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Toxicological information

Genetic toxicity: in vitro

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Administrative data

in vitro gene mutation study in mammalian cells
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
according to guideline
other: UKEMS Guidelines (1990) and ICH Tripartite Harmonised Guideline on Genotoxicity and ICH Requirements for Registration of Pharmaceuticals for HUman Use, Genotoxicity: a standard battery for genotoxicity testing of pharmaceuticals
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Details on test material:
Dapsone, batch no. 70522014, white powder, stored at 1-10 degress C, in the dark.
Source : Sigma Aldrich Co Ltd, Gillingham UK


Target gene:
Thymidine kinase gene
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
derived from the American Type Culture collection
Metabolic activation:
with and without
Metabolic activation system:
male Sprague Dawley rats liver enzymes induced with Aroclor 1254
Test concentrations with justification for top dose:
Cells were grown in RPMI 10 under 5 % v/v CO2 in the air, 10% Fetal Horse Serum
Vehicle / solvent:
DMSO final conc 1 %
Untreated negative controls:
Negative solvent / vehicle controls:
True negative controls:
Positive controls:
Details on test system and experimental conditions:
Expression period was 2 days, cell densitites were adjusted to 10E+4/ml, and samples were dilutes to 8 cells/ml for cell viability.
Evaluation criteria:
1) the acceptance criteria were met
2) the mutant frequency at one or more concentrations was significantly greater than the negative control
3) there was a significant dose-relationship as indicated by the linear trend analysis.
Statistical significance of mutant frequencies was carried out according to UK UKEMS guidelines Thus the control log frequency was compared with the log mutant frequency from each treatment concentration based on Dunnett's test for multiple comparison and secondly the data were checked for a linear trend in mutant frequencies with treatment concentration using weighted regression.

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
Vehicle controls validity:
Positive controls validity:
Additional information on results:
After 3-hour treatment the cytotoxocicity at 2000 micrograms/ml was too high, so that in the main experiment only 1000 micrograms/ml were tested and evaluates in the absence of S-9.
Thus, in experiment 1, dose levels of 62.5 - 750 microgram/ml were tested without S-9, and 2.5 - 1200 micrograms/ml with S-9.

Any other information on results incl. tables

The positive controls were -nitroquinoline1 -oxide and benzo-a-pyrene.

2 independent experiments were made, using the fluctuation protocol. Both were 3-hour incubation, with a 24-hour incubation.

In the 1st experiment, a survival rate at 1200 micrograms/ml was 20.47 % in absence of S-9 and 51.96% in presence of S-9 at 750 µg/ml.

In the second experiment a survival rate of 33.69% was found in the highest dose level in presence of S-9 (1200 µg/ml).

There was no increase in the mutant frequencies at any dose level, while the positive controls gave results as expected from the historical controls.

Applicant's summary and conclusion

Based on the results in this mammalian gene mutation test on the tk-locus of L5178 Y mouse lymphoma cells, it is concluded that Dapsone is not mutagenic in mammalian cells.
Executive summary:

Dapsone was assayed for its ability to induce mutation at the tk locus in mouse lymphoma cells using a fluctuation protocol. The study consisted of a cytotoxicity range-finding experiment followed by two independent experiments, in the absence and presence of metabolic activation (rat liver S9).

A 3-hour treatment incubation period was used for all experiments performed in the presence of S9. In the absence of S9, the rangefinder was performed using a 3 hour and 24-hour treatment incubation period, Experiment 1 was performed using a 3 hour treatment incubation and Experiment 2 was performed using a 24 hour treatment incubation.

In the cytotoxicity range-finding experiment, 3-hour treatment, six concentrations were tested, in the absence and presence of S9, with doses ranging from 62.5 to 2000 g/mL (limited by solubility). Extreme toxicity was observed at the top concentration tested, both with and without metabolic activation. The maximum concentration where cells survived treatment was 1000 g/mL, with a 5.79% and 14.47% relative survival in the absence and presence of S9 respectively.

In the 24h range finding experiment, nine concentrations were chosen in the absence of S9, with concentrations up to 2000 g/mL. Extreme toxicity was observed at the top two concentrations tested (1000 and 2000 g/mL), the top concentration where cells survived was 500 g/mL, which yielded 10.14% relative survival.

Accordingly, for the first experiment, six concentrations were chosen without S9, upto 750 g/mL and seven concentrations were chosen in the presence of S9, upto 1200 g/mL. Two days post treatment, all concentrations were selected to determine viability and TFT resistance. The top concentrations tested were 75 and 1200 g/mL, which yielded 51.96 % (absent of S9) and 20.47% (with S9).

In the second experiment, seven concentrations were tested in the absence of s9, ranging from 31.25 to 750 g/mL and seven concentrations were tested in the presence of S9 (3h) ranging from 62.5 to 1200 g/mL. Two days after the end of treatment all concentrations tested in the absence and presence of S9 were selected to determine TFT resistance. However, the top concentration tested in the absence of S9 (750 g/mL), was later rejected from analysis due to excessive heterogeneity. This concentration was also highly toxic, yielding less than 10% relative survival. The top concentrations analyzed were 500 g/mL in the absence of S9 and 1200 g/mL in the presence of, which yielded 27.69% and 33.69% relative survival respectively. The top concentration in all experiments was limited by test article solubility in tissue culture medium.

No statistically significant increase in mutant frequency was observed following treatment with dapsone at any concentration level tested, in the absence or presence of S9. Dapsone was not considered mutagenic in this test system.