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Diss Factsheets

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: genome mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study was conducted according to internationally accepted guideline and GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report date:
1996

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes
Type of assay:
bacterial gene mutation assay

Test material

Constituent 1
Reference substance name:
Dienone
IUPAC Name:
Dienone
Details on test material:
- Name of test material (as cited in study report):Dienone
- Substance type: off-white powder
- Physical state: solid
- Analytical purity: 99.5% (by DSC)
- Lot/batch No.: Y08696/002 C166/1
- Storage condition of test material: Ambient temperatures in the dark

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
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
5000, 2500, 1000, 500, 200, and 100 micrograms/plate
Vehicle / solvent:
DMSO
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Positive controls:
yes
Positive control substance:
mitomycin C
Positive controls:
yes
Positive control substance:
sodium azide
Positive controls:
yes
Positive control substance:
other: Acridine mutagen ICR191; 2-Aminoanthracene (2AA); Daunomycin HCl (DR)
Details on test system and experimental conditions:
Stock cultures of each strain were prepared and maintained in liquid nitrogen as described by Maron and Ames (1983). The routine source of inocula for overnight cultures was on set of frozen stocks, used and discarded on an approximately weekly basis. The overnight culture from each new frozen culture was screened for deep-rough characters, DNA repair deficiency and Ampicillin resistance as previously described (Maron and Ames, 1983) with one modification:

The presence of the uvrB deletion (Salmonella) and the uvrA mutation (E.coli) was confirmed by testing the sensitivity of each culture to mitomycin C (10uL of a 10ug/mL solution) in the same ammaer as sensitivity to crystal violet was tested. Damage to DNA caused by mitomycin C is repaired in normal bacteria by the uvr excision repair pathway, and is thus toxic to strains deficient at either the uvrA or uvrB loci.

When fresh frozen stocks were prepared, the strains were tested for amino acid requirement and for reversion properties using diagnostic mutagens as described by Ames et al (1975) and Maron and Ames (1983), except that the mutagens were incorporated in the top agar layer as in a standard experiment (see Section 4.6.1), rather than spot tested as described by Ames et al.


Metabolic activation system

The S9-mix was prepared as required, on each day of experimentation as follows:

Volume per 30ml S9-mix
S9 fraction: 3ml
Sucrose-Tris-EDTA buffer (S9 buffer): 7ml
Cofactor solution: 20ml

In tests without metabolic activation, the S9 fraction and cofactor solution were replaced by an equivalent volume of S9 buffer. Both the S9-mix and the S9 buffer were kept on ice until used.

S9 was prepared from male Sprague Dawley rats, dosed once daily (by oral gavage) for 3 days with a combined phenobarbital (80 mg/kg bodyweight) and beta-naphthoflavone (100 mg/kg) corn oil solution. The treated animals were sacrificed on the day following the third dose. A 25% w/v homogenate (the S9 fraction) was prepared according to the method given in Callender et al (1995).

The cofactor solution was prepared according to Maron and Ames (1983) as a single stock solution Na2HPO4, KCl, glucose-6-phosphate, NADP (Na salt) and MgCl2 (150:49. 5:7. 5:6:12 mM).


Methodology:

0.1ml aliquots of an overnight culture (10-12 hours) of each bacterial strain were dispensed by micropipette into the required number of sterile plastic bijou bottles fitted with screw caps and stored at room temperature.

Top agar consisting of 0.6%w/v and 0.5%w/v sodium chloride in deionised water was melted by brief autoclaving and stored at approximately 50degC until required.

Prior to testing the molten top agar was prepared by adding sterile 0.5mM histidine/0.5mM biotin stock solution (10ml solution:100ml agar) for Salmonella work, and by adding sterile tryptophan solution (10ml 0.5mM stock:100ml agar) for E.coli work. Separate agars were prepared for each species.

Plate incorporation protocol:

0.5ml S9-mix (or S9 buffer) was then added by dispensing syringe to the number of bijou bottles of one strain required for one concentration, followed by 0.1ml of the appropriate concentration of test substance preparation added by micropipette. Finally, 2.0 ml top agar was then added by syringe to each bijou: the force of addition was sufficient to mix the contents. The resulting mixture was then poured rapidly onto the surface of a prepared pre-labelled Vogel Bonner plate (9cm diameter vented Petri-dish prepared with 25ml Vogel Bonner minimal medium and containing 1.5% w/v agar and 2% w/v glucose) and allowed to gel. Plates were then incubated inverted at 37 deg C for 3 days in the dark.

