N-methoxy-1-(2,4,6-trichlorophenyl)propan-2-amine

Genetic toxicity: in vitro

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:

13 July 2015 to 29 September 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

methoxy[1-(2,4,6-trichlorophenyl)propan-2-yl]amine

EC Number:

941-634-6

Cas Number:

1228284-78-3

Molecular formula:

C10H12Cl3NO

IUPAC Name:

methoxy[1-(2,4,6-trichlorophenyl)propan-2-yl]amine

Test material form:

other: liquid

Details on test material:

- Physical state: Brown liquid
- Purity test date: 28 May 2015
- Stability under test conditions: Recertification date 31 May 2017.
- Storage condition of test material: Room temperature.

Method

Target gene:

This in vitro test is an assay for the detection of forward gene mutations at the autosomal thymidine kinase (TK) locus of heterozygous L5178Y/TK+/- cells to TKP-/- mutants.

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: Prior to mutagenicity testing the amount of spontaneous mutants was reduced by growing the cells for one day in RPMI 1640-HAT medium supplemented with:
Hypoxanthine 5.0 x 10-3 M
Aminopterin 2.0 x 10-5 M
Thymidine 1.6 x 10-3 M
Glycine 5.0 x 10-3 M

The incubation of the cells in HAT-medium was followed by a recovery period of 2 days in RPMI 1640 medium containing:
Hypoxanthine 1.0 x10-4 M
Thymidine 1.6 x 10-3 M

After this incubation the L5178Y cells were returned to normal RPMI 1640 medium (complete culture medium).

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Experiment I
without metabolic activation: 4.1; 8.2; 16.4; 32.9; 65.8, 132.0 µg/mL
with metabolic activation: 4.1, 8.2; 16.4; 32.9; 65.8, 132.0 µg/mL

Experiment II
without metabolic activation: 11.0; 22.0; 44.0, 66.0, 88.0, 110.0 µg/mL
with metabolic activation: 22.0; 44.0; 66.0, 88.0, 132.0, 176.0 µg/mL

Due to phase separation visible at the end of treatment only the following concentrations were evaluated:

Experiment I
without metabolic activation: 4.1; 8.2; 16.4; 32.9 and 65.8 µg/mL
with metabolic activation: 8.2; 16.4; 32.9; 65.8 and 132.0 µg/mL

Experiment II
without metabolic activation: 11.0; 22.0; 44.0 and 66.0 µg/mL
with metabolic activation: 22.0; 44.0; 66.0 and 88.0 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9 mix 19.5 µg/mL = 0.18 mM

Controls 2

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9 mix 3.0 µg/mL = 10.7 µM, 4.5 µg/mL = 16.1 µM

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 10-15 days

SELECTION AGENT (mutation assays): TFT (SERVA, 69042 Heidelberg, Germany)

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: After the expression period the cultures were selected. Cells from each experimental group were seeded into 2 microtiter plates so that each well contained approximately 4x10^3 cells in selective medium. The viability (cloning efficiency 2) was determined by seeding about 2 cells per well into microtiter plates (same medium without TFT). The plates were incubated at 37°± 1.5°C in 4.5% CO2/95.5% water saturated air for 10 - 15 days. Then the plates were evaluated. Colonies were counted manually. In accordance with their size the colonies were classified into two groups. The colony size distribution was determined in the controls and at all concentrations of the test substance. Criteria to determine colony size were the absolute size of the colony (more than 1/3 of a well for large colonies) and the optical density of the colonies (the optical density of the small colonies is generally higher than the large colonies).

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (main expt), relative suspension growth (pre-expt)

Evaluation criteria:

A test substance is classified as mutagenic if the induced mutation frequency reproducibly exceeds a threshold of 126 colonies per 10^6 cells above the corresponding solvent control or negative control, respectively.

A relevant increase of the mutation frequency should be concentration-dependent.

A mutagenic response is considered to be reproducible if it occurs in both parallel cultures.

However, in the evaluation of the test results the historical variability of the mutation rates in solvent controls and the mutation rates of all solvent controls of this study are taken into consideration.

Results of test groups are generally rejected if the relative total growth and the cloning efficiency 1 (survival) is less than 10% of the solvent control unless the exception criteria specified by the IWGT recommendations are fulfilled.

