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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:
2009-11-10 to 2010-03-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline 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
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
No. 440/2008 of 30 May 2008
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2,6-dimethyloct-7-en-2-ol
EC Number:
242-362-4
EC Name:
2,6-dimethyloct-7-en-2-ol
Cas Number:
18479-58-8
Molecular formula:
C10H20O
IUPAC Name:
2,6-dimethyloct-7-en-2-ol

Method

Target gene:
Thymidine kinase heterozygous system, TK +/- to TK -/- conversion
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
L5178Y TK +/- 3.7.2c mouse lymphoma cell line, obtained from Dr. J. Cole of the MRC Cell Mutation Unit, University of Sussex, Brighton, UK
Metabolic activation:
with and without
Metabolic activation system:
Rat liver microsomal S9
Test concentrations with justification for top dose:
Preliminary Toxicity Test:
0, 6.09, 12.19, 24.38, 48.75, 97.5, 195, 390, 780, 1560 µg/mL

Main Experiment, 4-Hour, Without S9:
0, 12.5, 25, 50, 100, 125, 150, 175, 200 µg/mL

Main Experiment, 4-Hour, With S9:
0, 25, 50, 100, 125, 150, 175, 200, 250 µg/mL

Main Experiment, 24-Hours, Without S9:
0, 6.25, 12.5, 25, 50, 75, 100, 125, 150 µg/mL
Vehicle / solvent:
Dimethyl sulfoxide
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Without S9 activation: Sigma batch 1419706 and 15108051 at 400 µg/mL (4 hour) and 150 µg/mL (24-hour)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With S9 activation: Acros batch A0164185 at 2 µg/mL
Details on test system and experimental conditions:
Cell Culture

Cells were stored in liquid nitrogen at -196°C. Cells were cultured at 37°C with 5% CO2 in air in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 μg/mL), Sodium pyruvate (1 mM), Amphotericin B (2.5 μg/ml) and 10% donor horse serum (giving R10 media). Cells had a generation time of approximately 12 hours and were sub-cultured accordingly. RPMI 1640 with 20% donor horse serum (R20) and without serum (R0) were used during the course of the study. Master cultures of stock cells were checked and found to be mycoplasma free.

Preparation of test and control materials

The test material was dissolved in dimethyl sulfoxide (DMSO) and the appropriate dilutions were made. The maximum dose level investigated in the preliminary toxicity test was 1560 μg/mL which was equivalent to approximately 10 mM. Analysis for concentration, homogeneity and stability of the test material preparations were not a requirement of the test method and were therefore not performed.

Vehicle and positive controls were used in parallel with the test material. Solvent (DMSO) treatment groups were used as the vehicle controls. Ethylmethanesulphonate (EMS) at 400 μg/mL and 150 μg/mL for the 4-hour and 24-hour exposures respectively, was used as the positive control in the absence of metabolic activation. Cyclophosphamide (CP) 2 μg/ml was used as the positive control in the presence of metabolic activation.

Preliminary toxicity test

A preliminary toxicity test was performed on cell cultures at 5 x 10^5 cells/mL, using a 4-hour exposure time both with and without metabolic activation (S9), and at 1.5 x 10^5 cells/mL using a 24-hour exposure without S9. The dose range used in the preliminary toxicity test was 6.09 to 1560 μg/mL for all three of the exposure groups. Following the exposure period the cells were washed twice with R10, resuspended in R20 medium, counted using a coulter counter and then serially diluted to 2 x 10^5 cells/mL.

The cultures were incubated and sub-cultured after 24 hours by counting and diluting to 2 x 10^5 cells/mL. After a further 24 hours, the cultures were counted and then discarded. The cell counts were then used to calculate Suspension Growth values (SG). The SG values were then adjusted to account for immediate post treatment toxicity, and a comparison of each treatment SG value to the concurrent vehicle control performed to give a % Relative Suspension Growth Value (%RSG).

Results from the preliminary toxicity test were used to set the test material dose levels for the mutagenicity experiment. Maximum dose levels were selected using the following criteria:

i) Maximum recommended dose level, 5000 μg/ml or 10 mM.
ii) The presence of excessive precipitate where no test material-induced toxicity was observed.
iii) Test material-induced toxicity, where the maximum dose level used should produce 10 to 20% survival (the maximum level of toxicity required).

Mutagenicity test (main test)

4-Hour exposures, with and without metabolic activation

An exponentially growing stock culture of cells was set up to provide an excess of cells on the morning of the experiment. The cells were counted and processed to give 1 x 10^6 cells/ml in 10 mL aliquots in R10 medium in sterile plastic universals. The treatments were performed in duplicate (A + B), both with and without metabolic activation (S9-mix) at eight dose levels of the test material (12.5 to 200 μg/mL in the absence of metabolic activation, and 25 to 250 μg/mL in the presence of metabolic activation), vehicle and positive controls. To each universal was added 2 mL of S9-mix if required, 0.2 mL of the treatment dilutions, (0.2 mL for the positive control) and sufficient R0 medium to bring the total volume to 20 mL.

