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Genetic toxicity: in vitro

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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:
03 March 2009 to 09 June 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed in accordance with OECD test guidelines in compliance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
not specified
Details on test material:
Identification: DUSANTOX 86Lot Number: 005/07 (Annex 2 to the Final report)Producer: Duslo a.s., Sal'a, Slovak RepublicStorage conditions and stability: Product is stable at room temperature, protected against sunlight. The test article has been stored approximately at 20°C in the laboratory No. 412A. The storage conditions were monitored according to (SOPs 11, 14 ). Life time of the test article is 24 months from the manufacturing date. Handling with tested compound according to SOP 14. The records from monitoring of temperature in laboratory No. 412A will be archivised according to Principle of GLP- Department of Biology 2008, chapter 10 (4).The test substance has been maintained in the Department of Microbiology during the study and until one year after finishing of study and remaining test article will be returned to the customer.Safety: The sponsor provided relevant occupational safety information known about the test article (Certificate of Safety Data from 1st June 2007).

Method

Target gene:
hypoxantine-phosphoribosyl transferase (HPRT)
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
V79 cells (Lot No. 05F013), originally derived from male Chinese hamster lung tissue by Ford and Yerganian in 1958, were used. The cells used in our experiments were obtained from ECACC (European Collection of Cell Cultures), England.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix, methylcholanthrene-induced rat liver
Test concentrations with justification for top dose:
100 to 1000 μg/ml
Vehicle / solvent:
dimethylsulfoxide (DMSO)
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
1% DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
DUSANTOX 86: Tested compound was dissolved in dimethylsulfoxide (DMSO) and added to the serum free culture medium to the desired concentrations. Final DMSO concentration in the medium never exceeded 1% and the control group was exposed to an equivalent concentration of this solvent. All solutions of tested compound were made up freshly before use.

