Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 Aug - 24 Nov 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: IWGT Recommendations
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Landesinstitut für Arbeitsschutz und Produktsicherheit, Munich, Germany)
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Reference substance name:
Reference substance 001
Cas Number:
675-62-7
Constituent 2
Chemical structure
Reference substance name:
Dichloromethyl(3,3,3-trifluoropropyl)silane
EC Number:
211-623-4
EC Name:
Dichloromethyl(3,3,3-trifluoropropyl)silane
Cas Number:
675-62-7
Molecular formula:
C4H7Cl2F3Si
IUPAC Name:
dichloro(methyl)(3,3,3-trifluoropropyl)silane
Test material form:
other: liquid

Method

Target gene:
Thymidine kinase gene
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 supplemented with: 10 % horse serum (HS), 100 U/100 µg/mL penicillin/streptomycin, 1 mM sodium pyruvate, 2 mM L-glutamine, 25 mM HEPES, 2.5 µg/mL amphotericin B
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
Pre-experiment I with metabolic activation: 0.1, 0.5, 2.5, 5.0, and 7.5 mM
Pre-experiment I without metabolic activation: 0.1, 0.5, 2.5, and 5.0 mM

Pre-experiment II without metabolic activation (24 h long-term exposure): 0.05, 0.1, 0,5, 1.0, 2.0, and 4.0 mM

Experiment I
with metabolic activation: (4 hours)
0.2, 0.5, 1.0, 2.0, 3.0, 4.0, 4.5, and 5.0 mM
and without metabolic activation: (4 hours)
0.05, 0.1, 0.2, 0.5, 1.0, 1.5, 2.4, and 2.8 mM

Experiment II
with metabolic activation: (4 hours)
0.7, 1.5, 2.5, 3.4, 4.2, 4.6, 5.2, and 5.6 mM
and without metabolic activation: (24 hours)
0.05, 0.1, 0.2, 0.4, 0.6, 0.7, 0.8, and 1.0 mM







Vehicle / solvent:
RPMI cell culture medium was used as solvent (RPMI + 5% HS).
- Justification for choice of solvent/vehicle: A solubility test was performed using different solvents and vehicles up to the maximum recommended concentration of 10 mM. Based on the results RPMI cell culture medium was chosen as solvent. The pH value was adjusted to physiological pH 7 with 1 M NaOH if necessary. RPMI medium is compatible with survival of the cells and activity of the S9 mix.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Ethylmethanesulfonate (EMS, 200 and 300 µg/ml, -S9); Methylmethanesulfonate (MMS, 10 µg/ml, -S9); Benzo[a]pyrene (B[a]P, 2.5 µg/ml, +S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: dissolved in medium
DURATION: 4 h (short-term exposure), 24 h (long-term exposure)
Expression time (cells in growth medium): 48 h
Selection time (if incubation with selection agent): about 14 days

SELECTION AGENT ( mutation assay) 5 µg/mL trifluorothymidine
NUMBER OF REPLICATIONS: two separate experiments (I+II) with single exposure; cells were seeded in 4 plates and evaluated
NUMBER OF CELLS SEEDED: 2000 cells per well

DETERMINATION OF CYTOTOXICITY: relative total growth (RTG), cloning efficiency
Evaluation criteria:
The test item is considered mutagenic if following criteria are met:
-The induced mutant frequency meets or exceeds the Global Evaluation factor (GEF) of 126 per 1E+6 cells
- A dose-dependent increase in mutant frequency is detected.

Besides, combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (≥40% of total colonies) is an indication for potential clastogenic effects and/or chromosomal aberrations.
According to the OECD guideline, the biological relevance is considered first for the interpretation of results. Statistical methods might be used as an aid in evaluation the test result.
A test item is considered to be negative if the induced mutant frequency is below the GEF and the trend test is negative.

Statistics:
The non-parametric Mann-Whitney test is applied to the mutation data to prove the dose groups for any significant difference in mutant frequency compared to the negative /solvent controls.

Results and discussion

Test results
Key result
Species / strain:
mouse lymphoma L5178Y cells
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:
RANGE-FINDING/SCREENING STUDIES:
Toxicity and precipitation were seen in the pre-experiments I with and without metabolic activation. Toxicity was seen by reduced relative suspension growth (RSG) at 2.5 mM and higher with and without metabolic activation. Precipitation was noted at 5.0 mM without metabolic activation and 7.5 mM with metabolic activation. In pre-experiment II (24 hours), toxicity was seen starting at 0.5 mM and no precipitation was observed.

