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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment

Data source

Reference
Reference Type:
publication
Title:
Assay of 855 Test Chemicals in Ten Tester Strains Using a New Modification of the Ames Test for Bacterial Mutagens.
Author:
McMahon R.E, Cline J.C and Thompson C.Z.
Year:
1979
Bibliographic source:
Cancer Reswearch 39, 682-693, March 1979

Materials and methods

Principles of method if other than guideline:
Gradient method, a modification of Ames Test for Bacterial Mutagens.
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100, TA 1538, G46, C3076, D3052
Species / strain / cell type:
E. coli, other: WP2 and WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Rat-liver metabolic fraction S9
Test concentrations with justification for top dose:
10000-fold concentration gradient of test compound:0.1 to 1000 μg/ml
Vehicle / solvent:
- Vehicle/solvent used: Dimethyl Sulfoxide (DMSO); when appropriate, water or dimethoxyethane was used instead of dimethyl sulfoxide.
Controls
Untreated negative controls:
yes
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
other: streptozotocin
Remarks:
Streptozoticin without metabolic activation; 2-AF with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)MEDIUMCultures are maintained in Trypticase soy broth (Baltimore Biological Laboratory) with 0.5 % yeast extract (Difco Laboratories) added.
The minimal agar medium was prepared as follows. Each liter of medium contains 14 g of K2HPO4, 6 g of KH2PO4, 2 g of (NH4)2SO4, 1 g of sodium citrate, 0.4 g of MgSO4 x 7H2O, 2.0 g of glucose, and 10 g of Difco agar (purified). Trace amounts of histidine, tryptophan, and biotin are added immediately before use.

DURATION
Exposure duration: 48 hr

SETTING UP OF THE TEST PLATES
Ten ml of minimal agar medium were poured into a square Petri dish (9 x 9 cm) which was tilted at a slight angle. The agar was then allowed to solidify into a wedge-shaped layer by standing at room temperature. Meanwhile, a 1000 μg/ml mixture of test compound in agar was prepared by adding 10 ml of minimal agar to 0.1 ml of a 100 mg/mI solution of test compound in DMSO. The cooled agar plates were then placed on a level surface, and an overlay of the 10 ml of agar containing the test compound was poured onto the plate to form a reversed wedge of agar on top of the first wedge. A concentration gradient of compound (approximately 100 to 1000 μg/ml) was produced by allow ing the compound in the upper wedge to diffuse into the lower layer for 2 hr at room temperature.Three additional plates with concentration ranges of 10 to 100 μg/ml, 1 to 10 μg/ml and 0.1 to 1 μg/ml were prepared. A streaking device consisting of 10 sterile 50 μl pipets was dipped into suspensions of the 10 test strains and was allowed to fill by capillary action. The pipets were touched to the upper edge of the gradient and were drawn across the plate. The minimal medium used in these studies was supplemented with tryptophan, histidine, and biotin in amounts sufficient to allow growth of only about 6 to 10 generations of auxotroph.

METABOLIC ACTIVATION
Bacteria were exposed to the test substance both in the presence and absence of the metabolic activation system. The final agar overlay mixture was made as follows: 10 ml of 1 % agar containing traces of histidine and tryptophan, and an excess of biotin at 45 ° were mixed in a test tube with 2 ml of the enzyme mixture and were immediately dispensed to 4 plates by pipets. Under these conditions, the microsomes were at a temperature about 40 °C for less than 1 min. This process was repeated for each multiple of 4 plates in sequence until all plates were overlayed. The plates were streaked in the usual manner and incubated for 48 hr at 37 °C.

COLONY COUNTING AND SCORING OF THE PLATES
After the incubation period the concentration range over which chemically induced mutant colonies were present was recorded. When the plates were read, the operator had a series of 4 plates covering a 10000-fold concentration range on which to base his judgment.
Evaluation criteria:
- In the cases of non mutagenicity occurunce, a very pale streak of bacterial growth was expected to be seen along the inoculation streak.
- In the cases of nonlethal mutagenic events occurunce, discrete colonies were expected to appear in the pale background lawn.
- The frequency of colony appearance was expected to increase along the increasing gradient to a concentration at which maximal mutation occurs. Conversely, frequency was expected to decrease along the decreasing gradient to a concentration below which only background lawn is observable. These upper and lower concentrations is the concentration range in which the compound is mutagenic under test conditions.
- The concentration range over which the test compound is too toxic to permit growth of the auxotroph could be observed by the absence of growth along the application streak (for example no background lawn is observable).
- If the test chemical is toxic to the auxotroph, minimal inhibitory concentrations could be observed as clear zones.
Statistics:
No statistical method available.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100, TA 1538, G46, C3076, D3052
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
E. coli, other: WP2 and WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
Based on the results of these experiments and on standard evaluation criteria, it is concluded that the substance and its metabolites did not induce gene mutations in the strains of Salmonella typhimurium nor in the strains of E.coli used.
Executive summary:

The substance was tested for mutagenic effects in vitro strains of Salmonella typhimunum (TA 98, TA 100, TA 1535, TA 1537, TA 1538, G46, C3076, D3052) and in tryptophan-requiring strains of E.coli (WP2 and WP2 urvA). The test was performed with and without the addition of rat-liver supernatant as an extrinsic metabolic activation system. The compound was tested at concentrations in the range of 0.1 - 1000 μg/ml. In order to confirm the results, the plates containing no compound were used as negative control. Each strain was additionally tested in the presence and in the absence of a metabolic activation system with a suitable, known mutagen as positive control.

In the tests performed with and without metabolic activation, the substance gave a negative response.

Conclusion

Based on the results of the test it is concluded that the substance and its metabolites did not induce gene mutations in the strains of Salmonella typhimurium nor in the strains of E.coli used.