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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:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Report lacks information on metabolic activation system, characterisation of test material, individual plate counts, mean number of revertant colonies per plate, and positive and negative controls used.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1980
Report Date:
1980

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
An additional Spot Plate Test was conducted against each indicator organism using the undiluted test material.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): TX-1968

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 1538
Metabolic activation:
with and without
Metabolic activation system:
rat liver induced by AROCLOR 1254
Test concentrations with justification for top dose:
0.5, 5, 100 and 500 ug/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: standard solvent
Controls
Untreated negative controls:
yes
Remarks:
Solvent control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide, 9-amino acridine, 2-nitrofluorene, 2-anthramine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar spot plate test and overlay plate test

Spot Plate Test
Approximately 10e8 cells from a 16-hour culture of each indicator strain are added to separate test tubes containing 2.0 ml of molten overlay agar supplemented with biotin and a trace of histidine. For nonactivation tests, the contents of the tube is then poured over the surface of a sterile petri dish containing approximately 30 ml of hardened base agar and allowed to solidify. In activation tests, just prior to pouring, an aliquot of the reaction mixture (0.5 ml containing the 9000 x g liver homogenate) is added to each of the overlay agar tubes which are then mixed and poured over the base agar. When all plates are hardened, the test materials, solvent and positive controls are spotted on individual platesof each bacterial strain, with and without activation, in 10 uL quantities. The plates are then covered to prevent photoreactivity of chemicals and within one hour the plates are transferred to a darkened incubator where they are held at 37 deg C for 48-72 hours.

Overlay Plate Test
Approximately 10e8 cells from a 16-hour culture of each indicator strain are added to separate test tubes containing 2.0 ml of molten agar supplemented with biotin and a trace of histidine. For non-activation tests, at least four dose levels of the test compound are added to the contents of the appropriate tubes and poured over the surfaces of selective agar plates. In activation tests, a minimum of four different concentrations of the test chemical are added to the appropriate tubes with cells. Just prior to pouring, an aliquot of reaction mixture (0.5 ml containing the 9000 x g liver homogenate) is added to each of the activation overlay tubes, which are then mixed and the contents poured over the surface of a minimal agar plate and allowed to solidify. The plates are incubated for 48-72 hours at 37 deg C and scored for the number of colonies growing on each plate.

The number of colonies on each plate are counted using a Model C111 Automated Colony Counter.
Evaluation criteria:
Because the procedures used to evaluate the mutragenicity of the test chemical are semi-quantitative, the criteria used to determine positive effects are inherently subjective and are based primarily on a historical database. Most data sets are evaluated using the following criteria :
1. Strains TA-1535, TA-1537, and TA-1538
If the solvent control value is within the normal range, a chemical that produces a positive dose response over three concentrations with the lowest increase equal to twice the solvent control value is considered t o be mutagenic.
2. Strains TA-98 and TA-100
If the solvent control value is within the normal range, a chemical that produces a positive dose response over three concentrations with the highest increase equal to twice the solvent control value for TA-100 and two to three times the solvent control value for strain TA-98 is considered to be mutagenic. For these strains , the dose response increase should start at approximately the solvent control value.
3. Pattern
Because TA-1535 and TA-100 were both derived from the same parental strain (G-46) and because TA-1538 and TA-98 were both derived from the same parental strain (03052), there is a built - in redundancy in the microbial assay. In general, the two strains of a set respond to the same mutagen and such a pattern is sought. It is also anticipated that if a given strain , e.g. TA-1537, responds to a mutagen in non-activation tests, it will generally do so inactivation tests. (The converse of this relationship is not expected). While similar response patterns are not required for all mutagens, they can be used to enhance the reliabilityof an evaluation decision.
4. Reproducibility
If a chemical produces, a response in a single test that cannot be reproduced in one or more additional runs, the initial positive test data loses significance.

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:
not applicable
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:
not applicable
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The test material, TX-1968, failed to exhibit mutagenic activity in both the activation and non-activation systems and is considered not to be mutagenic under the conditions employed.
Executive summary:

The test substance was prepared in DMSO at concentrations ranging from 0.5 - 500 ug/plate. The Ames bacterial assay was conducted both with and without metabolic activation. An additional Spot Plate test was conducted against each indicator organism using the undiluted test material. No evidence of genetic activaity was observed.