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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:
other: experimental study on similar substance
Adequacy of study:
key study
Study period:
1994
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented, test procedure in accordance with OECD 471 methodes, 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:
1994
Report Date:
1994

Materials and methods

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

Test material

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

Method

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:
Main Test: 33, 100, 333, 1000, 2500, 5000 µg/plate
Vehicle / solvent:
Distilled water
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylenediamine (NPD)
Remarks:
Ta 98 and TA1537 without S9 fraction
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA100 an TA1535 without S9 fraction
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
all strains with S9 fraction
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)PREPARATION OF FRACTION S9: The S9 liver microsomal fraction was obtained from the livers of 8 - 12 weeks old male rats strain Wistar Hanlbm* (BRL, CH-4414 Füllingsdorf; weight approx. 220 - 300 g) which received a single i.p. injection of 500 mg/kg b.w. Aroclor 1254 (Antechnika,D-76275 Ettlingen, F.R.G.) in olive oil 5 days previously.After cervical dislocation the livers of the animals were removed, washed in 150 mM KCl and homogenised. The homogenate was diluted 1+3 in KCl and centrifuged cold at 9,000 g for 10 minutes. A stock of the supernatant containing the microsomes was frozen in ampoules of 2, 3 or 5 ml and stored at -80° C. Smallnumbers of the ampoules are kept at -20° C for only one week before use. The standardisation of the protein content was made using the analysis kit of Bio-Rad Laboratories, D-80939 München: Bio-Rad protein assay, Catalogue 500 000 6 (6).The protein concentration in the S9 preparation was 45 mg/ml. PREPARATION OF S9 MIX: Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 co-factor solution. The amount of S9 supernatant was 15% v/v. The composition of the co-factor solution was concentrated to yield the following concentrations in the S9 mix:8 mM MgCl233 mM KCl5 mM glucose-6-phosphate5 mM NADPin 100 mM sodium-ortho-phosphate-buffer, pH 7.4.During the experiment the S9 mix was stored in an ice bath.PREPARATION OF PLATES: For each strain and dose level, incltiding the controls three plates were used as a minimum.The following materials were mixed in a test tube and poured onto the selective agar plates :100 ul Test solution at each dose level, solvent control, negative control, or reference mutagen solution (positive control),500 ul S9 mix (for test with metabolic activation) or S9 mix substitution-buffer (for test without metabolic activation),100 ul Bacteria suspension (cf. test system, pre-culture of the strains),2000 ul Overlay agarIn the pre-incubation assay 100 ul test solution, 500 ul S9 mix / S9 mix substitution buffer and 100 ul bacterial suspension were mixed in a test tube and incubated at 37°C for 60 minutes. After pre-incubation 2.0 ml overlay agar (45°) was added to each tube.The mixture was poured on minimal agar plates. After solidification the plates were incubated upside down for at least 4 8 hours at 37° C in the dark.DURATION- Expression time (cells in growth medium): 48 to 72 hours
Evaluation criteria:
Toxicity of the test article can be evidenced by a reduction in the number of spontaneous revertants, a clearing of the bacterial background lawn, or by degree of survival of treated cultures.
Statistics:
Kier, L.E., D.J. Brusick, A.E. Auletta, E.S. Von Halle, M.M. Brown, V.F. Simmon, V. Dunkel, J. McCann, K. Mortelmans, M. Privai, T.K. Rao and V. Ray (1986)The Salmonella typhimurium/mammalian microsomal assay A report of the U.S. Environmental Protection Agency Gene-Tox Program Mutation Res. 168, 69-240.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 98
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 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
Additional information on results:
The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls were tested in triplicate. The test article was tested at the following concentrations; 33.3; 100.0; 333.3; 1000.0; 2500.0; and 5000.0 ng/plate (active ingredient)Toxic effects, evidenced by a reduction in the number of revertants, occurred in strain TA 1535 at 5000.0 (ig/plate without S9 mix in experiment I and with S9 mix in experiment II. The plates incubated with the test article showed normal background growth up to 5000.0 ug/plate with and without S9 mix inall strains used.No substantial increases in revertant colony numbers of any of the four tester strains were observed following treatment with the tested substance at any concentration level, either in the presence or absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of significance.

Applicant's summary and conclusion

Conclusions:
negative
Executive summary:

No substantial increases in revertant colony numbers of any of the four tester strains were observed following treatment with the tested substance at any concentration level, either in the presence or absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of significance.Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies.In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore Acid Blue 113 is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.