Concentration range was selected according to preliminary cytotoxicity assay (determination of the cells growth). The used concentration range was from 100 to 1000 μg/ml for experiments without and with metabolic activation using 3 h treatment. Five different concentrations were evaluated in experiments.Positive and negative controls: In each experiment following positive and negative controls were used:a) negative control - cells were treated with medium containing 1% DMSO without test compounds - for determination of spontaneous mutations.For the verification of cell line mutability were used positive controls:b) positive control for experiments without metabolic activation: ethylmethanesulfonate (EMS) (Sigma Lot 125K1797)- 0.4 mg/ml was dissolved in DMSO and diluted with culture medium immediately prior to usec) positive control for experiments with microsomal fraction S9: 7,12-dimethyl-benz(a)anthracene (DMBA) (Sigma Lot 383844/1)- 0.003 mg/mlThe compound was dissolved in DMSO and diluted with culture medium immediately prior to use.Metabolic activation: All standard in vitro assays were performed in the absence and presence of a rat liver exogenous metabolic activation system (S9 mix). The post-mitochondrial fraction (S9) was derived from livers of adult Sprague-Dawley male rats (ANLAB, Czech republic), weighing approximately 200 g. The animals were pre-treated with the agent 20-methylcholanthrene (MCH), administered i.p. at 80 mg/kg 5 days prior to killing.The S9 fraction (batch MCH18032008, protein content 41.3 mg/ml, Preparation of S9 fraction - Protocol No. 1/2008) was prepared according to (SOP 2) and stored in liquid nitrogen (-196°C). The S9 mix was prepared with following composition and was added to the culture medium at a final concentration of 10%: 3ml of S9 traction; 1 ml of 40 mM NADP; 1 ml of 50 mM glucose-6-phosphate; 1 ml of 330 mM KCl; 1 ml of 50 mM MgCh; 2 ml of20 mM HEPES buffer; 1 ml of deionised H20 (total volume 10.0 ml) (SOP 12).Cell growth and maintenance: Chinese hamster lung V79 cells were grown in Dulbecco's Modified Eagle's medium (DMEM) with 4.5 g/L glucose, with L-glutamine supplemented with 10% v/v fetal bovine serum (FBS), penicillin (1 00 units/ml) and streptomycin (100 μg/ml).All cultures were incubated at 37°C in a humidified atmosphere of 95% air and 5% C02. For subculture (twice-three times a week), cells were detached by brief treatment with trypsin (1:250; 0.025%)/EDTA solution and counted in suspension by Btirker's chambers.TEST PERFORMANCEProtocol: Tests were performed in compliance with OECD 476 (1) according to (2) and (SOP 12). Cytotoxicity tests (PE) were performed according to (SOP 1).Treatment: Plating efficiency P E; Cells were treated with DUSANTOX 86 for 3 h in medium without serum. Then the cells were washed twice with serum free medium. After this the cells from each sample were trypsinized, diluted and plated in five Petri dishes (diameter 8 cm), at amounts of 3 x 102 cells/dish. Seven days later the colonies had grown, they were stained and the numbers of viable cells were determined.Estimation of mutant frequency; V79 cells (1 x 106) were seeded in Petri dishes containing 5 ml MEM with FBS and cultivated for 24 h. The medium was removed and the cells were treated with DUSANTOX 86 for 3 h in medium without serum, in absence or presence of an external metabolising enzyme system (S9 mix from rat liver). After treatment, cells were washed with FBS free medium and typsinized. The cells were plated into five Petri dishes (diameter 8 cm), at amounts of 3 x 102 cells/dish for determination of cytotoxicity (PE 1) and in two Petri dishes (diameter 10 cm) for mutant freqeuency. After the expression period of 7 days (two subcultures), cells were trypsinized and sampled for resistance to TG. Cells (2 x 103) were seeded in 10 Petri dishes (diameter 10 cm) containing 10 ml of MEM with 10% FBS and 10 μg TG to select for HPR T mutants. In order to determine cell viability (PE 2) at the time of mutant selection, 300 cells each were seeded in 8-cm Petri dishes containing 8 ml MEM with 10 % FBS but without TG. Surviving colonies were counted 7 days after plating and frequency of mutation to thioguanine resistance determined.Staining method: The colonies of viable cells were stained with I % methylene blue in deionized water; 0.2 ml of 1% methylene blue was added into Petri dishes with medium for 20 min.Analysis of samples: After air drying, colonies were counted and the plating efficiency, i.e. number of colonies per number of seeded cells, and mutation frequency, i.e. number of colonies per number of seeded cells times the plating efficiency, were calculated.Treatment of results: Data included cytotoxicity and viability determination, colony counts and mutant frequencies for the treated and control cultures. Plating efficiency (PE) was calculated as percent colonies in treated dishes relative to negative controls. Mutant frequency from 105 cells was expressed as number of mutant cells per number of surviving cells. All data were summarised in tabular form in protocols (SOP 1, 12).
Evaluation criteria:
Positive results for an in vitro mammalian cell gene mutation test indicate that the test substance induces gene mutations in the cultured mammalian cells used. Negative results indicate that under the test conditions, the test substance does not induce gene mutations in the cultured mammalian cells used.There are several criteria for determining a positive result, such as a concentration-related, or a reproducible increase in mutant frequency. Biological relevance of the results is considered first (1).A substance is classified as mutagenic if it induces a concentration-related increase in the number of mutants with at least three concentrations or a reproducible unequivocal increase in the number of mutants after treatment with at least one concentration. In the latter case, the induced number of mutants should be at least 3-fold as compared to the actual spontaneous mutation frequency of the solvent group (3).
Statistics:
Differences between treated and untreated cells were analyzed statistically by using nonparametric Kruskal-Wallis test followed by Multiple range test. Data are given as mean ± standard deviation (5, 6).

