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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:
2011-10-05 to 2012-02-09
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:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
Slightly yellowish, liquid
> 99% Neodecanoic acid, oxiranyl methyl ester, polymer with 4-methylbenzenesulfonic acid
<0.001% each isomer p-, m-, o-Xylene and ethyl benzene

Method

Target gene:
hypoxanthine-guanine phosphoribosyl transferase (HPRT)
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
Cells were maintained in Dulbecco's modified Eagle-Mediumsupplemented with 10% fetal calf serum, penicillin (100 U/mL) and streptomycin
(100 µg/mL) called DMEM-FCS; Cells were periodically checked for the absence of mycoplasma contamination by using the HOECHST stain 33258;
Spontaneous mutation rate was continuously monitored
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
post-mitochondrial supernatant fraction derived from livers of Aroclor 1254-treated rats (S9 mix)
Test concentrations with justification for top dose:
Five concentrations: 6.25; 12.5; 25; 50; 100µg/mL without metabolic activation
62.5, 125, 250, 500, 1000 µg/mL with metabolic activation
Vehicle / solvent:
Test item was completely dissolved in acetone. Preparations of the test item made on the day of use were employed.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: ethyl methanesulphonate (EMS) in direct mutagenicity experiment; 9,10-dimethyl-1,2-benzanthracene (DMBA) in S9 mix mediated assay; both EMS and DMBA were dissolved in DMSO. The applied concentrations were 600 or 700 µg EMS/mL medium or 20 or 30 µg DMBA/mL
Details on test system and experimental conditions:
CELLS AND TISSUE CULTURE MEDIA
- V79 cells were maintained in Dulbecco's modified Eagle-Mediumsupplemented with 10% fetal calf serum, penicillin 3 (100 U/mL) and streptomycin (100 µg/mL) called DMEM-FCS
- Incubation of cultures: at 37°C in a humidified atmosphere (90%) containing 10% CO2
- For subculturing, a trypsin (0.05%)-EDTA (ethylenediaminetetraacetic acid, 0.02%) solution in modified Puck's salt solution A was used.

METHOD OF APPLICATION:
- Exposure to the test item in the presence of S9 mix was performed in Dulbecco's phosphate buffered saline (PBS) which additionally contained 20 mM HEPES (N'-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid) pH 7.4 (PBS-HEPES).


DURATION (see tables below)
- Preincubation period:
* 1 day (in 30 mL DMEM-FCS)
- Exposure duration:
* 4 hours (1st experiment) and 24 hours (2nd experiment) without S9 mix, respectively;
* in the experiments with S9 mix, the medium was replaced by 18 mL S9 mix and the exposure limited to 4 hours.
* the negative control was treated with acetone (the vehicle) in the same way
* After removal of the test item and washing of the plates with PBS cells were trypsinised and a relative plating efficiency was determined for each
dose to obtain an accurate measure of the toxic effect of the chemical
- Expression time (cells in growth medium):
* Three replicate plates (60 mm diameter) were used with a known number of cells.
* Remaining cells were replated and the culture incubation continued until day 8 with 30 mL normal DMEM-FCS with one subcultivation on day 5.
* Afterwards cells were harvested by trypsinisation and replated at a density of 1 000 000 per 150mm diameter dish in DMEM-FCS containing
6-thioguanine (10 µg/mL) for selection of mutants (5 replicate plates), or at approx. 100 to 150 cells (exact number known) per 60 mm diameter
dish in medium without 6-thioguanine for the estimation of plating efficiencies (PE 2), (3 replicate plates).
- Fixation time:
* Plates were fixed and stained after about 8 days (plating efficiency plates) or 12 days (6-thioguanine plates).
- Positive control:
* ethyl methanesulphonate (EMS) in direct mutagenicity experiment;
* 9,10-dimethyl-1,2-benzanthracene (DMBA) in S9 mix mediated assay
both EMS and DMBA were dissolved in DMSO.
* The applied concentrations were 600 or 700 µg EMS/mL medium or 20 or 30 µg DMBA/mL


