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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation test in vitro:

Ames test:

Ames assay was performed to investigate the potential of Methyl-2-napthyl ether (CAS no. 93-04-9) to induce gene mutations in comparison to vehicle control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102. The assay was performed in two independent experiments both with and without liver microsomal activation(S9 mix). Each concentration, including the negative, vehicle and positive controls was tested in triplicate. Based on the pre-experiment results, the test item was tested with the following concentrations 0, 0.003, 0.008, 0.025, 0.079 and 0.250 mg/plate for main study, both in the presence of metabolic activation (+S9) and in the absence of metabolic activation (-S9). No substantial increase in revertant colony numbers in any of the tester strains were observed following treatment with Methyl-2-napthyl ether at any dose level in both the confirmatory trials, neither in the presence nor in the absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.The spontaneous reversion rates in the negative, vehicle and positive controlswerewithin the range of historical data. The positive controls used for various strains showed a distinct increase in induced revertant colonies in both the methods i.e. Plate incorporation method and Pre-incubation method. In conclusion, it is stated that during the described mutagenicity test and under the experimental conditions reported, Methyl-2-napthyl ether did not induce gene mutations by base pair changes or frame shifts in the genome of Salmonella typhimurium strains used.

In vitro mammalian chromosome aberration test:

An in vitro mammalian chromosome aberration test (OECD TG 473) was conducted to determine the chromosomal aberration induction potential of Methyl 2-napthyl ether in human peripheral blood lymphocyte cultures. Blood was drawn from a healthy volunteer, by venous puncture using heparinised syringe. The experiment was performed both in the presence and in the absence of metabolic activation system after 48 hours mitogenic stimulation. The test chemical was dissolved in DMSO and used at dose level of 0, 0.00025, 0.0005 and 0.001 mg/ml, in the presence and absence of S9 metabolic activation system in Trial 1 and Trial 2. Trial I was performed by short term treatment method both in the presence and absence of metabolic activation system (1 %, S9 mix). Phase II was performed either by short-term treatment (4 hours) as well as long-term treatment (24 hours) methods. Long term treatment was performed in absence of metabolic activation to confirm the negative results obtained in the absence of metabolic activation in Phase I. Short term treatment method was performed with increased metabolic activation (2%) condition to confirm the negative results obtained in the presence of metabolic activation in Phase I. The doses for the main study were based on the cytotoxicity study conducted both in the presence and absence of metabolic activation system. Three test concentrations (0.5, 1 and 2 mg/mL of culture media) based on the solubility, precipitation and pH test of the test item were used. Minimum 300 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides andthe percent aberrant cells was calculated. Before conducting the chromosome aberration study, Methyl 2-napthyl ether was evaluated for cytotoxicity both in the absence and presence of metabolic activation system (S9 mix, 1 %). A minimum of 1000 cells were counted in different fields of slide per culture and the number of metaphases were recorded for mitotic index (MI) calculation.In the cytotoxicity experiment, exposure to 0.001 mg/ml of test substance induced slight cytotoxicity and the mitotic index showed 41.8 % reduction in the absence of metabolic activation and 42.18 % in the presence of metabolic activation. In the mutagenicity test,no structural abnormality of chromosome and induction of ploidy cells were observed at any dose tested in both confirmatory trials. The percent aberrant cells were comparable in control and treated groups, both in the presence and in the absence of exogenous metabolic activation. Similarly, there was no reduction in mitotic index after exposure to the test substance at any concentration tested, in both trials.In conclusion, under the experimental conditions reported, Methyl 2 -napthyl ether did not induce structural and numerical chromosome aberration and, consequently it is not clastogenic up to the concentration of 0.001 mg/ml in human peripheral blood lymphocytes both in the presence (1-2 %) and absence of metabolic activation.