Whenever a test substance is considered mutagenic according to the above mentioned criteria, the ratio of small versus large colonies is used to differentiate point mutations from clastogenic effects. If the increase of the mutation frequency is accompanied by a reproducible and concentration dependent shift in the ratio of small versus large colonies clastogenic effects are indicated.

A test substance is classified as non-mutagenic if the induced mutation frequency does not reproducibly exceed a threshold of 126 colonies per 10^6 cells above the corresponding solvent control or negative control, respectively.

A test substance not meeting the conditions for a classification as mutagenic or non-mutagenic will be considered equivocal in this assay and may be considered for further investigation.

Statistics:

A linear regression (least squares) was performed to assess a possible concentration dependent increase of mutant frequencies using the validated R Script LM.Rnw statistics software. The number of mutant colonies obtained for the groups treated with the test substance was compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological relevance and statistical significance were considered together.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Expt I at 65.8 µg/mL (+/- S9 mix). Expt II at 66.0 µg/mL (- S9 mix) and ≥ 44.0 µg/mL (+ S9 mix). Phase separation noted +/- S9 mix in Expt I and -S9 mix in Expt II.

Vehicle controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

No substantial or reproducible concentration-dependent increase of the mutation frequency was observed in the main experiments with and without metabolic activation. The threshold was exceeded at 44.0 and 88.0 µg/mL in culture II of the second experiment with metabolic activation. However, this increase was judged as biologically irrelevant as it was not reproduced in the parallel culture under identical experimental conditions and no increase in mutation frequency was seen in the first experiment with metabolic activation.

 

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using R Script LM.Rnw statistics software. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was detected in the first culture of the first experiment with metabolic activation. This trend however, was judged as irrelevant as it actually was reciprocal, going down versus increasing concentrations. Another significant dose dependent trend was determined in the second culture of the second experiment with metabolic activation. This trend was judged as biologically irrelevant as it was not reproduced in the parallel culture under identical experimental conditions.

Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies, indicating that the tests were sensitive and valid.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

During the mutagenicity test described and under the experimental conditions reported the test substance did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation. Therefore, the test substance is considered to be non-mutagenic in this mouse lymphoma assay.

Executive summary:

The study was performed to investigate the potential of the test substance to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

 

The assay was performed in two independent experiments, using two parallel cultures each. Experiments I and II were performed with and without liver microsomal activation and a treatment period of 4 hours

          

 The maximum concentration of the pre-experiment was 2819 µg/mL, equal to approximately 10 mM, based on the molecular weight (268.6 g/mol) and the purity (95.3%) of the test substance. The concentration range of the main experiments was limited by cytotoxicity and solubility of the test item.

 

The main experiments I and II were evaluated at the following concentrations:

Experiment I

without metabolic activation:               4.1; 8.2; 16.4; 32.9; and 65.8 µg/mL
with metabolic activation:                8.2; 16.4; 32.9; 65.8; and 132.0 µg/mL

 

Experiment II

without metabolic activation:                    11.0; 22.0; 44.0; and 66.0 µg/mL
with metabolic activation:                         22.0; 44.0; 66.0; and 88.0 µg/mL

 

Relevant cytotoxic effects indicated by a relative total growth of less than 50% in both parallel cultures occurred in experiment I at 65.8 µg/mL without metabolic activation and at 132.0 µg/mL with metabolic activation. In experiment II cytotoxic effects as described above were noted at 66.0 µg/mL without metabolic activation and at 44.0 µg/mL and above with metabolic activation.

 

No substantial or reproducible concentration-dependent increase of the mutation frequency was observed in the main experiments with and without metabolic activation. The threshold was exceeded at 44.0 and 88.0 µg/mL in culture II of the second experiment with metabolic activation. However, this increase was judged as biologically irrelevant as it was not reproduced in the parallel culture under identical experimental conditions and no increase in mutation frequency was seen in the first experiment with metabolic activation.

 

A linear regression (least squares) was performed to assess a possible concentration dependent increase of mutant frequencies. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was detected in the first culture of the first experiment with metabolic activation. This trend however, was judged as irrelevant as it actually was reciprocal, going down versus increasing concentrations. Another significant dose dependent trend was determined in the second culture of the second experiment with metabolic activation. This trend was judged as biologically irrelevant as it was not reproduced in the parallel culture under identical experimental conditions.

In conclusion, it can be stated that during the mutagenicity test described and under the experimental conditions reported the test substance did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation. Therefore, the test substance is considered to be non-mutagenic in this mouse lymphoma assay.