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
EVALUATION OF CYTOTOXIC EFFECTS: Preliminary toxicity test was undertaken to define the dose range for V79/HPRT gene mutation test. The evaluation of the cytotoxic effect of DUSANTOX 86 on V79 cells was based on their growth activity. DUSANTOX 86 was tested in concentration range up to maximum dose 1000 μg/ml, limited by precipitation of test article. The cytotoxic effect wasn't manifested at any concentration. On the base of these results, the concentrations ranging from 100 to 1000 μg/ml, for in vitro mammalian cell gene mutation tests without and with metabolic activation were chosen.EVALUATION OF MUTATION FREQUENCY: The mutagenic effect of DUSANTOX 86 was evaluated as resistance to TG. Resistance to the purine analogue TG is a widely used genetic marker in a number of mammalian mutagenesis systems. Mutants resistant to TG arise mainly by alterations of the gene encoding the salvage pathway enzyme hypoxanthine-guanine phosphoribosyl-transferase (HPRT), which normally enables cells to utilize exogenous guanine, hypoxanthine, or their analogues for nucleotide synthesis. Since a functional enzyme is not essential for DNA synthesis, TG mutants may arise through base pair substitution, frameshift mutation, or deletion of the HPR T gene.According to the cytotoxicity of the test substance (first-pilot experiment), the concentration range was adjusted.Five concentrations of DUSANTOX 86 (100, 200, 400, 800, 1000 μg/ml) were evaluated in tests without metabolic activation system in three independent experiments and in tests with S9 in two independent experiments. In all experiments negative (solvent) control (1 % DMSO in medium) for determination of spontaneous mutations was used. For the verification of cell line mutability positive controls EMS (0.4 mg/ml) in tests without S9 and DMBA (0.003 mg/ml) in tests with S9 were used.In first experiment without metabolic activation DUSANTOX 86 did not induce statistically significant increase in mutation frequency in Chinese hamster lung cells at any concentration from range of 100 μg/ml – 1000 μg/ml.In second experiment without metabolic activation statistically significant increases in mutant frequency were found at concentration of 1000 μg/ml. Mutant frequency (10.21 mutants/105 cells) was found to be 1.37- fold higher when compared to concurrent negative control (7.47 mutants/105 cells).In these experiments test article at the top concentration of 1000 μg/ml did not produced reduction of the colony forming ability (PEl) (81.82 %, 102.71 %).Responsiveness of the test system was verified by exposing the cells to the direct-acting mutagen ethylmethanesulfonate (EMS). The well know genotoxic agents produced a high frequency of HPRT mutations (16.50 and 34.84 mutants/105 cells).In the first test with metabolic activation, statistically significant increases in mutant frequency were found at concentration of 1000 μg/ml. Mutant frequency (14.32 mutants/105 cells) was found to be 1.39- fold higher when compared to concurrent negative control (10.31 mutants/105 cells).In second experiment in presence of S9 metabolic system DUSANTOX 86 did not induce statistically significant increase in mutation frequency in Chinese hamster lung cells at any concentration from range of 100 μg/ml – 1000 μg/ml.In these experiments test article at the top concentration of 1000 μg/ml did not produced reduction of the colony forming ability (PEl) (100.68% and 92.07 %).Responsiveness of the test system as well as metabolic activity of S9-mix were verified by exposing the cells to the 7,12-dimethyl-benz(a)anthracene (DMBA). The positive control induced a high frequency of HPRT mutations (42.08 mutants/105 cells and 46.68 mutants/105 cells.In some cases in experiments without and with metabolic activation, the small differences between the test groups at given concentrations vs. control group both for mutants' count as well as frequency of survivals were identified as statistically significant at p<0.05. These differences being of a negligible biological meaning can be explained by low variability of the data in component sample groups.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Mutagenicity of DUSANTOX 86 in V79 assay measured by resistance to TG without metabolic activation (first test)

Substance

Concentration (μg/ml)

Plating efficiency (%)a

Mean mutants/105survivors

PE 1

PE 2

NC

-

100.00

100.00

5.13±1.92

Dusantox 86

100.00

93.11

80.51

6.61±1.79

200.00

78.68

98.40

6.04±1.45

400.00

98.75

89.78

5.34±1.29

800.00

86.83

87.86

5.07±1.38

1000.00

81.82

79.87

6.30±1.94

EMS

400.00

103.76

86.26

16.50±3.18*

Table 2: Mutagenicity of DUSANTOX 86 in V79 assay measured by resistance to TG without metabolic activation (second test)

Substance

Concentration (μg/ml)

Plating efficiency (%)a

Mean mutants/105survivors

PE 1

PE 2

NC

-

100.00

100.00

7.47±1.35

Dusantox 86

100.00

101.93

100.00

6.59±1.41

200.00

118.21

91.06

3.78±2.30

400.00

101.55

102.73

9.54±2.15

800.00

110.01

87.16

4.49±2.45

1000.00

102.71

93.78

10.21±3.18*

EMS

400.00

96.89

97.67

34.84±3.08*

 