In vitro mammalian gene mutation test:

An in vitro mammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of methyl 2-napthyl ether when administered to Chinese Hamster Ovary (CHO) cells. The test substance was administered to CHO cells for 3 hours at the dose levels of 0, 0.1, 0.25, 0.5 or 1.0 mM in the absence or presence of exogenous metabolic activation (S9 mix). 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.No mutant colonies were detected following the exposure to positive control of 7,12-dimethylbenz(a) anthracene and, therefore no conclusion could be drawn from experiments performed in the presence of exogenous metabolic activation. No clear indication of gene mutation was seen when CHO cells were treated with methyl 2-naphthyl ether at concentration of0, 0.1, 0.25, 0.5 or 1.0 mM for 3 hoursin the absence of S9 mix. One very diffuse colony was seen in one well out of four at the concentration 0.5 mM, but this colony was a small cluster of apoptotic cells as it was only mildly coloured by crystal violet and, consequently it was considered irrelevant tothepresenttest.This was 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.In addition, no cytotoxic effects were observed when CHO cells were exposed to the test substance at any concentration used. In conclusion, methyl 2-napthyl etherdoes not give rise to gene mutations when CHO cells are exposed to the test chemical at 0, 0.1, 0.25, 0.5 or 1.0 mM for 3 hours in the absence of exogenous metabolic activation (-S9 mix). In addition, methyl 2-napthyl ether does not induce cytotoxic effects at concentrations ≤ 10.0 mM. 

 

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from study report
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
This study was performed to investigate the potential of the test chemical to induce gene muta¬tions in comparison to vehicle control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
other:
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced S9 metabolic activation system
Test concentrations with justification for top dose:
0, 0.003, 0.008, 0.025, 0.079 and 0.250 mg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was solulble in DMSO
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-Nitro-o-phenylenediamine (TA 1537, TA 98, without S9); 2-Aminoanthracene (TA 1535, TA 1537, TA 98, TA 100 and TA 102, with S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation- Trial I); preincubation (Trial II)

DURATION
- Preincubation period: Trial I: Not applicable Trial II: 60 min
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data

SPINDLE INHIBITOR (cytogenetic assays): No data

STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: Each concentration, including the negative, vehicle and positive controls was tested in triplicate in two independent experiments performed

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Not applicable

NUMBER OF CELLS EVALUATED: No data

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): No data

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data
- Any supplementary information relevant to cytotoxicity: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): No data

- OTHER: No data
Rationale for test conditions:
No data
Evaluation criteria:
A test item is considered as a mutagen, if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100 and TA 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding vehicle/solvent control is observed.

A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.

An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.

A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative control and vehicle control such an increase is not considered biologically relevant.
Statistics:
No data
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: To evaluate the toxicity of the test item, a pre-experiment was performed with strains TA 98 and TA 100. Eight concentrations (0, 0.001, 0.003, 0.008, 0.025, 0.079, 0.250, 0.791 and 2.5 mg/plate ) were tested for toxicity and mutation induction with 3 plates each (triplicates). The experimental conditions in this pre-experiment were the same as described below for the Trial-I (Plate incorporation test).
Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

In the pre-experiment, the concentration range of the test item was 0.001 – 2.5 mg/plate based on the solubility and precipitation test. There was no reduction in colony count but reduction in background lawn was observed in treated concentrations 2.5 mg/plate (T8), 0.791 mg/plate (T7) and no reduction in colony count as well as in background lawn in treated concentrations (0.250 (T6) mg/plate – 0.001 (T1) mg/plate) both in absence and in the presence of metabolic activation. Based on the results of pre-experiment following doses were selected for the main study trials: (0, 0.003, 0.008, 0.025, 0.079 and 0.250 mg/plate , both in the absence (-S9) as well as in the presence of metabolic activation (+S9).

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: No data

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: No data
- Indication whether binucleate or mononucleate where appropriate: No data

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: No data
- Negative (solvent/vehicle) historical control data: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: No data
- Other observations when applicable: No data
Remarks on result:
other: No mutagenic potential

TABLE 4 -    MEAN REVERTANT COUNT IN PLATE INCORPORATION METHOD (TRIAL I)

Dose (mg/plate)

In the presence of Metabolic Activation (+S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

NC

(0.00)

5.33

0.58

10.67

1.15

21.33

1.15

124.67

4.16

242.67

10.07

VC

(0.00)

7.00

1.00

14.00

1.73

27.33

3.06

135.33

7.02

281.33

6.11

T1

(0.003)

5.67

0.58

11.33

1.15

23.33

3.06

125.33

1.15

251.33

11.02

T2

(0.008)

6.00

1.00

12.00

2.00

22.67

2.31

125.00

3.00

266.00

6.00

T3

(0.025)

6.67

0.58

11.67

1.53

24.00

2.00

125.67

1.15

264.00

6.00

T4

(0.079)

