1,3-diphenylguanidine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1979

Report date:

1979

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

Litton Bionetics Inc. standard protocol of Mouse Lymphoma Forward Mutation Assay

GLP compliance:

no

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1,3-diphenylguanidine

EC Number:

203-002-1

EC Name:

1,3-diphenylguanidine

Cas Number:

102-06-7

Molecular formula:

C13H13N3

IUPAC Name:

1,3-diphenylguanidine

Method

Target gene:

TK

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

no data

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 (male Fischer rats)

Test concentrations with justification for top dose:

16.4, 32.8, 65.6, 131, 188 µg/ml without S9,
32.8, 131, 188, 375, 525 µg/ml with S9

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Ethylmethan sulfonate (without S9), dimethylnitrosamine (with S9)

Details on test system and experimental conditions:

The solubility of the test chemical in growth medium and/or DMSO is first determined. Then a wide range of chemical concentrations is tested for cytotoxicity, starting with a maximum applied dose of 10 mg/ml for test chemicals soluble in media or 1 mg/ml for solutions in organic solvents. After an exposure time of four hours, the cells are washed and a viable cell count is obtained the next day. Relative cytotoxicities expressed as the reduction in growth compared to the growth of untreated cells are used to select seven to ten doses that cover the range from 0 to 50-90% reduction in 24-hour growth. These selected doses are subsequently applied to cell cultures prepared for mutagenicity testing, but only four or five of the doses will be carried through the mutant selection process. This procedure compensates for daily variations in cellular cytotoxicity and ensures the choice of four or five doses spaced from 0 to 50-90% reduction in cell growth.
1. Nonactivation Assay:Cultures exposed to the test chemical for four hours at the preselected doses are washed and placed in growth medium for two or three days to allow recovery, growth and expression of the induced TK-/- phenotype. Cell counts are determined daily and appropriate dilutions are made to allow optimal growth rates.

At the end of the expression period, 3 x 10e6 cells for each selected dose are seeded in soft agar plates with selection medium and resistant (mutant) colonies are counted after 10 days incubation. To determine the actual number of cells capable of forming colonies, a portion of the cell suspension is also cloned in normal medium (nonselective). The ratio of resistant colonies to total viable cell number is the mutant frequency.
2. Activation Assay : The activation assay can be run concurrently with the nonactivation assay. The only difference is the addition of the S9 fraction of rat liver homogenate and necessary cofactors (CORE) during the four-hour treatment period. CORE consists of NADP (sodium sait) and isocitric acid. The final concentrations of the activation system components in the cell suspension are: 2.4 mg NADP/ml; 4.5 mg isocitric acid/ml; and 50 pl S9/ml.

Evaluation criteria:

A compound is considered mutagenic in this assay if:
-A dose-response relationship is observed over 3 of the 5 dose levels employed.
-The minimum increase at the low level of the dose-response curve is at least 2.5 times greater than the solvent and/or negative control values.
-The solvent and negative control data are within the normal range of the spontaneous background for the TK locus.

All evaluations of mutagenic activity are based on consideration of the concurrent solvent and negative contrat values run with the experiment in question. Positive control values are not used as reference points, but are included to ensure that the current ceil population responds to direct and promutagens under the appropriate treatment conditions.
Occasionally, a single point within a concentration range will show an increase 2.5 times greater than the spontaneous background. If the increase is at the high dose, is reproducible, and if an additional higher dose level is not feasible because of toxicity, the chemical can be considered mutagenic. If the increase is interna] within the dose range and is not reproducible, the increase will normally be considered aberrant. If the internat increase is reproducible, several doses clustered around the positive concentration will be examined to either confirm or reject the reliability of the effect.

Statistics:

No data

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: >= 65.6 µg/ml without S9, >=188 µg/ml with S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

With and without activation, the test compound did not induce a significant increase in mutant frequency at any of the tested doses. A 2.5-fold increase over the background frequency (average of the solvent and untreated negative control values) is considered necessary to demonstrate mutagenesis at any given dose level. Even at the relatively toxic doses of 188 ug/ml without activation (13.5% relative growth) and 375 µg/ml with activation (26.6% relative growth) the mutant frequencies observed were comparable to the negative controls.

The validity of the mutagenesis assay can be assessed by the results obtained for the positive and negative controls. The cloning efficiencies for the negative (solvent and untreated) controls varied from 100% without activation to 99% with activation, which demonstrates excellent culturing conditions for the assay. The negative control mutant frequencies were all within the normal range for nonactivation and activation tests, and the positive control compounds yielded frequencies in the normal range that were greatly in excess of the negative control values.

Applicant's summary and conclusion

Conclusions:

DPG did not induce a significant increase in mutations at the TK locus in L5178Y mouse lymphoma cells at applied concentrations of 16.4 to 188.0 ug/ml without activation and at 32.8 to 525 ug/ml with microsomal activation. These concentration ranges included highly toxic treatments. Therefore, the test compound is considered to be inactive in the Mouse Lymphoma Forward Mutation Assay.

Executive summary:

This study evaluates DPG for its ability to induce forward mutation in the L5178Y TK+/- mouse lymphoma cell line, as assessed by colony growth in the presence of 5-bromo-2'-deoxyuridine (BrdU). DPG did not induce a significant increase in mutations at the TK locus in L5178Y mouse lymphoma cells at applied concentrations of 16.4 to 188.0 ug/ml without activation and at 32.8 to 525 ug/ml with microsomal activation. These concentration ranges included highly toxic treatments. Therefore, the test compound is considered to be inactive in the Mouse Lymphoma Forward Mutation Assay.