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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: non-GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1978
Report Date:
1978

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
A. Identification: 78-341
B. Date Received: August 29, 1978
C. Physical Description: Clear colorless, sticky semi-solid

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
9,000 xg supernatant from aroclor 1254-induced male rat liver
Test concentrations with justification for top dose:
10 ug
100 ug
500 ug
1000 ug
1500 ug
2000 ug
Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: see table 1
Details on test system and experimental conditions:
Plate Test (Agar Incorporation)
Approximately 108 cells from an overnight culture of each indicator strain were added to separate test tubes containing 2.0 ml molten agar supplemented with biotin and a trace of histidine. For nonactivation tests, at least 4 dose levels of the test compound were added to the contents of the appropriate tubes and poured over the surfaces of selective agar plates. In activation tests, at least 4 dose levels of the test chemical were added to the
appropriate tubes with cells. Just prior to pouring, an aliquot of reaction mixture (0.5 ml containing the 9,000 x g liver homogenate) was added to each of the activation overlay tubes, which were then mixed, and the contents poured over the surface of a minimal agar plate and allowed to solidify. The plates were incubated for 48 hrs at 37°C and scored for the number of colonies growing on each plate. 04 yeast plates were incubated at 30°C
for 3-5 days and then scored. Positive and solvent controls using both directly active positive chemicals and those that require metabolic activation were run with each assay.

Recording and Presenting Data
The numbers of colonies on each plate were counted and recorded on printed forms. These raw data were analyzed in a computer program and reported on a printout. The results are presented as revertants (or convertants for D4) per plate for each indicator strain employed in the assay. The positive and solvent controls are provided as reference points.

Control Tests
Positive and negative control assays are conducted with each experiment and consist of direct-acting mutagens for nonactivation assays and mutagens that require metabolic biotransformation in activation assays. Negative controls consist of the test compound solvent in the overlay agar together with the other essential components. The negative control plate for each strain gives a reference point to which the test data are compared. The positive control assay is conducted to demonstrate that the test systems are functional with known mutagens.

Evaluation Criteria for Ames Assay
Because the procedures used to evaluate the mutagenicity of the test chemical are s~niquantitative, the criteria used to determine positive effects are inherently subjective and are based primarily on a historical data base. 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 to be mutagenic.
2. Strains TA-98, TA-100 and D4
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 2-3 times the solvent control value for strains TA-98 and 04 is considered to be
mutagenic. For these strains, the dose-response increase should start at approximately the solvent control va1ue.
3. Pattern
Because TA-1535 and TA-100 are both derived from the same parental strain (G-46) and because TA-1538 and TA-98 are 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 nonactivation tests, it will generally do so in activation 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 reliability of 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 lose significance.

The preceding criteria are not absolute, and other extenuating factors may enter into a final evaluation decision. However, these criteria are applied to the majority of situations and are presented to aid those individuals not familiar with this procedure. As the data base is increased, the criteria for evaluation can be more firmly established.
Evaluation criteria:
Plate test data consist of direct revertant colony counts obtained from a set of selective agar plates seeded with populations of mutant cells suspended in a semisolid overlay. Because the test chemical and the cells are incubated in the overlay for 2 days, and a few cell divisions occur during the incubation period, the test is semiquantitative in nature. Although these features of the assay reduce the quantitation of results, they provide certain advantages not contained in a quantitative suspension test:
• The small number of cell divisions permits potential mutagens to act on replicating DNA, which is often more sensitive than nonreplicating DNA.
• The combined incubation of the compound and the cells in the overlay permits constant exposure of the indicator cells for 2 days.
A. Surviving Populations
Plate test procedures do not permit exact quantitation of the number of cells surviving chemical treatment. At low concentrations of the test chemical, the surviving population on the treatment plates is essentially the same as that on the negative control plates. At high concentrations, the surviving population is usually reduced by some fraction. Our protocol normally employs several doses ranging over 2 or 3 log concentrations, the highest of these doses being selected to shew slight toxicity as determined by subjective criteria.

The demonstration of dose-related increases in mutant counts is an important criterion in establishing mutagenicity. A factor that might modify dose-response results for a mutagen would be the selection of doses that afe too low (usually mutagenicity and toxicity are related). If the highest dose is far lower than a toxic concentration, no increases may be observed over the dose range selected. Conversely, 1f the lowest dose employed is highly cytotoxic, the test chemical may kill any mutants that are induced, and the compound will not appear to be mutagenic.

Results and discussion

Test results
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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: positive control material
strain non-activation activation
TA-1535 N-methyl,N-nitro,N-nitrosoguanidine 2-anthramine
TA-1537 9-aminoacridine 2-anthramine
TA-1538 2-nitrofluorene 2-anthramine
TA-98 2-nitrofluorene 2-anthramine
TA-100 N-methyl,N-nitro,N-nitrosoguanidine 2-anthramine
D4 N-methyl,N-nitro,N-nitrosoguanidine 2-anthramine

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The test compound, 78-341, did not demonstrate genetic activity in any of the assays conducted in this evaluation and was considered not mutagenic under these test conditions.
Executive summary:

The test compound was examined for mutagenic activity in a series of in vitro microbial assays employing Salmonella and Saccharomyces indicator organisms. The compound was tested directly and in the presence of liver microsomal enzyme preparations from Aroclor induced rats. The compound was tested over a series of concentrations such that there was either quantitative or qualitative evidence of some chemically-induced physiological effect at the highest dose level. The low dose in all cases was below a concentration that demonstrated any toxic effect. The dose range employed for the evaluation of this compound was from 10 ug to 2000 ug per plate. The results of the test conducted on the compound were all negative in the absence or presence of a metabolic activation system. The test compound, 78-341, did not demonstrate genetic activity in any of the assays conducted in this evaluation and was considered not mutagenic under these test conditions.