6.33

0.58

12.67

1.15

24.67

1.15

128.33

2.52

266.67

13.61

T5

(0.250

6.67

0.58

13.33

1.53

25.67

1.15

131.00

2.65

274.00

8.00

PC

170.67

14.05

514.67

28.10

1269.33

24.44

1424.00

28.84

1725.33

28.10

 

Dose

(mg/plate)

In the Absence of Metabolic Activation (-S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

NC

(0.00)

5.00

0.00

10.33

1.15

18.00

3.00

108.00

2.00

238.00

9.17

VC

(0.00)

7.33

0.58

14.00

1.73

25.33

1.15

127.33

5.03

291.33

7.02

T1

(0.003)

5.67

0.58

10.67

1.15

19.67

2.89

110.67

1.15

242.00

6.00

T2

(0.008)

5.33

0.58

11.33

1.15

20.33

1.15

110.00

4.00

250.67

6.11

T3

(0.025)

6.00

0.00

12.00

0.00

20.00

3.46

112.67

1.15

252.67

7.02

T4

(0.079)

6.33

1.15

11.67

1.53

20.67

2.31

114.00

4.00

258.00

12.49

T5

(0.250

6.67

0.58

12.67

2.52

21.33

2.31

121.33

3.06

267.33

8.08

PC

173.33

22.03

1093.33

40.27

992.00

16.00

1136.00

16.00

1712.00

48.00

NC= Negative Control,VC= Vehicle Control,T =Test concentration (T5: Highest, T1: Lowest),SD= Standard Deviation

PC= Positive control

2-Aminoanthracene [2.5μg/plate]: TA 1537, TA 1535, TA 98, TA 100                  Methyl methanesulfonate [4μl/plate]: TA 102

2-Aminoanthracene [10μg/plate]:TA 102                                           

Sodium azide [10μg/plate]: TA 1535, TA 100

4-Nitro-o-phenylenediamine: TA 1537[50μg/plate], TA 98 [10μg/plate]


TABLE 5 -    MEAN REVERTANT COUNT IN PRE-INCUBATION METHOD (TRIAL II)

Dose

(mg/plate)

In the presence of Metabolic Activation (+S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

NC

(0.00)

5.33

0.58

10.00

0.00

17.33

1.15

102.00

5.29

250.67

10.07

VC

(0.00)

7.00

1.00

12.67

1.15

25.33

3.06

112.67

3.06

286.00

4.00

T1

(0.003)

5.00

1.00

10.67

1.15

18.00

2.00

103.33

3.06

253.33

6.11

T2

(0.008)

5.67

0.58

11.33

0.58

19.33

1.15

105.33

4.62

257.33

9.02

T3

(0.025)

6.00

1.00

11.00

1.00

20.67

1.15

106.67

5.03

264.00

5.29

T4

(0.079)

6.33

0.58

11.67

0.58

22.00

2.00

108.67

4.62

272.00

8.00

T5

(0.250

6.67

0.58

12.00

0.00

24.00

2.00

109.33

4.62

276.00

6.00

PC

170.67

10.07

464.00

21.17

1096.00

28.84

1216.00

32.00

1418.67

37.81

 

Dose

(mg/plate)

In the Absence of Metabolic Activation (-S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

NC

(0.00)

4.67

0.58

10.33

0.58

16.67

1.15

107.33

3.06

245.33

6.11

VC

(0.00)

6.67

0.58

14.33

2.08

23.33

1.15

120.00

2.00

272.67

11.37

T1

(0.003)

5.33

0.58

10.67

1.15

17.33

2.31

108.67

3.06

247.33

3.06

T2

(0.008)

5.00

0.00

11.33

1.15

18.00

2.00

109.33

6.11

258.67

6.11

T3

(0.025)

5.67

0.58

11.67

0.58

18.67

1.15

112.00

5.29

260.67

12.06

T4

(0.079)

6.00

1.00

11.00

1.00

19.33

1.15

114.67

3.06

261.33

6.11

T5

(0.250

6.33

1.15

12.00

2.00

22.67

1.15

115.33

4.16

266.00

6.00

PC

168.67

13.01

1368.00

24.00

816.00

32.00

1322.67

28.10

1541.33

40.27

NC= Negative Control,VC= Vehicle Control,T =Test concentration (T5: Highest, T1: Lowest),SD= Standard Deviation

