4-methoxy-N-phenyl-o-toluidine

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:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from study report

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Principles of method if other than guideline:

The purpose of this study was to assess toxic and genotoxic effects of Methyl 2-napthyl ether on Chinese Hamster Ovary (CHO) cells by using several different in vitro-based assays, including genotoxicity tests based on the OECD Guideline No. 476 “In Vitro Mammalian Cell Gene Mutation Test”.

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Methyl 2-naphthyl ether

EC Number:

202-213-6

EC Name:

Methyl 2-naphthyl ether

Cas Number:

93-04-9

Molecular formula:

C11H10O

IUPAC Name:

2-methoxynaphthalene

Constituent 2

Reference substance name:

Methyl 2-napthyl ether

IUPAC Name:

Methyl 2-napthyl ether

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): Methyl 2-napthyl ether
- Molecular Formula: C11H10O
- Molecular Weight: 158.20 g/mol
- Substance type: Organic
- Physical state: Solid
- Analytical purity: 99%
- Consistency: White crystalline powder
- Activity (Clinical Indication): Industrial Chemical
- Stability under test conditions: Stable
- Storage condition of test material: As per requirements mentioned in guidance for safe use

Specific details on test material used for the study:

- Name of test material: Methyl 2-napthyl ether
- IUPAC name: 2-methoxynaphthalene
- Molecular Formula: C11H10O
- Molecular Weight: 158.20 g/mol
- Substance type: Organic
- Physical state: Solid
- Analytical purity: 99%

Method

Target gene:

Cells deficient in hypoxanthine-guanine phosphoribosyl transferase (HPRT) due to the mutation HPRT+/- to HPRT-/- are resistant to cytotoxic effects of 6-thioguanine (TG). HPRT proficient cells are sensitive to TG (which causes inhibition of cellular metabolism and halts further cell division since HPRT enzyme activity is important for DNA synthesis), so mutant cells can proliferate in the presence of TG, while normal cells, containing hypoxanthine-guanine phosphoribosyl transferase cannot.

This in vitro test is an assay for the detection of forward gene mutations at the in hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus on the X chromosomes of hypodiploid, modal No. 20, CHO cells. Gene and chromosome mutations are considered as an initial step in the carcinogenic process.
The hypodiploid CHO cells are exposed to the test item with and without exogenous metabolic activation. Following an expression time the descendants of the treated cell population are monitored for the loss of functional HPRT enzyme.
HPRT catalyses the transformation of the purine analogues 6-thioguanine (TG) rendering them cytotoxic to normal cells. Hence, cells with mutations in the HPRT gene cannot phosphoribosylate the analogue and survive treatment with TG.

Therefore, mutated cells are able to proliferate in the presence of TG whereas the non-mutated cells die. However, the mutant phenotype requires a certain period of time before it is completely expressed. The phenotypic expression is achieved by allowing exponential growth of the cells for 7 days.

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Cell line used: Chinese Hamster Ovary (CHO) cells
- Type and identity of media: Ham's F-12K (Kaighn's) Medium containing 2 mM L-Glutamine supplemented with 10% Fetal Bovine Serum and 1% Penicillin-Streptomycin (10,000 U/mL).
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Not applicable
- Periodically checked for karyotype stability: Not applicable

Additional strain / cell type characteristics:

other: Hypodiploid, modal No. 20

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

S9 liver microsomal fraction obtained from Arcolor 1254-induced male Sprague-Dawley rats

Test concentrations with justification for top dose:

0, 0.1, 0.25, 0.5 or 1 mM

Vehicle / solvent:

Vehicle(s)/solvent(s) used: Ethanol
Justification for choice of solvent/ vehicle: Methyl 2-napthyl ether was easily dissolved in ethanol.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

N-ethyl-N-nitrosourea (ENU) was the positive control substance in the tests done without S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: In medium with pre-incubation

DURATION
- Preincubation period: One week involving 3 days of incubation with Hypoxanthine-aminopterin-thymidine (HAT) in medium as a mutant cleansing stage, followed by overnight incubation with hypoxanthine-thymidine (HT) in medium prior to a 3-4 days incubation in regular cell medium. After seeding and prior to treatment, the mutant-free cells were incubated for an additional of 24 hours
- Exposure duration: 3 hours
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 14 days
- Fixation time (start of exposure up to fixation or harvest of cells): 7 days (harvest of cells)

SELECTION AGENT (mutation assays): 6-thioguanine (TG)
SPINDLE INHIBITOR (cytogenetic assays): Not applicable
STAIN (for cytogenetic assays): Crystal violet

NUMBER OF REPLICATIONS: A minimum of 2 replicates per dose concentration including negative and positive control.

NUMBER OF CELLS EVALUATED: 5 x 10 E5 cells were plated 7 days after treatment and whatever cells left, after 14 days of incubation with the selection medium, were evaluated.

DETERMINATION OF CYTOTOXICITY
- Cytotoxicity test
After being exposed to the test chemical for 3 hours, in the absence or presence of S9, cells were trypsinized and 0.5 x 10 E5 cells per well was seeded in duplicates from two parallel duplicate cultures into 6-well plates in fresh medium. The relative total growth and cytotoxicity was evaluated 24 and 48 hours after seeding.

