Registration Dossier

Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14-July-2011 - 27-March-2012
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
21 July 1997
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:
C7 H16 S5
Test material form:
solid - liquid: suspension
Details on test material:
Batch No. DMPT-EC4-24062011


Target gene:
L5178Y (TK+/-)
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 -induced rat S9 fraction

Type and composition of metabolic activation system:
- source of S9: carried out by LPT according to MARON & AMES (1983)
- method of preparation of S9 mix: The enzymes were contained in a 9000 x g supernatant from liver homogenate prepared from male rats treated with 500 mg/kg of Aroclor 1254 five days prior to sacrifice. The treatment with Aroclor 1254 was used to induce mixed function oxidase enzymes capable of transforming chemicals to more active forms. The S9 was retained frozen at below -80°C until used.
- concentration or volume of S9 mix and S9 in the final culture medium: The S9 fraction was tested for protein content (according to LOWRY), and relative P-448/P-450 activity. The protein content of the S9 fraction was 34.5 or 34.2 mg/mL S9, cytochrome P-450: 0.43 or 0.36 nmol/mg protein.
Test concentrations with justification for top dose:
In the preliminary experiment without and with metabolic activation a complete cytotoxicity (decreased survival) was noted at concentrations of 10 µg/mL and higher. Hence, in the main experiments without or with metabolic activation a concentration range of 0.625 to 10 µg/mL were used.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: established vehicle for substances with low water solubility.
Untreated negative controls:
Negative solvent / vehicle controls:
True negative controls:
Positive controls:
Positive control substance:
Details on test system and experimental conditions:
- Number of cultures per concentration single
- Number of independent experiments: two

- Cell density at seeding (if applicable): 5E+05 cells/mL
- Test substance added in medium

- Exposure duration/duration of treatment: 3h or 24h
- Harvest time after the end of treatment (sampling/recovery times): 18-19 days

- Expression time (cells in growth medium between treatment and selection): 1 week
- Selection time (if incubation with a selective agent): 11-12 days
- Fixation time (start of exposure up to fixation or harvest of cells):
- Method used: microwell plates for the mouse lymphoma assay.
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure. 3 µg/mL TFT (final concentration) was added and incubated for 11-12 days
- Criteria for small (slow growing) and large (fast growing) colonies: The ratio of small : large colonies will be calculated from the results of the determination of small to large colonies.
If the test item is positive, the ratio of small to large colonies for the test item will be compared with the corresponding ratios of the positive and negative controls. Based on this comparison the type of the mutagenic properties (i.e. basepair substitutions, deletions or large genetic changes frequently visible as chromosomal aberrations) of the test item will be discussed.

- Method: b cloning efficiency
Evaluation criteria:
Please refer to any other information on materials and methods.

Results and discussion

Test results
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
Vehicle controls validity:
Untreated negative controls validity:
not applicable
Positive controls validity:

Applicant's summary and conclusion

According to the evaluation criteria for this assay, DMPT neither induce mutations nor have any chromosomal aberration potential up to a cytotoxic concentration of 10 µg/mL medium in the absence and presence of metabolic activation.
Executive summary:

DMPT was assayed in a gene mutation assay in cultured mammalian cells (L5178Y TK+/-) both in the presence and absence of metabolic activation by a liver post-mitochondrial fraction (S9 mix) from Aroclor 1254-induced rats. The test was carried out employing two exposure times without S9 mix: 3 and 24 hours, and one exposure time with S9 mix: 3 hours; this experiment with S9 mix was carried out in two independent assays.

DMPT was completely dissolved in DMSO. As the content of the test item was only 95% a correction factor of 1.05 was used.

In the preliminary experiment without and with metabolic activation pronounced to complete cytotoxicity (decreased survival) was noted at concentrations of 10 µg/mL, and higher.

Hence, in the experiments without or with metabolic activation the concentration-ranges of 0.625 to 10 µg/mL were used.

In the main study, cytotoxicity (decreased survival) was noted immediately after treatment (plating efficiency 1) and in the following plating for 5-trifluoro-thymidine resistance (plating efficiency 2) in the presence and absence of metabolic activation at the top concentrations of 10 µg/mL.

Methylmethanesulfonate (MMS) at 10 or 15 µL/mL was employed as a positive control in the absence of exogenous metabolic activation and 3-Methylcholanthrene (3-MC) at 2.5 or 4.0 µg/mL in the presence of exogenous metabolic activation.

The mean values of mutation frequencies of the negative controls ranged from 66.46 to 87.02 per 106 clonable cells in the experiments without metabolic activation and from 55.92 to72.40 per 106 clonable cells in the experiments with metabolic activation and, hence, were well within the historical data-range.

The mutation frequencies of the cultures treated with DMPT ranged from71.21 to 98.78 per 106 clonable cells (3 hours exposure) and from 78.62 to 102.82 per 106 clonable cells (24 hours exposure) in the experiments without metabolic activation and from 58.29 to 82.17 per 106 clonable cells (3 hours exposure, first assay) and from 60.98 to 82.85 per 106 clonable cells (3 hours exposure, second assay) in the experiments with metabolic activation. These results were within the range of the negative control values and, hence, no mutagenicity was observed according to the criteria for assay evaluation.

In addition, no change was observed in the ratio of small to large mutant colonies, ranging from 0.53 to 1.19 for DMPT treated cells and from 1.00 to 1.22 for the negative controls.

The positive controls MMS and 3-MC caused pronounced increases in the mutation frequency ranging from 1238.32 to 1884.11 per 106 clonable cells in the case of MMS and ranging from 696.56 to 1230.27 per 106clonable cells in the case of 3-MC. In addition, the colony size ratio was moderately shifted towards an increase in small colonies, ranging from 1.60 to 2.78 in the case of MMS.