PC= Positive control

2-Aminoanthracene [2.5μg/plate]: TA 1537, TA 1535, TA 98, TA 100

2-Aminoanthracene [10μg/plate]: TA 102

Sodium azide [10μg/plate]: TA 1535, TA 100

4-Nitro-o-phenylenediamine: TA 1537[50μg/plate] TA 98[10μg/plate]

Methyl methanesulfonate: [4μl/plate]: TA 102

Conclusions:
The test chemical, Methyl-2-napthyl ether did not induce gene mutations by base pair changes or frame shifts in the genome of the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutagen as per the criteria mentioned in CLP regulation.
Executive summary:

Ames assay was performed to investigate the potential of Methyl-2-napthyl ether (CAS no. 93-04-9) to induce gene mutations in comparison to vehicle control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102. The assay was performed in two independent experiments both with and without liver microsomal activation(S9 mix). Each concentration, including the negative, vehicle and positive controls was tested in triplicate. Based on the pre-experiment results, the test item was tested with the following concentrations 0, 0.003, 0.008, 0.025, 0.079 and 0.250 mg/plate for main study, both in the presence of metabolic activation (+S9) and in the absence of metabolic activation (-S9).

No substantial increase in revertant colony numbers in any of the tester strains were observed following treatment with Methyl-2-napthyl ether at any dose level in both the confirmatory trials, neither in the presence nor in the absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.The spontaneous reversion rates in the negative, vehicle and positive controlswerewithin the range of historical data. The positive controls used for various strains showed a distinct increase in induced revertant colonies in both the methods i.e. Plate incorporation method and Pre-incubation method.

In conclusion, it is stated that during the described mutagenicity test and under the experimental conditions reported, Methyl-2-napthyl ether did not induce gene mutations by base pair changes or frame shifts in the genome of Salmonella typhimurium strains used.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from study report
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Principles of method if other than guideline:
This in vitro assay was performed to assess the potential of the test chemical to induce structural / numerical chromosomal aberrations in one experiment (phase I). The induction of cytogenetic damage in human lymphocytes was assessed with and without metabolic activation. Due to the negative result in phase I, a second experiment (phase II) was performed.
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
No data
Species / strain / cell type:
lymphocytes: human peripheral blood lymphocytes
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Human blood
- Suitability of cells: No data
- Cell cycle length, doubling time or proliferation index:
- Sex, age and number of blood donors if applicable:Age: 25-30 years age
- Whether whole blood or separated lymphocytes were used if applicable: Separated lymphocytes were used
- Number of passages if applicable: No data
- Methods for maintenance in cell culture if applicable: No data
- Modal number of chromosomes: No data
- Normal (negative control) cell cycle time: No data

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Blood cultures were set up in medium containing RPMI-1640, Fetal Bovine Serum, Phytohaemagglutinin, Heparin solution, Whole Blood and Antibiotic Solution
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: No data
- Periodically checked for karyotype stability: No data
- Periodically 'cleansed' against high spontaneous background: No data
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
S9 metabolic activation system
Test concentrations with justification for top dose:
0.00 (NC), 0.00025 (T1), 0.0005 (T2) and 0.001 (T3) mg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): A volume of 7.92 mL of proliferating culture was dispensed to individual sterile culture tubes/flasks

DURATION
- Preincubation period: No data
- Exposure duration: Phase 1: 4 hrs (with and without metabolic activation system)
Phase 2: 4 hrs (with metabolic activation system) and 24 hrs (without metabolic activation system)
- Expression time (cells in growth medium): Phase 1: 20 hrs (with and without metabolic activation system)
Phase 2: 20 hrs (with metabolic activation system)
- Selection time (if incubation with a selection agent):No data
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hrs