NUMBER OF REPLICATIONS: three
NUMBER OF CELLS EVALUATED: 1 500 000

DETERMINATION OF CYTOTOXICITY (same procedure was used as employed for the mutagenicity experiments, except that no mutant selection was carried out)
- Method: survival
- A concentration of the test item which produces a low level of survival (10 to 20%) would be used as highest concentration and the survival in the
lowest concentration being approximately the same as that in the negative control.
- Five adequately spaced concentrations are employed
- In this preliminary experiment without and with metabolic activation test item precipitation was noted at the top concentration of 5000 µg
test item/mL. Cytotoxicity in form of decreased plating efficiency was noted starting at concentrations of 100 or 1000 µg test item/mL in the
experiment without and with metabolic activation, respectively. Hence, 100 µg test item/mL were employed as the top concentration for the
mutagenicity tests in the absence and 1000 µg/mL in the presence of metabolic activation.
Rationale for test conditions:
The study was carried out according to the following guidelines: Council Regulation (EC) No 440/2008 method B.17. “Mutagenicity – In vitro mammalian cell gene mutation test” (published in the Official Journal of the European Union L 142, dated May 31, 2008); and OECD Guideline for Testing of chemicals No. 476: In vitro mammalian cell gene mutation test (adopted July 21, 1997).
Evaluation criteria:
The following predetermined descriptive criteria are used for interpretation of the results:
- If in both independent experiments solvent and positive controls show results within the norm and if the test item does not increase the mutation
frequency 2-fold above the mean of the solvent controls under any condition, or if the mutation frequency is always lower than 40 x 10^-6 and if at least 1 000 000 cells per condition have been evaluated, the item is considered as negative in the test.
- In case of a dose-dependent increase of the mutation frequency in both independent experiments (at similar concentrations) to at least 2-fold
solvent control and at least 40 x 10^-6 both in the presence and/or absence of S9 mix, the item is considered as positive in the test.
Statistics:
No satisfactory mathematical methods are available for the statistical analysis of mammalian cell mutagenicity experiments. See evaluation criteria

Results and discussion

Test results
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
precipitation were noted at concentrations of 5000 µg test item/mL. Cytotoxicity in form of decreased plating efficiency: 100 µg/mL without S9mix, 1000 µg /mL with metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES (Preliminary cytotoxicity test):
In the preliminary experiment without and with metabolic activation test item precipitation was noted at the top concentration of 5000 µg test
item/mL. Cytotoxicity in form of decreased plating efficiency was noted starting at concentrations of 100 or 1000 µg/mL in the experiment without and with metabolic activation, respectively. Hence, 100 µg test item/mL were employed as the top concentration for the mutagenicity tests in the
absence and 1000 µg/mL in the presence of metabolic activation.
COMPARISON WITH HISTORICAL CONTROL DATA:
The historical background mutation frequency in this system has been reported to be 1 to 44 mutants per 106 survivors in non-activation solvent
controls and 6 to 46 per 106 survivors in S9 activation solvent controls [1]. The background data obtained at LPT are given at the end of this
chapter. The spontaneous mutation frequency may be variable from experiment to experiment, but should normally lie within the above-mentioned range. The positive controls EMS (600 and 700 µg/mL) and DMBA (20 and 30 µg/mL) should cause a 10-fold or greater increase in mutation
frequency.
The background mutation frequency at LPT ranges from 1.30 to 38.36 x 10-6 clonable cells for the negative controls. The mutation frequency of the positive controls at LPT ranges from 112.1 to 1708.4 x 10 6 clonable cells for EMS and 130.0 to 2693.3 x 106 clonable cells for DMBA
(see table below).
.


TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH:
- Effects of osmolality:
- Evaporation from medium:
- Water solubility:
- Precipitation:
- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES:

COMPARISON WITH HISTORICAL CONTROL DATA:

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Criteria for assay acceptance

Solvent control: As the total number of colonies is normally low and as a single mutation may cause several colonies due to cell division during the expression period, a relatively large variation of the mutation frequency may result. This is especially true, if a low spontaneous mutation frequency is forced by cloning (in order to achieve a high sensitivity of the test).

The historical background mutation frequency in this system has been reported to be 1 to 44 mutants per 106survivors in non-activation solvent controls and 6 to 46 per 106survivors in S9 activation solvent controls [1]. The background data obtained atLPTare given at the end of this chapter. The spontaneous mutation frequency may be variable from experiment to experiment, but should normally lie within the above-mentioned range. The positive controls(600 and 700 µg/mL) and DMBA (20 and 30 µg/mL) should cause a 10-fold or greater increase in mutation frequency.

The background mutation frequency atLPTranges from 1.30 to 38.36 x 10-6clonable cells for the negative controls. The mutation frequency of the positive controls atLPTranges from 112.1 to 1708.4 x 10-6clonable cells forand 130.0 to 2693.3 x 10-6clonable cells for DMBA (see table below).


