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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
Study period:
1986
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: guideline study performed under GLP-like quality control conditions with QAU statement.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Report Date:
1986

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: solid
- Analytical purity: commercial grade

Method

Target gene:
histidine
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction of rat liver induced with Aroclor 1254
Test concentrations with justification for top dose:
0.08 - 5000 µg/0.1 ml, in the toxicity test
20, 78, 313, 1250 and 5000 ug/0.l ml, in the mutagenicity test
Vehicle / solvent:
- Vehicle used: acetone
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: TA 98: daunorubicin- HCl; TA 100: 4-nitroquinoline-N-oxide; TA 102: mitomycin-C; TA 1535: sodium azide; TA1537: 9(5)-aminoacridine hydrochloride monohydrate
Remarks:
without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: TA 98, TA 100, TA 102, TA 1537: 2 - aminoanthracene; TA 1535: cyclophosphamide
Remarks:
with metabolic activation
Details on test system and experimental conditions:
Each Petri dish contained: approx. 20 ml of minimum agar (plus salts and glucose), 0.1 ml of a solution of the test substance or the vehicle and 0.1 ml of a bacterial culture in 2.0 ml of soft agar. In the experiments in which the substance was metabolically activated, 0.5 ml of an activation mixture was added also. 1 ml activation mixture contained: 0.3 ml S9 fraction of liver from rats (Tif:RAIf(SPF)) induced with Aroclor 1254 and 0.7 ml of a solution of co-factors.
In the experiments without and with the addition of microsomal activation mixture three Petri dishes were prepared per strain and per group (i.e. per concentration or per control group). The plates were incubated for about 48 hours at 37 +/- 1.5 °C in darkness.
The experiment was repeated for confirmation.
Evaluation criteria:
The test substance is generally considered to be nonmutagenic if the colony count in relation to the negative control is not doubled at any concentration.
Statistics:
When the colonies had been counted, the arithmetic mean was calculated.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
At the concentrations of 1250 µg/0.1 ml and above the substance precipitated in soft agar.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Without S9 -Mix

Concentration µg/0.1 ml TA 98 TA 100 TA 102 TA 1535 TA 1537
I II I II I II I II I II
Solvent Control 26 37 238 160 362 322 18 17 7 7
Positive Control 873 992 959 1123 1163 1221 1289 1364 1069 1287
20 22 25 241 140 362 311 17 17 6 7
78 25 28 220 152 347 329 18 15 11 7
313 24 26 212 133 347 282 13 16 11 7
1250 23 26 176 158 315 217 16 16 7 7
5000 30 18 185 111 354 245 13 13 7 3

I = experiment 1

II = experiment 2

Positive controls:

daunorubicin-HCl (10 µg/0.1 ml) for TA 98;

4 -nitroguinoline-N-oxide (0,25 µg/0.1 ml) for TA 100;

mitomycin-C (1 µg/0.1 ml) for TA 102

sodium azide (5.0 µg/0.1 ml) for TA 1535

9 (5) aminoacridine hydrochloride (100 µg/0.1 ml) for TA 1537

With S9 -Mix

Concentration µg/0.1 ml TA 98 TA 100 TA 102 TA 1535 TA 1537
I II I II I II I II I II
Solvent Control 47 37 137 135 359 334 17 20 15 13
Positive Control 1098 1048 1401 1623 1413 1424 330 547 119 197
20 40 44 133 115 339 348 15 11 14 16
78 46 56 138 113 271 253 8 15 13 12
313 48 39 126 115 322 237 14 16 11 9
1250 34 39 127 110 258 286 15 16 15 7
5000 37 31 121 109 329 256 15 10 9 5

I = experiment 1

II = experiment 2

Positive controls:

2 -aminoanthracene (5 µg/0.1 ml) for TA 98, TA 100 and TA 1537;

2 -aminoanthracene (20 µg/0.1 ml) for TA 102;

cyclophosphamide (250 µg/0.1 ml) for TA 1537

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

During the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Executive summary:

The test substance was tested for mutagenic effects on histidine-auxotrophic mutants of Salmonella typhimurium. The investigations were performed on strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 with the following concentrations of the trial substance without and with microsomal activation: 20, 78, 313, 1250 and 5000 µg/0.l ml. In order to confirm the results the experiments were repeated. Any mutagenic effects of the substances are demonstrable on comparison of the number of bacteria in the treated and control cultures that have undergone back-mutation to histidine-prototrophism. In the experiments performed without and with microsomal activation, comparison of the number of back-mutants in the controls and the cultures treated with the various concentrations of the test substance revealed no marked deviations. No evidence of the induction of point mutations by the test substance or by the metabolites of the substance formed as a result of microsomal activation was detectable in the strains of S. typhimurium used in these experiments.