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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 August 2018 - 17 September 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Test material

Constituent 1
Chemical structure
Reference substance name:
4,4'-methylenebis[N-sec-butylaniline]
EC Number:
226-122-6
EC Name:
4,4'-methylenebis[N-sec-butylaniline]
Cas Number:
5285-60-9
Molecular formula:
C21H30N2
IUPAC Name:
4,4'-methylenebis[N-sec-butylaniline]
Test material form:
liquid
Details on test material:
Appearance: Slight yellow liquid
Storage conditions: At room temperature protected from light

Method

Target gene:
Thymidine kinase (TK) locus in L5178Y mouse lymphoma cells
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Source of cells: L5178Y/TK+/- -3.7.2C mouse lymphoma cells from American Type Culture Collection, (ATCC, Manassas, USA), (2001)
- Suitability of cells: recommended test system in international guidelines

MEDIA USED
- Type and identity of media
Horse serum: inactivated by incubation at 56°C for at least 30 minutes
Basic medium: RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/mL and 50 μg/mL, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin
Growth medium: basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
Exposure medium: 3 hr exposure: basic medium, supplemented with 5% (v/v) heat-inactivated horse serum (=R5 medium); 24 hr exposure: basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
Selective medium: basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium) and 5 μg/mL trifluorothymidine (TFT)
Non-selective medium: basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium)
- Properly maintained: yes, stock cultures of the cells were stored in liquid nitrogen (-196°C).
- Periodically checked for Mycoplasma contamination: yes
- Cleansed against high spontaneous background: yes

ENVIROMENTAL CONDITIONS
- All incubations were carried out in a humid atmosphere All incubations were carried out in a humid atmosphere (80 - 100%, actual range 52 -99%) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.1 - 37.5°C).
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg body weight).
Test concentrations with justification for top dose:
Dose-range finding test (with and without S9-mix, 3 hour treatment; without S9-mix, 24 hour treatment): 9.8, 20, 39, 78, 156, and 313 μg/mL

Main experiment 1
3 hour treatment (Without S9-mix): 0.31, 0.63, 1.25, 2.5, 5, 10, 20, 30, 35 and 40 μg/mL exposure medium.
3 hour treatment (With S9-mix): 2.5, 5, 10, 20, 40, 60, 80, 90, 100, 120 and 140 μg/mL exposure medium.

Main experiment 1a
3 hour treatment (without S9-mix): 0.63, 1.25, 2.5, 5, 10, 20, 30, 35, 40, 50, 60, 70 and 80 μg/mL exposure medium

Main experiment 2
24 hour treatment (without S9): 1.25, 2.5, 5, 10, 15, 20, 25, 30, 35, 40, 45 and 50 μg/mL exposure medium.

Vehicle / solvent:
- Vehicle used: DMSO
- Justification for choice of vehicle: Based on a solubility test, DMSO was selected as vehicle.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Without S9; in DMSO, 15 µg/mL for the 3 hours treatment period and 5 µg/mL for the 24 hours treatment period.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9; 7.5 µg/mL in Hanks’ balanced salt solution without calcium and magnesium.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding: below 1 x 10^6 cells/mL

TEST ITEM PREPARATION
The test item was dissolved in DMSO. Test item concentrations were used within 1 hour after preparation.


DURATION
- Prior to dose-range finding and mutagenicity testing, the mouse lymphoma cells were grown for 1 day in R10-medium containing 10^-4 M hypoxanthine, 2 x 10^-7 M aminopterine and 1.6 x 10^-5 M thymidine (HAT-medium) to reduce the amount of spontaneous mutants, followed by a recovery period of 2 days on R10-medium containing hypoxanthine and thymidine only. After this period cells were returned to R10-medium for at least 1 day before starting the experiment.
- Exposure duration: 3 hours (experiment 1; with and without S9-mix); 24 hours (experiment 2; without S9-mix).
- Expression time: 2 days in which at least 4 x 10^6 cells were subcultured every day
- Selection time (if incubation with a selection agent): 11 or 12 days

SELECTION AGENT (mutation assays): 5 µg/mL trifluorothymidine (TFT)
STAIN: 0.5 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)

NUMBER OF REPLICATIONS:
- test concentrations: 1
- positive control: 1
- solvent control: 2

DETERMINATION OF THE MUTATION FREQUENCY
For determination of the cloning efficiency the cell suspensions were diluted and seeded in wells of a 96-well dish. One cell was added per well (2 x 96-well microtiter plates/concentration) in non-selective medium.
For determination of the mutation frequency (MF) a total number of 9.6 x 10^5 cells per concentration were plated in five 96-well microtiter plates, each well containing 2000 cells in selective medium (TFT-selection), with the exception of the positive control groups where a total number of 9.6 x 10^5 cells/concentration were plated in ten 96-well microtiter plates, each well containing 1000 cells in selective medium.
The microtiter plates for cloning efficiency and MF were incubated for 11 or 12 days. After the incubation period, the plates for the TFT-selection were stained for 1.5-2 hours, by adding 0.5 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to each well. The plates for the cloning efficiency and MF were scored with the naked eye or with the microscope.

ACCEPTABILITY CRITERIA
A mutation assay was considered acceptable if it met the following criteria:

a) The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120% in order to have an acceptable number of surviving cells analyzed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 10^6 survivors and ≤ 170 per 10^6 survivors.
c) The suspension growth (SG) over the 2-day expression period for the solvent controls should be between 8 and 32 for the 3 hour treatment, and between 32 and 180 for the 24 hour treatment.
d) The positive control should demonstrate an absolute increase in the total mutation frequency, that is, an increase above the spontaneous background MF (an induced MF (IMF)) of at least 300 x 10^-6. At least 40% of the IMF should be reflected in the small colony MF. And/or, the positive control has an increase in the small colony MF of at least 150 x 10^-6 above that seen in the concurrent solvent control (a small colony IMF of 150 x 10^-6).
Evaluation criteria:
EVALUATION CRITERIA:
a) Any increase of the mutation frequency should be evaluated for its biological relevance including comparison of the results with the historical control data range.
b) The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.