The treatment vessels were incubated at 37°C for 4 hours with continuous shaking using an orbital shaker within an incubated hood.

24-Hour exposures, without metabolic activation

Cells were counted and processed (as above) to give 0.3 x 10^6 cells/mL in 10 mL duplicate cultures established in 25 cm2 tissue culture flasks. To each culture was added 2 mL of the treatment dilutions (0.2 mL for the positive control) and sufficient R10 medium to give a final volume of 20 mL. The dose range of the test material was 6.25 to 150 μg/mL. The treatment vessels were incubated at 37°C with continuous shaking using an orbital shaker for 24 hours.

Cell processing following treatment periods

At the end of the treatment periods for each experiment, the cells were washed twice using R10 medium then resuspended in R20 medium at a cell density of 2 x 10^5 cells/mL. The cultures were incubated and subcultured every 24 hours for the expression period of two days, by counting and dilution to 2 x 10^5 cells/mL.

On Day 2 of the experiment, the cells were counted, diluted to 10^4 cells/mL and plated for mutant frequency (2000 cells/well) in selective medium containing 4 μg/mL 5-trifluorothymidine (TFT) in 96-well microtitre plates. Cells were also diluted to 10 cells/mL and plated (2 cells/well) for viability (%V) in non-selective medium. The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post treatment toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (%V) data a Relative Total Growth (RTG) value.

Plate scoring

Microtitre plates were scored using a magnifying mirror box after ten to fourteen days incubation. The number of positive wells (wells with colonies) was recorded together with the total number of scorable wells (normally 96 per plate). The numbers of small and large colonies seen in the TFT mutation plates were also recorded. Colonies were scored manually by eye using qualitative judgment. Large colonies were defined as those covering approximately ¼ to ¾ of the surface of the well and were generally no more than one or two cells thick. As a rule of thumb, all colonies less than 25% of the average area of the large colonies were scored as small colonies. Small colonies were normally observed to be more than two cells thick. To assist the scoring of the TFT mutant colonies 0.025 ml of MTT solution (2.5 mg/mL in PBS) was added to each well of the mutation plates. The plates were incubated for approximately two hours. the MTT vital stain was taken up by viable cells to give a brown/black color, thus aiding the visualization of the mutant colonies, particularly the small colonies.
Evaluation criteria:
The normal range for mutant frequency per survivor is 50-200 x 10^-6 for the TK+/- locus in L5178Y cells at this testing laboratory. Vehicle control results should ideally be within this range, although minor errors in cell counting and dilution or exposure to the metabolic activation system may cause this to be slightly elevated. Experiments where the vehicle control values are markedly greater than 250 x 10^-6 mutant frequency per survivor are not normally acceptable and will be repeated. Positive control chemicals should induce at least three to five fold increases in mutant frequency greater than the corresponding vehicle control.

For a test material to demonstrate a mutagenic response it must produce a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value. A Global Evaluation Factor (GEF) value was set following International Workshop (Moore et al., 2003; Moore et al, 2006) at 126 x 10^-6 for the microwell method. Therefore any test material dose level that has a mutation frequency value that is greater than the corresponding vehicle control by the GEF of 126 x 10^-6 will be considered positive. However, if a test material produces a modest increase in mutant frequency, which only marginally exceeds the GEF value and is not reproducible or part of a dose-related response, then it may be considered to have no toxicological significance. Conversely, when a test material induces modest reproducible increases in the mutation frequencies that do not exceed the GEF value then scientific judgment will be applied. If the reproducible responses are significantly dose-related and include increases in the absolute numbers of mutant colonies then they may be considered to be toxicologically significant.
Statistics:
Small significant increases designated by the UKEMS statistical package will be reviewed using the above criteria, and may be disregarded at the Study Director's discretion.

Robinson W D et al (1989) Statistical evaluation of bacterial/mammalian fluctuation tests. In: Statistical Evaluation of Mutagenicity Test Data, UKEMS sub-committee on guidelines for mutagenicity testing (Kirkland D J Ed.), Cambridge University Press Report part III, pp102-140.

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
Remarks:
The maximum dose level used was limited by test material toxicity.
Vehicle controls validity:
valid
Untreated negative controls validity:
not valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Preliminary toxicity test

The results for Relative Suspension Growth (%RSG) are as shown in Table 1:

A precipitate of the test material was observed at and above 195 μg/mL in the 4-hour exposure group in the absence of metabolic activation, and at and above 390 μg/ml in both the 4-hour exposure group in the presence of metabolic activation and the 24-hour exposure group in the absence of metabolic activation. The precipitate was observed to become greasy and oily in appearance at 1560 μg/mL in both of the 4-hour exposure groups.

