterile, ultra-pure water.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

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/ EXPOSURE:
- Test substance added in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 2 days.

Evaluation criteria:

A test article was considered positive if there was either a dose-related response or a reproducible effect in independent tests. A significant mutagenic response was recorded, if at least a doubling of the mean concurrent vehicle control values was observed in strains TA 1535, TA 1537, TA 1538 and TA 98 at some concentrations of the test substances, and a 1.5 fold increase over the control value in strain TA 100.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Slight toxicity to bacteria was observed at 10000 µg/plate with and without S9. Precipitation of the test substance was noted at 10000 mg/plate in all strains with and without metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Very slight toxicity was also noted at 10000 mg/plate. Precipitation of the test substance was noted at 10000 mg/plate in all strains with and without metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Very slight toxicity was also noted with and without S9 at 10000 mg/plate. Precipitation of the test substance was noted at 10000 mg/plate in all strains with and without metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Very slight toxicity was also noted with and without S9 at 10000 mg/plate. Precipitation of the test substance was noted at 10000 mg/plate in all strains with and without metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Slight toxicity to bacteria was observed at 10000 µg/plate with and without S9. Precipitation of the test substance was noted at 10000 mg/plate in all strains with and without metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

No mutagenic activity was observed in any of the 5 bacterial strains used, with or without S9.

Any other information on results incl. tables

See the Results' tables in the attched file in "Overall remarks, attachments"

Applicant's summary and conclusion

Conclusions:

Desmedipham was not mutagenic in the Salmonella typhimurium strains used in the study at doses up to 10000 µg/plate. The study was acceptable.

Executive summary:

Desmedipham technical was tested for mutagenic activity in Salmonella tvphimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 at concentrations ranging from 33 µg to 10000 µg per plate.

The tests were conducted on agar plates in the presence and absence of induced rat liver preparation and co-factors (S9 mix) required for mixed-function oxidase activity. 

Concurrent positive controls demonstrated the sensitivity of the assay and the metabolising activity of the S9 mix. 

Desmedipham technical did not induce mutagenic activity in any of the 5 bacterial strains used, either in the presence or absence of S9 mix.

A slightly thin background lawn of microcolonies was noted in Salmonella typhimurium TA 1535 and TA 100 at 10000 µg per plate in the presence and absence of S9 mix. A thin lawn was noted in TA 1537, TA 1538 and TA 98 at 10000 µg per plate in the presence and absence of S9 mix.

Precipitation of the test substance was observed at 10000 µg per plate in all 5 bacterial strains, in both activation conditions.

It was concluded that Desmedipham technical was not mutagenic to Salmonella typhimurium when tested in dimethylsulphoxide at concentrations extending into the toxic range.