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Genetic toxicity: in vitro

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Administrative data

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:
12 May to 13 June, 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Proprietary GLP guideline-compliant study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

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 (incl. certificate)
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
The test substance was 2,3-dichloro-1,3-butadiene, batch number Partie Q101 Proben-Nr 1129, purity 95.2670% (GC), molecular weight 123 g/mol. Dose calculations were adjusted to purity. The substance was given internal test item number S1090911. The substance was stored in the refrigerator at 2-8°C, and had an expiry date of 01 July 2010.

Method

Target gene:
HPRT locus of V79 cells
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
Frozen batches of cells are screened for mycoplasma contamination and checked for karyotype stability and spontaneous mutant frequency.
Metabolic activation:
with and without
Metabolic activation system:
S9 mix prepared from the livers of phenobarbital/ß-naphthoflavone induced 8-12 week old male Wistar rats
Test concentrations with justification for top dose:
Preliminary toxicity study: 10.2-1300 µg/ml
Mutagenicity experiment 1: with S9 - 1.25, 2.5, 5.0, 10.0, 15.0, 20.0 and 30.0; without S9 - 2.5, 5.0, 10.0, 20.0, 40.0, 60.0 and 80.0.
Mutagenicity experiment 2: with S9 - 0.63, 1.25, 2.5, 5.0, 10.0, 15.0 and 20.0; without S9 - 10.0, 20.0, 40.0, 80.0, 160.0, 240.0 and 320.0.
Vehicle / solvent:
Immediately before treatment the test material was dissolved in DMSO. The solvent was chosen according to its solubility properties and relative nontoxicity to the cells. The final concentration of DMSO in culture medium was 0.5% v/v.
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Migrated to IUCLID6: Without S9; 0.15 mg/ml
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
Migrated to IUCLID6: With S9; 1.1 µg/ml
Details on test system and experimental conditions:
Large stocks of the V79 cell line (obtained from the Laboratory for Mutagenicity Testing, Germany) are stored in liquid nitrogen in the cell bank of Harlan CCR allowing repeated use of the same batch. Before freezing the level of spontaneous mutants was depressed by treatment with HAT-medium. Thawed stock cultures were propagated at 37°C in 80 cm² plastic flasks. About 5x10E5 cells are seeded into each flask with 15 ml of MEM (minimal essential medium) supplemented with 10% FBS (fetal bovine serum) and 1% neomycin. The cells were sub-cultured twice weekly. Cultures were incubated at 37°C in a 4.5% CO2 atmosphere. For the selection of mutants, the medium was supplemented with 11 µg/ml 6-thioguanine (6-TG).

Preliminary toxicity test
The general culture/experimental conditions were the same as those described for the mutagenicity experiment below. Approximately 500 single cells (duplicate cultures per concentration) were treated with the test material and compared to the controls. Toxicity was indicated by a reduction of the cloning efficiency (CE). The experiment was performed with S9 (4 h treatment) and without S9 (4 h and 24 h treatment). Concentrations ranging from 10.2 to 1300 µg/ml were tested. The highest concentration was chosen with regard to the purity and molecular weight of the test substance.

Mutagenicity study
The mutagenicity study was performed in two independent experiments, using identical procedures. In the first experiment the treatment period was 4 hours with and without S9 mix. In the Second experiment the treatment time was 4 hours with S9 and 24 hours without.
For seeding and treatment of the cell cultures the complete culture medium was MEM containing Hank's salts, Neomycin (5 µg/ml) and Amphotericin B (1%). For selection of mutant cells the complete medium was supplmented with 11 µg/ml thioguanine. All cultures were incubated at 37°C in a humidified atmosphere with 1.5% CO2. Three days (exp. 1) or 2 days (exp. 2) after sub-cultivation stock cultures were trypsinised at 37°C for 5 minutes. Complete culture medium was then added and a single cell suspension prepared. The trypsin concentration for all subculturing steps was 0.2% in Ca-Mg-free salt solution. Prior to the trypsin treatment the cells were rinsed with Ca-Mg-free salt solution containing 200 mg/l EDTA.
The cell suspension was seeded in plastic culture flasks and approx. 1.5x10E6 (single culture) and 5x10E2 cells (in duplicate) were seeded in MEM with 10% FBS for the determination of mutation rate and toxicity, respectively.
After 24 h the medium was replaced with serum-free medium containing the test material, either with or without 50 µl/ml S9 mix. Concurrent solvent and positive controls were treated in parallel. After 4 h this medium was replaced with complete medium following two washing steps with "saline G". In the second experiment the cells were exposed to the test item for 24 h in complete medium, supplemented with 10 % FBS, in the absence of metabolic activation.
The colonies used to determine the cloning efficiency (survival) were fixed and stained approx. 7 days after treatment. Three days (exp. 1) or 4 days (exp. 2) after treatment 1.5x10E6 cells per experimental point was subcultivated in 175 cm² flasks containing 30 ml medium. Following the expression time of 7 days five 80 cm² cell culture flasks were seeded with about 3-5x10E5 cells each in medium containing 6-TG. Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability. The cultures were incubated at 37°C in a humidified atmosphere with 1.5% CO2 for about 8 days. The colonies were stained with 10% methylene blue in 0.01% KOH solution. The stained colonies with more than 50 cells were counted, and if in doubt the colony size was checked with a preparation microscope.
Evaluation criteria:
A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test item producing neither a concentration- related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-muta¬genic in this system.
Statistics:
Least squares linear regression.

