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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:
key study
Study period:
1976
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Non-GLP study equivalent to OECD guideline 471.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1976
Report date:
1976

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 480 (Genetic Toxicology: Saccharomyces cerevisiae, Gene Mutation Assay)
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of divinylbenzene and ethylstyrene
EC Number:
910-757-7
Cas Number:
N/A
Molecular formula:
Divinylbenzene: C10H10 Ethylstyrene: C10H12
IUPAC Name:
Reaction mass of divinylbenzene and ethylstyrene
Details on test material:
lot# 05135 (DVB-55)

Method

Target gene:
TA1535- hisG46, TA1537- hisC3076, TA1538- hisD3052, TA98- hisD3052, TA100-hisG46
Species / strain
Species / strain / cell type:
other: S. typhimurium TA1535, TA1537, TA1538, TA98 and TA100; yeast S. cerevisiae
Metabolic activation:
with and without
Metabolic activation system:
Livers from adult male mice induced with a polychlorinated biphenyl (Aroclor 1254)
Test concentrations with justification for top dose:
Bacteria - 0.5, 1, 5, 10, 50, 100 or 500 µg/plate
Yeast - 0.01 or 0.1 (w/v or v/v)
Vehicle / solvent:
not reported
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 4-o-tolylazo-o-toluidine and N-methyl-N'-nitro-N-nitrosoguanidine
Details on test system and experimental conditions:
S. typhimurium
The S. typhimurium strains used at SRI were obtained from Dr. Bruce Ames of the University of California at Berkeley. All are histidine auxotrophs (his-) by virtue of mutations in the histidine operon. All the indicator strains are stored at -80 C. For each experiment, an inoculum from frozen stock culture is grown overnight at 37°C in a nutrient broth consisting of 1% tryptone and 0.5% yeast extrract. After stationary overnight growth, the cultures are shaken for 3 to 4 hours to ensure optimal growth. Each culture is checked for sensitivity to crystal violet. The presence of the rfa- mutation makes the indicator + strains sensitive to this dye, whereas the parent strain, rfa , is not sensitive to the dye. However, the mutation is reversible, leading to the accumulation of rfa+ cells in the culture. Therefore, the cells must be tested routinely to ensure their sensitivity to crystal violet. Each culture also is tested by specific mutagens known to revert each test strain (positive controls) .

To a sterile 13 x 100 test tube placed in a 43°C heating block, add in the following order:
(1) 2 mL of 0.6% agar*
(2) 0.1 mL of indicator organism
(3) 0.15 mL of metabolic activation mixture (optional)
(4) Up to 100 µL of a solution of the test chemical

For negative controls, use steps 1, 2 , and 3 (optional) and use 100 µL of the solvent used for the test chemical. This mixture was stirred gently and then poured onto minimal agar plates. After the soft agar had set, the plates were incubated at 37°C for two days. The number of hisf revertants (colonies that grow on plates lacking a sufficient amount of histidine to support colony formation) were counted and recorded. Some of the revertants were routinely tested to be sure that they are his+, require biotin, and were sensitive to crystal violet (rfa).

Saccharomyces Cerevisiae D3: The yeast cerevisiae D3 is a diploid and is heterozygous for a mutation in an adenine-metabolizing enzyme. The Saccharomyces test strain from the liquid nitrogen was grown overnight at 30°C with aeration in 1.0% tryptone and 0.5% yeast extract. The cells were washed twice in 0.067 M PO4, buffer (pH 7.4) and resuspended in the same buffer at a concentration of 10E 8 cells/mL. The in vitro yeast mitotic recombination assay in suspension consists of 5E7 washed, stationary-phase yeast cells in 1 mL of 0.067 M PO4 buffer (pH 7.4) and 50 mg/mL of the test chemical (or a fraction of the concentration required to give 50% killing). The suspension was incubated at 30°C for 4 hours. After incubation, the sample was diluted serially in sterile saline and plated on tryptone yeast agar plates. Plates of a dilution were incubated for 2 days at 30°C, followed by 2 days at 4°C to enhance the development of red pigment that is indicative of adenine-negative homozygosity. Plates were screened for red colonies or red sectors by scanning the plates with a dissecting microscope at 10 x magnification. Plates of a dilution were incubated for 2 days at 30°C for determination of the total number of colony-forming units. The in vitro yeast mitotic recombination assay in suspension with metabolic activation was carried out as above with the addition of the metabolic activation system to the incubation mixture.

Metabolic Activation: Adult male mice were given a single intraperitoneal injection of a polychlorinated biphenyl (Aroclor 1254) at a dosage of 500 mglkg. Four days after the injection, the food was removed. On the fifth day, the mice were sacrificed. The livers were removed aseptically and placed in preweighed, sterile glass beakers. The organ weight was determined, and all subsequent operations to the metabolic activation step were conducted in an ice bath. The organ was washed in an equal volume of cold, sterile 0.15M KCl (1 mL/g of wet organ), minced with sterile surgical scissors in three volumes of 0.15 KCl, and homogenized with a Potter-Elvehjem apparatus. The homogenate was centrifuged for 10 minutes at 9000 x g, and the supernate was removed and stored in liquid nitrogen. To the postmitochondrial supernate were added MgCl2, KCl, glucose-6-phosphate, TPN, and sodium phosphate (pH 7.4).
Evaluation criteria:
No data
Statistics:
No data

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
not determined
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Key result
Species / strain:
Saccharomyces cerevisiae
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
Key result
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
Key result
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
Key result
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:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
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
Key result
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:
other: S. typhimurium TA1535, TA1537, TA1538, TA98 and TA100; yeast S. cerevisiae
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:
Divinylbenzene was not mutagenic in the S. typhimurium assay and was negative in the mitotic recombination assay with S. cerevisiae D3.

Applicant's summary and conclusion

Conclusions:
DVB-55 was not mutagenic in the S. typhimurium assay with and without activation and was negative in the mitotic recombination assay with S. cerevisiae D3.
Executive summary:

Seventeen compounds were examined for mutagenic activity with five strains of the bacteria Salmonella typhimurium (TA98, TA100, TA1535, TA1537, and TA1538) and with the yeast Saccharomyces cerevisiae D3. Each assay was performed in the presence and in the absence of a metabolic activation system. None of the seventeen compounds were mutagenic in the bacterial assay with Salmonella typhimurium, either in the presence or in the absence of the liver metabolic activation system. One of the compounds, S-2 increased mitotic recombination in the Saccharomyces cerevisiae D3 assay, whereas S-4, S-5, and S-10 gave marginally positive responses. Compounds S-3, S-6, S-7, S-8, S-9, S-11 (divinylbenzene), S-12, S-13, and S-14 did not increase mitotic recombination. Assays S. cerevisiae with S-1, S-16, and S-17 were incomplete at the time of reporting.