Mutagenicity test results

4-Hour exposures with and without metabolic activation (Table 2):

There was evidence of toxicity following exposure to the test material in both the absence and presence of metabolic activation, as indicated by the %RSG and RTG values. There was no evidence of any significant reductions in (%V) viability in either the absence or presence of metabolic activation, therefore indicating that no residual toxicity had occurred. Near optimum levels of toxicity were achieved in the absence of metabolic activation. Optimum levels of toxicity were not achieved in either the absence or presence of metabolic activation due to the sharp onset of toxicity, despite using a very narrow dose interval. However, a dose level that exceeded the usual acceptable upper limit of toxicity was plated for viability and TFT resistance for each of the exposure groups. It was therefore considered that with no evidence of any toxicologically significant increases in mutant frequency at any of the dose levels, including the dose levels that exceeded the usual upper limit of acceptable toxicity, or in the 24-hour exposure group where optimum levels of toxicity were achieved, the test material had been adequately tested. The excessive toxicity observed at 200 μg/mL in the absence of metabolic activation, and at 250 μg/mL in the presence of metabolic activation, resulted in these doses not being plated for viability or 5-TFT resistance. Acceptable levels of toxicity were seen with both positive control substances.

24-Hour exposure without metabolic activation (Table 3):

As was seen in the Preliminary Toxicity Test, there was evidence of a marked reduction in % RSG and RTG values in cultures dosed with the test material. There was also evidence of a modest reduction in (%V) viability, therefore indicating that residual toxicity had occurred. Optimum levels of test material-induced toxicity were achieved. The positive control induced acceptable levels of toxicity.

The 24-hour exposure without metabolic activation (S9) treatment, demonstrated that the extended time point had an effect on the toxicity of the test material. The vehicle control mutant frequency value was within the acceptable range of 50 to 200 x 10^-6 viable cells. The positive control produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily.

The test material induced a small statistically significant dose related (linear-trend) in the mutant frequency x 10^-6 per viable cell (Table 9). However, statistically significant increases in mutant frequency were not observed at any of the individual dose levels, the GEF was not exceeded at any of the individual dose levels, and the mutant frequency values observed were within the acceptable range for vehicle controls. Therefore, the response was considered to be spurious and of no toxicological significance. Precipitate of the test material was not observed at any of the dose levels.

Any other information on results incl. tables

Table 1: Preliminary Toxicity Test Results


 




















































































Dose


(mg/mL)



% RSG (-S9)


4-Hour Exposure



%RSG (+ S9)


4-Hour Exposure



%RSG (-S9)


24-Hour Exposure



 



 



 



 



0



100



100



100



6.09



93



103



94



12.19



87



116



81



24.38



87



111



80



48.75



90



103



62



97.5



84



101



34



195



0



28



0



390



0



0



0



780



0



0



0



1560



0



0



0



 



 



 



 



 


 


Table 2: Experimental Results


 
























































































































































































4-Hour Test Without S9



 



 



 



 



 



 



Treatment


(mg/mL)



%RSG



RTG



MF



 



 



 



 



0



100



1.00



118.34



12.5*



95



 



 



25



92



1.16



91.83



50



88



1.05



101.08



100



84



0.99



104.88



125



73



0.81



120.90



150



37



0.38



122.92



175**



1



0.03



143.07



200***



0



-



-



Linear trend



 



 



NS



EMS



 



 



 



400



74



0.60



684.79



4-Hour Test With S9



 



 



 



 



 



 



0



100



1.00



149.96



12.5*



92



 



 



25



90



0.98



91.37



50



83



0.88



147.57



100



83



0.97



150.12



125



77



0.81



106.33



150



50



0.50



116.61



175**



9



0.12



111.16



200*



0



-



-



Linear trend



 



 



NS



CP



 



 



 



2



48



0.24



781.04



* Not plated for viability or 5-TFT resistence.


** Treatment excluded from statistics due to toxicity.


 


Table 3: Experimental Results


 





































































































24-Hour Test Without S9



 



 



 



 



 



 



Treatment


(mg/mL)



%RSG



RTG



MF



 



 



 



 



0



100



1.00



115.95



6.25*



104



 



 



12.5



108



0.92



91.35



25



99



0.96



98.55



50



71



0.66



121.85



75



46



0.43



101.71



100



30



0.16



149.79



125



11



0.10



158.82



150*



2



-



-



Linear trend



 



 



p < 0.05



EMS



 



 



 



150



66



0.45



1007.15



* Not plated for viability or 5-TFT resistence.


 

Applicant's summary and conclusion

Conclusions:
The test material did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and was not considered to be mutagenic under the conditions of the test.
Executive summary:

The GLP compliant study was conducted according to a method that was designed to assess the potential mutagenicity of the test material on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method used meets the requirements of the OECD (476), Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008. One main experiment was performed. In this main experiment, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material at eight dose levels, in duplicate, together with vehicle (DMSO) and positive controls. The exposure groups used were as follows: 4-hour exposures both with and without metabolic activation, and 24 hours without metabolic activation. The dose range of test material was selected following the results of a preliminary toxicity test and was 12.5 to 200 |jg/ml for the 4-hour exposure group in the absence of metabolic activation, 25 to 250 |jg/ml for the 4-hour exposure group in the presence of metabolic activation, and 6.25 to 150 \ig/m\ for the 24-hour exposure group in the absence of metabolic activation. The maximum dose level used was limited by test material-induced toxicity. Precipitate of test material was not observed at any of the dose levels in the Mutagenicity Test. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system. The test material did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in any of the three exposure groups. The test material was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

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