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see additional information on results
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Preliminary toxicity test: Strong cytotoxic effects occurred at 40.6 µg/ml and above following 4 hours treatment and at 325.0 µg/ml and above following 24 hours treatment in the absence of metabolic activation. In the presence of metabolic activation no cells survived at 20.3 µg/ml and above.
The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) before the test item was removed. Phase separation was observed by the unaided eye at 650.0 µg/ml and above after 4 hours treatment with and without metabolic activation and at 325.0 µg/ml and above after 24 hours treatment without metabolic activation.

Mutagenicity study
The top dose was limited by strong toxicity in all experiments. Cytotoxic effects indicated by a relative cloning efficiency of less than 50% of the solvent control at 60 µg/ml and above without S9 (exp 1) and at 10.0-15.0 µg/ml and above with S9 following 4 hours incubation (exp 2). Following the 24 hour incubation with S9 cytotoxic effects were observed at 240 µg/ml.

No relevant and reproducible increase in mutant colony numbers/10E6 cells was observed following 4 hours treatment without S9. The relative cloning efficiency at 80 µg/ml was 24.5 and 20.8% in cultures I and II, respectively. Several mutant frequecies exceeded the historical range of solvent controls, however this effect was considered to have no relevant impact on the outcome of the study as the induction factor was not exceeded.

Following 4 hours treatment with 15.0 µg/ml with S9 the total growth was reduced to 16.5% in exp 1, culture II and to 26.7% of solvent control in exp 2, culture I. In exp 1 following treatment with a test item concentration of 15.0 µg/ml no cells were detected for and the cell density was decreased to 6.0% of the respective solvent control. However, this dose group was chosen for mutagenicity evaluation as microbial contamination occurred in the lowest dose group and the evaluation of at least four dose groups is required by the OECD guideline. In culture II of exp 2 with S9 the relative cloning efficiency was reduced to 7.3% following treatment with 15.0 µg/ml. The next higher test item concentration of 20.0 µg/ml was continued for mutagenicity evaluation. However, both, relative survival and cell denstity were below 10 % . Therefore, the mutagenicity data generated at 20.0 µg/ml in the second culture of exp 2 with S9 were considered invalid. No relevant and reproducible increase in mutant frequencies was observed. The induction factor remained well within the threshold of three times the corresponding solvent control in both cultures. However, several mutant frequencies slightly exceeded the historical range of solvent controls. This effect was considered to have no relevant impact on the outcome of the study as the induction factor was not exceeded in either dose group and the effect was not reproducible in the second culture.

The relative cloning efficiency was reduced to 19.9% of the respective controls at a concentration of 320 µg/ml, following 24 h treatment without S9. In the second culture relative cloning efficiency was reduced to 41.4% of the control. No relevant and reproducible increase in mutant frequencies was observed. However the induction factor was increased above the threshold of three times the corresponding colvent control (3.0 and 4.5). This effect was not reproducible in the parallel culture, and was therefore considered to have no relevant impact on the outcome of the study.

A linear regression analysis was performed to assess a possible dose dependent increase of mutant frequency. A single significant dose dependent trend was observed in the second culture of exp 2 with S9. All mutation frequences were well within the historical range of solvent controls at the test facility, this statistical significant result was not considered to be biologically relevant.

In both experiments with and without S9, the range of solvent controls was 5.9 to 30.5 mutants per 10E6 cells. The range of treated groups was 7.1 to 57.5 mutants per 10E6 cells.

The positive controls showed distinct increases in induced mutant colonies.
Remarks on result:
other: strain/cell type: V79
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

The test substance did not induce gene mutations at the HPRT locus in Chinese Hamster V79 cells in vitro, and is therefore considered to be non-mutagenic under the conditions of the study.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activatio

2,3-dichloro-1,3-butadiene is non-mutagenic to mammalian cells in vitro, when tested with and without metabolic activation.
Executive summary:

The mutagenic potential of 2,3 -dichloro-1,3 -butadiene was determined in Chinese hamster V79 cells by investigating gene mutations at the HPRT locus.

An initial cytoxicity assay was performed to determine dose levels for the main study. In the main study, two independent experiments were performed each using two parallel cultures. The first mutagenicity experiment was performed with and without metabolic activation (S9 mix) and a treatment period of 4 hours. The second mutagenicity experiment was performed with a treatment time of 4 hours with metabolic activation, and 24 hours without. The highest concentration tested was 1300 µg/ml.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in either mutagenicity experiment. Positive controls induced a distinct increase in mutant colonies.

In conclusion, 2,3 -dichloro-1,3 -butadiene did not induce gene mutations at the HPRT locus in V79 cells under the conditions of the study and therefore is considered to be non-mutagenic in this assay.