Dapsone

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Summary of Available Studies

In vitro

AMES – OECD 471

A bacterial Ames test according to OECD guideline 471 and the EC guidelines was performed with the Salmonella strains TA 98, TA 100, TA 1535, and TA 1537, as well as with the  Escherichia coli strains WP2 pKM 101, and WP2 uvrA pKM 101. None of the strains showed a significant increase of revertant colonies (mutants) neither in the presence nor in the absence of the metabolic rat-enzymes. No potential for gene mutation was detected using the Ames assay.

Mouse Lymphoma TK assay – OECD 476

Dapsone was assayed for its ability to induce mutation at the tk locus in mouse lymphoma cells in an OECD TG 476 in vitro mammalian cell gene mutation study. 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. No potential for gene mutation was detected using the mouse lymphoma thymidine kinase assay.

Chromosome Aberration in human lymphocyte cells – OECD 473

The effect of Dapsone on the number of chromosome aberrations in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system, was tested in a procedure designed according to OECD guideline 473.
Dapsone did not induce a statistically significant increase in the number of cells with chromosome aberrations after 3 hours treatment in the presence of S9-mix. After 3 hours treatment, in the absence of S9-mix, Dapsone induced a statistically significant increase in the number of cells with chromosome aberrations at the highest cytotoxic concentration tested, both when gaps were included and excluded. The number of cells with chromosome aberrations is above the historical control data range and therefore biologically relevant.

No biologically relevant effects of Dapsone on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. It can be concluded that 4,4’-DDS does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described.

It is concluded that Dapsone is clastogenic in vitro after 3 hours treatment in the absence of S9-mix in human lymphocytes under the experimental conditions described.

The positive result in cytogenicity testing in mammalian cells (OECD 473), required in vivo testing as a follow up to confirm the potential for clastogenicity.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

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

Target gene:

Thymidine kinase gene

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:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

yes

Positive controls:

yes

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.

Statistics:

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.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

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.

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.

Conclusions:

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.

 

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29-JUl-2011 to 10-Jan-2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ICH. Topic S2A Genotoxicity: Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals; CPMP/ICH/141/95 Step 5 Guideline (Into Operation April 1996).

Qualifier:

according to guideline

Guideline:

other: ICH. Topic S2B Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals; CPMP/ICH/174/95 Step 5 Guideline (Into Operation March 1998).

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: Cultured peripheral human lymphocytes

Details on mammalian cell type (if applicable):

- Type and identity of media:
Blood samples
Blood samples were collected by venapuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started.
- Culture medium
Culture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 µg/mL respectively) and 30 U/mL heparin.
- Lymphocyte cultures
Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 ml (9 mg/mL) phytohaemagglutinin was added.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: not applicable, immediately after blood collection lymphocyte cultures were started.
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: not applicable, immediately after blood collection lymphocyte cultures were started.

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone.

Test concentrations with justification for top dose:

Dose range finding test:
Without and with S9-mix, 3hr exposure; 24 hr fixation: 33, 100, 333, 1000, 2483 µg/mL
Without S9-mix, 24/48hr exposure; 24/48 hr fixation: 33, 100, 333, 1000, 2483 µg/mL

First cytogenetic test:
Without S9-mix, 3 h exposure time, 24 h fixation time: 500, 750 and 1000 µg/mL
With S9-mix, 3 h exposure, 24 h fixation time: 100, 750 and 1000 µg/ mL
Second cytogenetic test:
Without S9-mix, 24 hr exposure; 24 hr fixation: 30, 70 and 100 µg/mL
Without S9-mix, 48 hr exposure; 48 hr fixation: 10, 30 and 150 µg mL
With S9-mix, 3 hr exposure; 48 hr fixation: 750, 1000 and 1250 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Test compound was soluble in DMSO and DMSO has been accepted and approved by authorities and international guidelines

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

mitomycin C

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hr
- Exposure duration: 3 hr (with and without S9-mix), 24 and 48 hr (without S9-mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 and 48 hr

SPINDLE INHIBITOR (cytogenetic assays): colchicine
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicates in two independent experiments

NUMBER OF CELLS EVALUATED: 100 metaphase chromosome spreads per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of each culture was determined by counting the number of metaphases per 1000 cells

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.

A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.

Statistics:

The incidence of aberrant cells (cells with one or more chromosome aberrations, gaps included or excluded) for each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Chi-square statistics.