Following the total incubation period the plates were examined for the lack of microbial contamination and evidence that the test was valid: ie there was a background lawn on the solvent control plates and on the plates for (at least) the concentrations of test substance, and that the positive controls had responded as expected. All plates were counted by an automatic colony counter (AMS 40-10) with the discrimination adjusted appropriately to permit the optimal counting of mutant colonies. Obviously contaminated plates were recorded as such without counting.


Pre-incubation period:

The assay procedure was as for the plate-incorporation protocol described above, except that

a) each compound/solvent dose was added in 0.02ml volumes, with the total volume made up to 0.1ml with phosphate buffered saline;

b) before adding the top agar, each compound/strain group of bijoux were placed on an orbital shaker (at approximately 140 rpm) for 60 minutes (at 37 deg C)
Evaluation criteria:
Test data from individual experiments are considered valid if:
a) the concurrent solvent control data are acceptable;
b) the positive control data show unequivocal positive responses;

Failure of one or more tester strain/S9 combinations does not invalidate the data for the remainder of a concurrent experiment.

A positive response in a (valid) individual experiment is achieved when one or both of the following criteria are met:

a) a statistically significant dose-related increase in the mean number of revertant colonies is obtained;

b) a two fold or greater increase in the mean number of revertant colonies (over that observed for the concurrent solvent control plates) which is statistically significant, is observed at one or more concentrations.

A negative result in a (valid) individual experiment is achieved when:
a) there is no statistically significant dose-related increase in the mean number of revertant colonies per plate observed for the test substance; and

b)in the absence of any such dose response, no increase in colony numbers is observed (at any test concentration) with exceeds 2x the concurrent solvent control.

For a positive response in an individual experiment to be considered indicative of an unequivocal positive, ie-mutagenic, result for that strain/S9 combination, then the observed effect must be consistently reproducible.

All derived calculations (ie mean colony count/plate; standard deviation, etc) shown in the results tables were carried out by computer. Counts from contaminated plates are not included in these calculations.

Statistics:
An assessment of statistical significance was carried out using a one-tailed Student’s t-test. The corresponding probability for each dose level was derived by computer using the appropriate degrees of freedom. Values of p<0.1 are treated as significant, with values of 0.01 ,p,0.05 being indicative of a positive effect.


Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
not specified
Cytotoxicity / choice of top concentrations:
not specified
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:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In two separate assays, the test substance did not induce any significant, reproducible increases in the observed number of revertant colonies in any of the tester strains used, either in the presence or absence of S9-mix.

The positive controls for each experiment induced the expected responses, indicating the strains were responding satisfactorily in each case.
Remarks on result:
other: strain/cell type:
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

It is concluded that, under the conditions of this assay, Dienone gave a negative, ie non-mutagenic response in S. typhimurium strains TA1535, TA1537, TA98 and TA100 and E. coli strains WP2P and WP2P uvrA in both the presence and absence of S9-mix. By read-across, didenac is also considered to be non-mutagenic.
Executive summary:

There are no available genetic toxicity studies on didenac. Results of a study conducted with a structurally similar compound (dienone) are reported and used for read across.

Dienone was evaluated in a bacterial mutagenicity assay over a range of concentrations using four strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and two strains of Eschericia coli (WP2P and WP2P uvrA) in the presence and absence of a rat liver-derived metabolic activation system (S9-mix).

 

In two separate experiments, the test substance did not induce any significant, reproducible increases in the observed numbers of revertant colonies in any of the tester strains used, either in the presence or absence of S9-mix. 

 

The sensitivity of the test system, and the metabolic activity of the S9-mix, were clearly demonstrated by the increases in the numbers of revertant colonies induced by positive control substances. 

 

It is concluded that, under the conditions of this assay, Dienone gave a negative, ie non-mutagenic response in S. typhimurium strains TA1535, TA1537, TA98, and TA100 and E.coli strains WP2P and WP2P uvrA in both the presence and absence of S9-mix.

By read across, didenac gives a negative, non-mutagenic response under the conditions of the test.