 NC, negative control (DMEM containing 1% DMSO); DMSO, Dimethylsulfoxide; DUSANTOX 86, tested compound (100 – 1000 μg/ml); EMS, Ethylmethanesulfonate, positive control (400 μg/ml);

a5 plates were counted for each (PE 1) and (PE 2) point; (PE 1), (PE 2)-plating efficiency relative to the negative control;

* p<0.05 significantly different from the negative control in Kruskal-Wallis test

 

Table 3: Mutagenicity of DUSANTOX86 in V79 assay measured by resistance to TG with metabolic activation (first test)

Substance

Concentration (μg/ml)

Plating efficiency (%)a

Mean mutants/105survivors

PE 1

PE 2

NC

-

100.00

100.00

10.31±2.74

Dusantox 86

100.00

67.01

107.72

9.22±2.26

200.00

79.72

108.46

12.05±2.72

400.00

108.59

102.21

12.95±1.99

800.00

71.47

108.10

10.25±2.02

1000.00

100.68

105.89

14.32±2.85*

DMBA

3.00

66.66

96.33

42.08±3.90*

Table 4: Mutagenicity of DUSANTOX 86 in V79 assay measured by resistance to TG with metabolic activation (second test)

Substance

Concentration (μg/ml)

Plating efficiency (%)a

Mean mutants/105survivors

PE 1

PE 2

NC

-

100.00

100.00

3.44±1.49

Dusantox 86

100.00

82.06

96.34

1.75±0.77

200.00

88.27

102.99

3.53±1.21

400.00

85.17

96.01

2.85±1.23

800.00

92.41

95.68

4.01±1.61

1000.00

92.07

101.99

2.74±1.01

DMBA

3.00

85.52

85.72

46.68±3.98*

 

NC, negative control (DMEM containing 1% DMSO); DMSO, dimethylsulfoxide; DUSANTOX 86, tested compound (100 – 1000 μg/ml); DMBA – 7,12-dimethyl-benz(a)anthracene, positive control ( 3 μg/ml);

a 5 plates were counted for each (PE 1) and (PE 2) point; (PE 1), (PE 2) – plating efficiency relative to the negative control

* p<0.05 significantly different from the negative control in Kruskal-Wallis test

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):negative with and without metabolic activationResults showed that DUSANTOX 86 did not induce a concentration-related increase in mutation frequency at the HPR T locus in V79 cells, both in the absence and presence of metabolic activation.
Executive summary:

DUSANTOX 86 was examined for mutagenicity in Chinese hamster V79 cells at concentrations from 100 to 1000 μg/ml in the absence and in the presence of methylcholanthrene-induced rat liver S9, with cofactors for NADP generation. Incubation time with test article was 3 h in both test systems. The protocol examined the induction of hypoxantine-phosphoribosyl transferase HPRT - deficient mutants and the mutagenicity was measured as 6-tioguanine (TG) resistance.

Validity of the test method was ascertained by positive controls: ethylmethanesulfonate (EMS) in experiments without exogenous activation and 7,12-dimethyl-benz(a)anthracene (DMBA) in experiments with exogenous activation. For each experimental condition 2 independent experiments were performed (2 without S9 fraction and 2 with S9 microsomal fraction) to show the response to induce HPRT mutations.

DUSANTOX 86 in the mammalian cell gene mutation (V79/HPRT) test in the absence and presence of metabolic activation at five tested doses from the concentration range from 100 to 1000 μg/ml did not induced increase in the mutation frequency more than 3 times of the negative control value. At the tested concentration of 1000 μg/ml the statistical significant increase was detected but without biological relevance. Reproducible increase in mutation frequencies wasn't observed.

Our results showed that DUSANTOX 86 did not induce a concentration-related increase in mutation frequency at the HPRT locus in V79 cells, both in the absence and presence of metabolic activation.

DUSANTOX 86 had no ability to produce a reproducible increases in number of mutants after treatment of V79 cells at any concentration from concentration range of 100 – 1000 μg/ml in the absence of metabolic activation and in the presence of S9.

The statistically significant increases of mutation freqeuency in some cases can by caused by low variability of the data in component sample groups and they have a negligible biological meaning.

This study satisfied the requirements for Test Guidelines OPPTS 870.5300, OECD 476 for in vitro mammalian cell gene mutation test and the Principles of Good Laboratory Practice (GLP).