Any other information on results incl. tables

Table 1: Main Experiment I - Data with metabolic activation

 Concentration [mM]  Cloning efficiency [%]  relative total growth [%]  Mutant frequency [mutants/1E+06 cells] Induced mutant frequency # [mutants/1E+06 cells]  % small colonies
 0 (control1)  100.0  100.0  97.0  -  16.7
 0 (control2)  100.0  100.0  114.1 -  21.3
 0.2  111.4  121.3  100.8  -4.8  -
 0.5  100.7  95.8  121.8  16.2  -
 1.0  104.3  93.9  129.1  23.5  -
 2.0  98.6  84.8  119.3  13.7  -
 3.0  105.7  53.0  99.1  -6.5  -
 4.0  102.9  24.7  178.3  72.7*  34.3
 4.5  113.6  18.1  186.7  81.1*  36.4
 5.0  110.0  11.8  193.0  87.4*  31.7
 B[a]P (2.5 µg/ml)  90.0  57.6  949.9  844.3*  46.2

Table 2: Main Experiment I - Data without metabolic activation

 Concentration [mM]  Cloning efficiency [%]  relative total growth [%]  Mutant frequency [mutants/1E+06 cells] Induced mutant frequency # [mutants/1E+06 cells]  % small colonies
 0 (control1)  100.0  100.0  139.5 -  17.3
 0 (control2)  100.0  100.0  89.4 -  22.2
 0.05  104.2  110.4  116.6  2.2  15.4
 0.1  102.8  101.8  98.9  -15.5  -
 0.2  106.3  101.5  102.7  -11.7  -
 0.5  93.0  84.4  111.1  -3.4  -
 1.0  100.0  78.6  114.4  -0.1  -
 1.5  98.6  39.8  112.2  -2.3  15.4
 2.4  106.3  19.7  129.8  15.3  31.4
 2.8  100.0  6.9  140.8  26.4  40.0
 EMS (300 µg/ml)  90.9  75.1  926.8  812 .3*  -
 MMS (10 µg/ml)  85.3  62.9  872.1  757.7*  59.2

Table 3: Main Experiment II - Data with metabolic activation

 Concentration [mM] Cloning efficiency [%]   relative total growth [%] Mutant frequency [mutants/1E+06 cells] Induced mutant frequency # [mutants/1E +06 cells] % small colonies 
 0 (control1)  100.0  100.0  80.9 -  26.4
 0 (conrol2)  100.0  100.0  77.7 -  21.8
 0.7  91.0  81.6  103.2  23.9  -
 1.5  95.0  87.0  90.7  11.4  -
 2.5  95.7  70.5  72.4  -6.9  -
 3.4 92.4  49.8  117.5  38.2*  -
 4.2  91.7  33.7  103.4  24.1*  -
 4.6  99.7  32.2  112.9  33.6*  29.7
 5.2  99.0  21.1  91.6  12.3  35.0
 5.6  88.4  8.6  104.8  25.5*  29.1
 B[a]P (2.5 µg/ml)  88.4  57.8  630.1  550.8*  47.2

Table 4: Main Experiment II - Data without metabolic activation

 Concentration [mM]  Cloning Efficiency [%]  relative total growth [%]  Mutant frequency [mutants/1E+06 cells]  Induced mutant frequency # [mutants/1E+06 cells]  % small colonies
 0 (control1)  100.0  100.0  83.1 -  6.0
 0 (control2)  100.0  100.0  98.2 -  15.9
 0.05  95.2  82.7  94.7  4.1  -
 0.1  104.1  78.7  69.5  -21.2  -
 0.2  95.2  66.3  56.8  -33.9*  -
 0.4  82.1  58.3  139.0  48.3  -
 0.6  100.7  46.2  95.3  4.6  
 0.7  96.6  26.2  112.0  21.3  10.9
 0.8  97.9  24.7  101.0  10.3  15.0
 1.0  100.0  9.8  86.2  -4.4  20.4
 EMS (200 µg/ml)  46.9  20.9  3689.7  3599.0*  -
 MMS (10 µg/ml)  46.9  19.5  1889.6  1798.9*  44.1

* Significantly different to solvent control, p<0.05

# Induced mutant frequency = mutant frequency sample - mean value mutant frequency corresponding controls

Applicant's summary and conclusion

Conclusions:
Dichloromethyl(3,3,3-trifluoropropyl)silane has been tested for mutagenicity to mammalian cells in a study conducted according to OECD 476 and GLP. No increase in mutant frequency was observed in the presence or absence of metabolic activation. It is concluded that, under the experimental conditions reported, the test substance is non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.