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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study well documented, test procedure in accordance with OECD 471 methods, meets generally accepted scientific principles, acceptable for assessment and GLP compliant. The test has been performed on only four Salmonella strains.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1985
Report Date:
1985

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Method

Target gene:
Salmonella typhimurium
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 98
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 1535
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 1537
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from rats
Test concentrations with justification for top dose:
For both mutagenicity test the treatment-levels are:
Strain TA98/TA100/TA1535/TA1537 with and without S9 mix: 20, 80, 320, 1280 and 5120 µg/plate
Vehicle / solvent:
distilled water
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-anthramine
Remarks:
all strains with S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
TA1537 wihtout S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: MNNG (N-methyl-N-nitro-N-nitrosoguanidine
Remarks:
TA100 and TA1535 without S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: daunomycine
Remarks:
TA98 without S9 mix
Details on test system and experimental conditions:
BACTERIAL STRAINS INFORMATION:
The bacterial strains of Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 were kept as frozen broth cultures, in aliquots of 0.5 ml at -70°C with 0.8 % of DMSO.
Fresh cultures were prepared by adding 0.5 ml of a thawed stock culture to 25 ml of nutrient broth (Normal Difco nutrient broth for strains TA1535 abd TA100, and a double strenght Difco Nutrient broth for TA1537 and Ta98, in 0.5 % NaCl.
A nutrient agar (0.8 % Difco nutient broth, 1.5 % Difco Agar, 0.5 % NaCl) was also streaked.
The broth was incubated in the dark in a shaking water bath at 37°C for 16 hours, while the plate was incubated at 37°C overnight. Plates were then transfereed to the refrigerator for up to one week.

CHECKING OUT TESTER STRAINS
All strains were tested for the presence of thei mutations.
a)The mutation in the histidine operon, basic to the test syste, was tested by checking for growth in the presence and absence of histidine on a minimal medium-agar base.
Bacteria of the nutrient agar were streaked on a minimal medium plate supplemented with 0.1 ml of 0.1 M L-histidine and 0.1 ml of 0.5 mM biotin as a growth requirement.
The plates were incubated at 37°C overnight. Growth, for all trains, was seen only where histidine and biotin were present.

b)Sensitivity to crystal violet is a check for the presence of deep rough (rfa) mutation which causes partial loss of the lipopolysaccharide barrier that coats the surface of the bacteria and increases permeability to large molecules that do not penetrate the normal bacteria cell wall.
Three drops of the broth culture were spread on the surface of a nutrient agar plate. A sterile filter paper disc containing crystal violet (10 µl of a 1 mg/ml solution) was placed on this usrfac. A zore of inhibition around the disc, after 24 hours incubation, shows the presence of the rfa mutation.

c) Two of the tested strains (TA98 and TA100) contain a plasmid expressing resistance to ampicillin (R factor). To check for the presence of the lasmid, a sterile filter paper disc conatinign ampicillin (10 µl og 8 mg/ml in 0.02 N NaOH) was placed on a nutrient agar plate spread with the broth.
In strains TA1535 and TA 1537 a zone of inhibition occurs around the disc but for TA98 and TA100 where the R factor is present, no zone of inhibition is seen.

d) The uvr (uvrB for Salmonella typhimurium) the loss of teh excision repair system, makes bacteria sensitive to UV irradiation.
One drop of the broth was cross-streaked in a utrient agar plate. One half of the plate was iradiated for 20 seconds under a15 watt UV lamp at a distance of approx. 30 cm.
After 24 hours incubtaion, growth was found only on the unirradiated parte of the streak for all strains.

METHOD OF APPLICATION: in agar (plate incorporation)

PREPARATION OF S9 mix: The S9 mix consists of induced enzymatic systems contained in rat liver post-mitochondrial fraction (S9 fraction) and the cofactors necessary for their function.
S9 was prepared with the following procedure: three male Sprague-Dawley rats weighting about 200 gr were given an i.p. injection of Aroclor 1254 (Monsanto) in peanut oil at a dose of 500 mg/kg.
Five dayes after the injection, the rats were anaesthetized with ether, decapitaed and the ral livers removed in a sterile manner. The livers were homogenized in three times their volume of a sterile 0.14 M KCl, pooled together and centrifuged for 15 minutes at 9000xg (Beckman refrigerated ultracentrifuge).
The supernatant (termed S9-fraction) was aliquoted into sterile tubes and frozen at - 70°C.
The composition of 0.5 ml of S9 mix was:
4 µ moles MgCl2
16.5 µ moles KCl
2.5 µ moles G-6-P
2.0 µ moles NADP
50 µ moles Sodium phosphate buffer pH 7.4
150 µl liver homogenate

Evaluation criteria:
A test article is considered as positive if either a significant dose-related increase in the number of revertants or a significant and reproducible increase for at least one test concentration is induced.
A test article producing neither a significant dose-related increase in the number of revertants nor a significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium 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:
S. typhimurium TA 1535
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:
S. typhimurium TA 97
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:
S. typhimurium TA 1538
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:
Preliminary toxicity test:
The test item was freely soluble in the vehicle (water for injections) at 50 mg/mL.
Consequently, with a treatment volume of 100 pL/plate, the dose-levels were 10, 100, 500, 1000,2500 and 5000 µg/plate.
No precipitate was observed in the Petri plates when scoring the revertants at all dose-levels. An orange coloration of agar was observed in all test item treated plates at dose-levels > 100 µg/plate.
No noteworthy toxicity was noted towards the four sfrains used, with and without S9 mix, except a decrease in the number of revertants at
dose-levels > 1000 µg/plate, for the TA 98 sfrain with S9 mix.

Mutagenicity experiments:
The number of revertants for the vehicle and positive confrols was as specified in the acceptance criteria. The study was therefore considered valid.
Since the test item was freely soluble and generally non-toxic, the highest dose-level was 5000 pg/plate, according to the criteria specified in the international guidelines.
The selected freatment-levels were: 312.5, 625, 1250, 2500 and 5000 µg/plate, for both mutagenicity experiments with and without S9 mix, except for the first experiment with the TA98 and TA 1537 strains with S9 mix where the following dose-levels were tested: 156.3, 312.5, 625, 1250, 2500 and 5000 µg/plate.
No precipitate was observed in the Petri plates when scoring flie revertants at all dose-levels.
A moderate to marked toxicity was noted in the second experiment with S9 mix (preincubation method), in tiie TA 1535, TA 1537, TA 98 and TA 100 sfrains, generally at dose-levels > 2500 µg/plate.
The test item did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of the six sfrains.
Remarks on result:
other: strain/cell type: TA98

Applicant's summary and conclusion

Conclusions:
negative

The test item did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in strains TA100, TA 1535 and TA1537. A mutagenic effect was observed with the strain TA98 in the absence of metabolic activation.
Executive summary:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article

is considered to be non-mutagenic in this Bacterial reverse mutation assay.