A test item is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.

A test item is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.

A test item is considered negative (not mutagenic) in the mutation assay if: none of the tested concentrations reaches a mutation frequency of MF(controls) + 126

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:
DOSE-RANGE FINDING STUDY

Precipitation observed:
78 μg/mL and above directly after adding.
156 μg/mL and above after 3 hours treatment.
313 μg/mL after 24 hours treatment.

3 hours treatment without S9-mix
Relative suspension growth was 14% at a dose level of 39 μg/mL compared to the relative suspension growth of the solvent control.
No significant cell survival was observed at test item concentrations of 78 μg/mL and above.

3 hours treament with S9-mix
Relative suspension growth was 29% at a dose level of 78 μg/mL compared to the relative suspension growth of the solvent control.
No significant cell survival was observed at test item concentrations of 156 μg/mL and above.

24 hours treatment without S9-mix
The relative suspension growth was 5% at the test item concentration of 39 μg/mL compared to the relative suspension growth of the solvent control.
No significant cell survival was observed at test item concentrations of 78 μg/mL and above.

MAIN EXPERIMENT 1

In the 3 hours treatment without S9-mix, no dose level with a cell survival below 46% was reached, therefore this experiment was rejected and replaced by experiment 1a.

The dose levels of 0.63 to 20 μg/mL showed no cytotoxicity. Therefore, the dose levels of 2.5 and 20 μg/mL were not regarded relevant for mutation frequency measurement. The dose levels of 35 and 40 μg/mL showed similar cell growth delay. Therefore, the dose level of 40 μg/mL was not regarded relevant for mutation frequency measurement. The dose levels of 70 and 80 μg/mL were not used for mutation frequency measurement, since these dose levels were too toxic for further testing.

In the presence of S9-mix, the dose levels of 2.5 to 40 μg/mL showed no cytotoxicity. Therefore, the dose levels of 2.5 and 10 μg/mL were not regarded relevant for mutation frequency measurement. The dose levels of 120 and 140 μg/mL showed similar cell growth delay. Therefore, the dose level of 120 μg/mL was not regarded relevant for mutation frequency measurement.

The dose levels selected to measure mutation frequencies at the TK-locus were:
Without S9-mix: 0.63, 1.25, 5, 10, 30, 35, 50 and 60 μg/mL exposure medium.
With S9-mix: 5, 20, 40, 60, 80, 90, 100 and 140 μg/mL exposure medium.

Precipitation observed:
Without S9-mix: no precipitation
With S9-mix: 140 μg/mL.

The relative total growth of the highest test item concentration was 12% compared to the total growth of the solvent controls both in the absence and presence of S9-mix.

No biologically relevant increase in the mutation frequency at the TK locus was observed after treatment with the test item either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the test item treated cultures were comparable to the numbers of small and large colonies of the solvent controls.


MAIN EXPERIMENT 2

The dose levels of 25 and 30 μg/mL showed similar cell growth delay. Therefore, the dose level of 25 μg/mL was not regarded relevant for mutation frequency measurement. The dose levels of 40 to 50 μg/mL were not used for mutation frequency measurement, since these dose levels were too toxic for further testing.

The dose levels selected to measure mutation frequencies at the TK-locus were:
1.25, 2.5, 5, 10, 15, 20, 30 and 35 μg/mL exposure medium.

The relative total growth of the highest test item was 15% compared to the total growth of the solvent control.

No biologically relevant increase in the mutation frequency at the TK locus was observed after treatment with the test item. The numbers of small and large colonies in the test item treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

CONTROLS

The mutation frequency of one of the solvent control cultures in two experiments were recorded to be outside the range of ≥ 50 per 10^6 survivors and ≤ 170 per 10^6 survivors (41 and 184 per 10^6 survivors in the first and second main experiment, respectively). The duplicate solvent controls were within the acceptability criteria (68 and 165 per 10^6 survivors in the first and second main experiment, respectively). According to the guideline a single solvent control group is sufficient. Hence determination of the mutagenicity of the test item with only one solvent control had no effect on the reliability of the results in the experiment.
The suspension growth over the two-day expression period for cultures treated with DMSO was between 15 and 23 (3 hour treatment) and 80 and 85 (24 hour treatment).
Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Applicant's summary and conclusion

Conclusions:
An in vitro mammalian cell gene mutation test was performed according to OECD guideline 490 and GLP principles with 4,4'-methylenebis-N-sec-butylaniline. Based on the results, 4,4'-methylenebis-N-sec-butylaniline is not mutagenic with and without metabolic activation in the TK mutation test system under the experimental conditions described in this report.
Executive summary:

An in vitro Mammalian Cell Gene Mutation Test using the Thymidine Kinase Gene was done according to OECD guideline 490 and GLP principles in order to evaluate the mutagenic potential of 4,4’-methylenebis-N-sec-butylaniline. The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay. In addition, positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation demonstrating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. In the absence of S9-mix, 4,4’-methylenebis-N-sec-butylaniline did not induce a biologically relevant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modification in the duration of treatment. In the presence of S9-mix, 4,4’-methylenebis-N-sec-butylaniline did not induce a biologically relevant increase in the mutation frequency. In conclusion, 4,4’-methylenebis-N-sec-butylaniline is not mutagenic in the mouse lymphoma L5178Y test system.

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