Key result

Species / strain:

other: Cultured peripheral human lymphocytes

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH:
Solvent control: 7.78
1000 µg/mL: 7.73
- Effects of osmolarity: No
Solvent control: 424 mOsm/kg
1000 µg/mL: 394 mOsm/kg

- Precipitation: Precipitation in the exposure medium was observed at the top dose of 2483 µg/mL (= 0.01 M)

RANGE-FINDING/SCREENING STUDIES:
- Toxicity was observed at dose levels of 333 µg/ml and above in the absence and presence of S9, 3 hr treatment/24 hr fixation; at dose levels of 100 µg/ml and above in the absence of S9 for the continuous treatment of 24 and 48 hr .


COMPARISON WITH HISTORICAL CONTROL DATA:
- The number of cells with chromosome aberrations found in the solvent and positive control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide induced appropriate responses.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Appropriate toxicity was reached at the dose levels selected for scoring.

Conclusions:

A chromosome aberration study with 4,4’-DDS was performed according to OECD 473 guideline and GLP principles, in cultured peripheral human lymphocytes in two independent experiments. It is concluded that 4,4’-DDS is clastogenic after 3 hours treatment in the absence of S9-mix in human lymphocytes.

Executive summary:

This study describes the effect of 4,4’-DDS on the number of chromosome aberrations in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (phenobarbital and ß-naphthoflavone induced rat liver S9-mix). The possible clastogenicity of 4,4’-DDS was tested in two independent experiments.

4,4’-DDS was soluble in dimethyl sulfoxide, at concentrations of 248 mg/ml and below.
In the first cytogenetic assay, 4,4’-DDS was tested up to 1000 µg/ml for a 3 hour exposure time with a 24 hours fixation time in the absence and presence of 1.8% (v/v) S9-fraction. Appropriate toxicity was reached at this dose level.
In the second cytogenetic assay, 4,4’-DDS was tested up to 100 µg/ml for a 24 hour continuous exposure time with a 24 hour fixation time and up to 150 µg/ml for a 48 hour continuous exposure time with a 48 hour fixation time in the absence of S9-mix. Appropriate toxicity was reached at these dose levels. In the presence of S9-mix 4,4’-DDS was tested up to 1250 µg/ml for a 3 hour exposure time with a 48 hour fixation time. 4,4’-DDS precipitated in the culture medium at this dose level.
The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.
After 3 hours treatment, in the absence of S9-mix 4,4’-DDS induced a statistically significant increase in the number of cells with chromosome aberrations at the highest cytotoxic concentration tested, both when gaps were included and excluded. The number of cells with chromosome aberrations is above the historical control data range and therefore biologically relevant.
4,4’-DDS did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations after 3 hours treatment in the presence of S9-mix, in either of the two independently repeated experiments and after 24 and 48 hours treatment in the absence of S9-mix.
No biologically relevant effects of 4,4’-DDS on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that 4,4’-DDS does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.
Finally, it is concluded that this test is valid and that 4,4’-DDS is clastogenic after 3 hours treatment in the absence of S9-mix in human lymphocytes under the experimental conditions described in this study.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: UKEMS Guidelines (1990), Japanese MHW (1989) and MAFF (1985) Guidelines and ICH Harmonised Tripartite Guideline (1997)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Tryptophan and Histidine-requring genes in Salmonella and Escherichia

Species / strain / cell type:

E. coli, other: WP2 pKM101

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

Araclor 1254-induced rat liver post-mitochondrial fraction (S-9)

Test concentrations with justification for top dose:

8, 40, 200, 1000, 5000 micrograms/plate in the range-finder experiment
8, 40, 200, 1000, 5000 and 4, 20, 100, 500, 2500 micrograms /plate were used in the main experiments

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Remarks:

treatment with solvent

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

treatment with appropriate stock positive control solution

Positive control substance:

sodium azide

Remarks:

other positive controls used were 9-aminoacridine (AAC), 2-aminoanthracene (AAN), 4-nitrochinoline (NQO), and 2-nitrofluorene(2NF)

Details on test system and experimental conditions:

the plating assay was chosen for all experiments.
Incubation was for three days in the dark at 37 degrees Celcius.

Statistics:

M-statistics to check whether ther data were Poisson-distributed,
Dunnetts test to compare the counts of each data point with the control.
Linear regression analysis

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

No positive result has been observed in any of the strains .
In addition to E.coli strain E. coli WP2 uvr A pKM 101 also E.coli strain WP2 pKM 101 was tested.