OTHER EXAMINATIONS: Not applicable

Rationale for test conditions:

No data

Evaluation criteria:

The plates were scored for total number of colonies

Statistics:

No data

Results and discussion

Test results

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not valid

Additional information on results:

No data

Remarks on result:

other: No mutagenic potential

Any other information on results incl. tables

Table 1A.Effect of Methyl 2-napthyl ether exposure on gene toxicity in CHO cells. After being exposed to the test chemical for 3 hrs, cells was washed with sterile PBS and then incubated for 7 days at 37°C, 5% CO₂. After 7 days, cells were re-seeded in new 6-well plates in the absence or presence of 10mM TG as a selection agent and returned to the incubator for 14 days at 37°C, 5% CO₂. On day 15, all 6-well plates were stained with crystal violet and the number of colonies were counted manually. The results are presented as the total number of colonies found in the number of independent wells analyzed (e.g. 0 colonies in 4 wells will give 0/4) (n = 2 samples from 2 independent cultures).

 

	With S9		Without S9
	with TG	without TG	with TG	without TG
Neg. control	0/4	195/4	0/4	180/4
Pos. control	0/4	199/4	27/4	142/4
0.1 mM	2^a/4	229/4	0/4	186/4
0.25 mM	0/4	209/4	0/4	142/4
0.5 mM	0/4	186/4	1/4^b	205/4
1.0 mM	0/4	179/4	0/4	137/4

 

^a)Two very diffuse colonies were found in one single well.

^a)One very diffuse colony was found in one single well.

 

 

Table 1B.Mutation frequency in CHO cells after 3 hrs of exposure to Methyl 2-napthyl ether in the absence or presence of 4% S9 liver microsomal fraction. N/A, no colonies present in the samples selected with TG, i.e. no mutation frequency could be determined.

 

	With S9	Without S9
Neg. control	N/A	N/A
Pos. control	N/A	5.35x10^-4
0.1 mM	N/A^a	N/A
0.25 mM	N/A	N/A
0.5 mM	N/A	N/A^b
1.0 mM	N/A	N/A

 

^a)Since two very diffuse colonies were found in one single well (see Table 1A), the diffuse colonies was not regarded as reliable and true colonies since the cells seemed to be apoptotic.

^b)Since only one diffuse colony were found in one single well (see Table 1A), the diffuse colony was not regarded as a reliable and true colony since the cells seemed to be apoptotic.

Applicant's summary and conclusion

Conclusions:

In a gene toxicity test, Chinese Hamster Ovary (CHO) cells were exposed to Methyl 2-napthyl ether in the concentration of 0, 0.01, 0.25, 0.5 or 1 mM and without S9-induced metabolic activation. The results showed no evidence of gene toxicity when cells were exposed to Methyl 2-napthyl ether. Therefore, it is considered that Methyl 2-napthyl ether in the concentration of 0, 0.01, 0.25, 0.5 or 1 mM does not cause genetic mutation(s) in the presence or absence of metabolic activation.

Executive summary:

An in vitro mammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of Methyl 2-napthyl ether (CAS No. 93-04-9) when administered to Chinese Hamster Ovary (CHO) cells.

In the genotoxicity test, methyl 2-napthyl ether was administered to CHO cells for 3 hrs at the dose levels of 0, 0.1, 0.25, 0.5 or 1.0 mM and in the absence or presence of exogenous metabolic activation. CHO cells representing the negative controls were exposed to the vehicle. Positive controls, such as N-ethyl-N-nitrosourea (ENU) experiments without metabolic activation and 7,12-dimethylbenz(a) anthracene in experiments with metabolic activation, were also included in each test.

The positive control ENU gave a clear indication of gene mutations occurring while no other treatment gave rise to gene toxicity. One very diffuse colony was seen in one well out of four at the concentration of 0.5 mM and in the absence with 4% S9 liver microsomal fraction, and two very diffuse colonies were detected in one well out of four at the concentration of 0.1 mM and in the presence with 4% S9 liver microsomal fraction. These diffuse colonies are not regarded to be relevant since the three spots were only mildly colored by crystal violet, thus indicating that it were small clusters of apoptotic cells taking their last breath instead of cells surviving the TG-selection. This is further supported by the results of the higher tested concentrations of methyl 2-napthyl ether, i.e. these concentrations did not show any evidence of diffuse or clear colonies present.

When the mutation frequency was determined, a frequency of 5.35 x 10^-4 was shown after a 3 hour exposure of ENU as the positive control and in the absence of S9 liver microsomal fraction. Since no other tested concentration of methyl 2-napthyl ether in the absence or presence of S9 liver microsomal fraction resulted in colonies, we conclude that methyl 2-napthyl ether does not give rise to gene mutations when CHO cells are exposed in vitro to the test chemical at 0, 0.1, 0.25, 0.5 or 1.0 mM for 3 hrs.

Conclusion

Based on the results of the current study, we conclude that methyl 2-napthyl ether does not give rise to gene mutations when CHO cells are exposed to the test chemical in vitro at 0, 0.1, 0.25, 0.5 or 1.0 mM for 3 hrs, in the presence or absence of metabolic activation.