The mutation frequencies of the solvent controls andthe positive controls without and with metabolic activation for the last 58 experiments (most recent background data, not audited by the QAU-department) are given as follows:

Mutation frequency per 106 clonable cells

 

Without metabolic activation

(24-h exposure)

With metabolic activation

(4-h exposure)

Solvent control (n = 58)

mean

14.11

14.88

SD

7.42

8.20

range

1.30 - 34.80

2.18 - 38.36

Positive control (µg/mL) (n = 58)

 

EMS

(600)

EMS

(700)

DMBA

(20)

DMBA

(30)

mean

449.1

468.4

347.1

563.8444.2

SD

444.2

268.6

241.8

700.1

range

112.1 – 1708.4

152.0 – 976.9

  130.0 – 844.8

151.3 – 2693.3

SD     = Standard deviation

EMS   = ethyl methanesulfonate

DMBA = 9,10-dimethyl-1,2-benzanthracene

Applicant's summary and conclusion

Conclusions:
Under the present test conditions, the test item was tested up to cytotoxic concentrations in the experiments without and with metabolic activation
was negative in the HPRT-V79 mammalian cell mutagenicity test under conditions where positive controls exerted potent mutagenic effects.
Executive summary:

The test item was tested for mutagenic potential in a gene mutation assay in cultured mammalian cells (V79, genetic marker HPRT) both in the presence and absence of metabolic activation by a rat liver post-mitochondrial fraction (S9 mix) from Aroclor 1254-induced animals. The duration of the exposure with the test item was 4 hours or 24 hours in the experiments without S9 mix and 4 hours in the experiments with S9 mix.

The test item was completely dissolved in acetone.

The concentrations employed were chosen based on the results of a cytotoxicity study. In this preliminary experiment without and with metabolic activation test item precipitation was noted at the top concentration of 5000 µg test item/mL. Cytotoxicity in form of decreased plating efficiency was noted starting at concentrations of 100 or 1000 µg test item/mL in the experiment without and with metabolic activation, respectively. Hence, 100 µg test item/mL were employed as the top concentration for the mutagenicity tests in the absence and 1000 µg/mL in the presence of metabolic activation.

 

Main study

Five concentrations ranging from 6.25 to 100 or 62.5 to 1000 µg test item/mL were selected for the experiments without and with metabolic activation.

 

Cytotoxicity

In the main study cytotoxicity in form of decreased plating efficiency (PE1) and (PE2 )was noted in the first and second experiments at the top concentrations 100 or 1000 µg/mL in the absence and presence of metabolic activation, respectively.

 

Experiments without metabolic activation

The mutation frequency of the negative control acetone was 8.25 and 9.09 x 10 -6 clonable cells. Hence, the negative controls were well within the expected range (see below).

The mutation frequency of the cultures treated with concentrations of 6.25, 12.5, 25, 50 or 100 µg test item/mL culture medium ranged from 6.28 to 13.87 x 10 -6 clonable cells. These results are within the normal range of the negative controls.

 

Experiments with metabolic activation

The mutation frequency of the negative control acetone was 9.52 and 11.50 x 10 -6 clonable cells. Hence, the negative controls were well within the expected range (see below).

The mutation frequency of the cultures treated with concentrations of 62.5, 125, 250, 500 or 1000 µg test item/mL culture medium ranged from 7.59 to 13.73 x 10 -6 clonable cells. These results are within the normal range of the negative controls.

The positive controls EMS (ethyl methanesulfonate) in the direct test and DMBA (9,10-dimethyl-1,2-benzanthracene), a compound which requires metabolic activation, caused a pronounced increase in the mutation frequencies ranging from 221.62 to 288.38 x 10-6clonable cells in the case of EMS and ranging from 155.43 to 486.69 x 10 -6 clonable cells in the case of DMBA, indicating the validity of this test system.

The background mutation frequency at LPT ranges from 1.30 to 38.36 x 10 -6 clonable cells for the negative controls. The mutation frequency of the positive controls at LPT ranges from 112.1 to 1708.4 x 10 -6 clonable cells for and 130.0 to 2693.3 x 106 clonable cells for[S214501] DMBA.

 

Conclusion

Under the present test conditions, test item tested up to cytotoxic concentrations in the experiments without and with metabolic activation was negative in the HPRT-V79 mammalian cell mutagenicity test under conditions where positive controls exerted potent mutagenic effects.