Remarks on result:

other: all strains/cell types tested

Conclusions:

Dapsone did not induce reverse gene mutation in bacteria (4 Salmonella strains and 2 E.coli strains). The concentrations applied showed at the high end some toxicity in the absence and presence of S-9 activating enzymes.

Executive summary:

Dapsone was assayed for mutation in four strains of Salmonella typhimurium, and two strains of Escherichia coli, both in the absence and presence of metabolic activation by rat liver S-9 in two separate experiments.

In the range finding study, conducted using strain TA100, concentrations of dapsone used were 8, 40, 200, 1000 and 5000 ug/plate plus negative and positive controls. Evidence of toxicity was observed following both maximum test dose treatments.

Experiment 1 treatments of the remaining strains used the same doses as the range-finder experiment. Evidence of toxicity was observed in all strains following maximum test dose treatments in the absence and in the presence of S9. Toxic signs were also observed in some strains in the absence and or presence of S9, following treatments of 1000 ug/plate. In these cases, insufficient data for a thorough assessment of mutagenicity were obtained and repeat treatments in these strains were performed.

Experiment 1 repeat treatment of strains TA1535 and WP2 pKM101 employed a reduced maximum test dose of 2500 ug/plate. For strain WP2 uvrA pKM101 and WP2 uvrA pKM101 were performed with the maximum test dose reduced to 800 ug/plate, a dose shown to be within the toxic range in these stains. A narrowed dose range was employed to investigate more thoroughly those concentrations of Dapsone considered most likely to induce any mutagenic activity. An increased number of test doses was used to ensure sufficient does suitable for mutagenicity assessment were obtained. Evidence of toxicity was apparent following all treatments of strains TA98, TA100 and TA1537 at 2000 and 5000 ug/plate. In strains WP2 pKM101 and WP2 uvrA pKM101 toxic signs were evident at test doses of 320 and 800 ug/plate.

Precipitation of test article was observed on test plates treated in the presence of S9 at 5000 ug/plate when a pre-incubation was employed. Mean numbers of revertant colonies on negative control plates were within acceptable ranges and significantly elevated by positive control treatments. Dapsone did not induce reverse gene mutation in bacteria (4 Salmonella strains and 2 E.coli strains). The concentrations applied showed at the high end some toxicity in the absence and presence of S-9 activating enzymes.

Genetic toxicity in vivo

Description of key information

In vivo

In vivo mammalian erythrocyte micronucleus test in mice – OECD 474

The potential for Dapsone to induce structural chromosomal damage and aneuploidy was assessed by determining the frequency of micronucleated polychromatic erythrocytes in mouse bone marrow.

Mice treated with Dapsone at all doses exhibited frequencies of micronucleated PCE which were similar to the values for the vehicle control group and also fell into the normal range. There were no instances of a statistically significant increase in micronucleus frequency for any of the groups receiving Dapsone. The mean PCE to NCE ratios of the groups given Dapsone were similar to the negative control value and fell within the historical negative control range.

 

Dapsone did not introduce micronuclei in the polychromatic erythrocytes of the bone marrow in mice treated up to 175 mg/kg/day, a dose at which limited mortality and clinical signs of toxicity were observed. The concern for potential clastogenicity which was raised by a positive in vitro chromosomal aberration assay has been addressed by this negative in vivo micronucleus. The absence of clastogenicity potential under in vivo conditions is of higher relevance in the overall assessment of the potential gentoxicity of dapsone to human and therefore it can be concluded that overall, dapsone can be considered as not having genotoxicity potential under in vivo conditions. 

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River-UK Ltd
- Age at study initiation: 7 weeks
- Weight at study initiation: 26-33g
- Diet (e.g. ad libitum): availabe ad-libitum
- Water (e.g. ad libitum): availabe ad-libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-23 deg C
- Humidity (%): 52-61%
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle

Route of administration:

oral: gavage

Vehicle:

1% methylcellulose in water

Details on exposure:

Groups of 8 male mice (as there was no evidence of sex-related difference in toxicity in the range finding test) were used for each dose groups and one control group. Dose levels were 0, 43.75, 87.5, 175 mg/kg bw/day in the main experiment. A positive control group of 8 male mice received 40 mg/kg cyclophosphamide once on day 2 of the experiment.

Duration of treatment / exposure:

2 consecutive days

Frequency of treatment:

daily once

Post exposure period:

none

No. of animals per sex per dose:

8 males per dose (as no evidence of sex-related differences in toxicity was revealed in the range finding test)

Control animals:

yes, concurrent no treatment

Positive control(s):

cyclophosphamide, 40 mg/kg bw once on day 2

Tissues and cell types examined:

Bone marrow were analysed for numbers of micronucleated polychromatic erythrocytes.

Details of tissue and slide preparation:

femours were excised and the content washed out with a syringe into 1% fetal bovine serum in RPMI medium

Evaluation criteria:

- Slides had to have at least 1000 scorable cells (PCE and NCE).
Acceptance Criteria:
The assay was considered valid if the following criteria are met:
1) the incidence of micronucleated PCE in the vehicle control group falls within or close to the historical vehicle control range
2) at least seven animals out of each group are available for analysis, and
3) the positive control chemical (CPA) induces a statistically significant increase in the frequency of micronucleated PCE.
Evaluation Criteria:
A test article is considered positive in this assay if:
1) A statistically significant increase in the frequency of micronucleated PCE occurs at least at one dose, and
2) the frequency of micronucleated PCE at such point exceeds the historical vehicle control range.
the frequency of PCE is higher than the historical control range.

Statistics:

Statistics was applied

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Mice treated with Dapsone at all doses exhibited frequencies of micronucleated PCE which were similar to the values for the vehicle control group and also fell within the normal range. There were no instances of statistically significant increases in micronucleus frequency for any of the groups receiving Dapsone. The mean PCE to NCE ratios of the groups given Dapsone were similar to the negative control value and fell within the historical negative control range.

Conclusions:

Dapsone did not introduce micronuclei in the polychromatic erythrocytes of the bone marrow in mice treated up to 175 mg/kg/day, at a dose which limited mortality and clinical signs of toxicity were observed.

Executive summary:

Dapsone was assessed for in vivo gentoxicity in a mouse bone marrow micronucleus assay. The potential of Dapsone to induce structural chromosomal damage and aneuploidy, by determining the frequency of micronucleated polychromatic erythrocytes in mouse bone marrow after exposure to 0 (negative control), 43.75, 87.5 and 175 mg/kg bw/day given on two consecutive days by oral gavage to groups of 8 male mice.

In a range finding test, doses from 500 to 1000 mg/kg Dapsone were administered orally by gavage as a suspension in 1% methylcellulose. Groups of three male and three female out-bred, virus-free, CD-1 mice were dosed on two consecutive days (mortality permitting). Observations were made over a two day period following the first administration and signs of toxicity recorded. No difference in toxicity was seen between males and females.

Based on the results of the range finding test, doses of 0, 43.75, 87.5 and 175 mg Dapsone/kg/d were given on two consecutive days to groups of 8 male mice. All surviving animals were killed 24 hours after receiving their final dose and femoral bone marrow smears examined. Slides from all animals were analyzed from the negative control, positive control and Dapsone-treated groups.

All animals were observed after dosing, with clinical signs and mortalities recorded. In the main study, bone marrow smears were analyzed for numbers of micronucleated polychromatic erythrocytes. The ratio of polychromatic erythrocytes to normochromatic (mature) erythrocytes was also recorded.

Negative and positive control group results were all confirmed as valid. Mice treated with Dapsone at all doses exhibited frequencies of micronucleated PCE which were similar to the values for the vehicle control group, and fell into the normal range. There were no instances of statistically significant increased in micronucleus frequency for any of the groups receiving Dapsone. The mean PCE to NCE ratios of the groups given Dapsone were similar to the negative control value and fell within the historical negative control range.

Dapsone did not introduce polychromatic erythrocytes of the bone marrow in mice treated up to 175 mg/kg/day. Limited mortality and clinical signs were observed at this dose.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

Dapsone is clastogenic in vitro but not in vivo. The absence of clastogenicity potential under in vivo conditions is of higher relevance in the overall assessment of the potential genotoxicity to human. Overall, Dapsone is considered as not having genotoxicity potential under in vivo conditions. This is further supported by the absence of carcinogenicity potential in human.

Justification for classification or non-classification

Based on the negative in vitro gene mutation studies (OECD 471 and OECD 476), and a negative in vivo micronucleus test in mice (OECD 474), there is no sufficient evidence to trigger the classification of Dapsone for mutagenicity according to UN GHS (Rev8 2019) 3.7.2.1./ CLP regulation (Regulation EC No. 1272/2008).