SELECTION AGENT (mutation assays): No data

SPINDLE INHIBITOR (cytogenetic assays): Colcemid

STAIN (for cytogenetic assays): Giemsa stain in phosphate buffer

NUMBER OF REPLICATIONS: No data

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The cultures were incubated at 37 ± 2 °C for duration (exposure period) as mentioned. For Phase I, after incubation cells were spun down by gentle centrifugation at 1500 rpm for 10 minutes. The supernatant with the dissolved test item was discarded and the cells were re-suspended in Phosphate Buffer Saline (PBS). The washing procedure was repeated once again. After washing the cells were re-suspended in complete culture medium (RPMI-1640 with 10 % serum) and cultured at 37 ± 2 °C for 1.5 normal cell cycle lengths (22 - 25 hours). The cultures were harvested at the end of incubation of 24 hours after treatment. Before 3 hours of harvesting, 240 µL of colcemid (10 µg/mL) (final concentration: 0.3 µg/mL) was added to each of the culture tube, and kept under incubation at 37 ± 2 °C. The cultures were harvested 24 hours after beginning of treatment by centrifugation at 1500 rpm for 10 minutes. The supernatant was discarded and the cells were re-suspended in 7 mL of freshly prepared, pre-warmed (37 ± 2 °C) hypotonic solution of potassium chloride (0.075 M KCl). Then the cell suspension was allowed to stand at 37 ± 2 °C for 30 minutes in water bath. After hypotonic treatment, the culture was centrifuged and supernatant was removed. After that 5 mL of freshly prepared, chilled Carnoy’s fixative (3:1 methanol: acetic acid solution) was added and left for 5 min. The cells were collected by centrifugation and washed twice with Carnoy’s fixative. After the final centrifugation, the supernatant was removed completely, and the cell pellet resuspended in 0.5 mL of Carnoy’s fixative. The slides were prepared by dropping the cell suspension onto a clean ice-chilled microscope slide. The labelled slides were dried over a slide warmer at 50°C and labelled. At least one slide was made from each sample. The cells were stained with 5 % fresh Giemsa stain in phosphate buffer and mounted using DPX mountant.

NUMBER OF CELLS EVALUATED: A minimum of 1000 cells were counted in different fields of slide per culture and the number of metaphases were recorded for mitotic index (MI) calculation.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 300 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides.

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Mitotic index
- Any supplementary information relevant to cytotoxicity: Cytotoxicity was assessed at the concentrations of 0.00 (NC), 0.00025 (T7), 0.0005 (T8) and 0.001 (T9) mg/mL of culture media.

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: Yes
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): No data

- OTHER: No data
Evaluation criteria:
A test item can be classified as clastogenic if:
 At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent vehicle control
 If the increase is dose-related
 Any of the results are outside the historical negative control range
A test item can be classified as non – clastogenic if:
 None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control
 If there is no dose-related increase
 All results are within the historical negative control range
Statistical significance was confirmed by means of the non-parametric Mann Whitney Test. However, both biological and statistical significance should be considered together.

If the above mentioned criteria for the test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.
Statistics:
Statistical significance at the p < 0.05 was evaluated by means of the non-parametric Mann-Whitney test
Species / strain:
lymphocytes: Human perpheral blood lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
In the cytotoxicity experiment III the highest test concentration 0.001 (T9) mg/ mL of culture media show 41.8 % reduction in absence of metabolic activation and 42.18% in the presence of metabolic activation indicates slight cytotoxicity of test item
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH of test item in culture medium was assessed at 0 h and 4 h after incubation at 37 ± 2 °C. Significant change in pH was not observed at 0 h and 4 h when compared with negative controls.
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: There was no precipitation observed at 0.0625 mg/mL concentration
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: To evaluate the toxicity of the test item a cytotoxicity assay was performed both in the presence and absence of metabolic activation system. Three test concentrations (0.00025, 0.0005 and 0.001 mg/mL of culture media) based on the solubility, precipitation and pH test of the test item were tested. Cytotoxicity was determined by reduction in the mitotic index in comparison with vehicle control. The procedure for conducting cytotoxicity was the same as main experiment phase I up to the scoring of the mitotic index, except slide coding.

Before conducting the chromosomal aberration study, Methyl-2-napthyl ether (CAS no. 93-04-9) was evaluated for cytotoxicity both in the absence and presence of metabolic activation system (1%). Cytotoxicity was assessed at the concentrations of 0.016 (T1), 0.0312 (T2) and 0.0625 (T3) mg/mL at initial cytotoxicity experiment (cytotxicity experiment I). All the tested concentrations at intial cytotoxicity experiment were cytotoxic. A second cytotoxicity experiment (cytotoxicity experiment II) was conducted with 0.002 (T4), 0.004 (T5) and 0.008 (T6) mg/mL of culture media. In second cytotoxicity experiment all tested concentrations were cytotoxic.

Hence one more cytotoxicity experiment (cytotoxic experiment III) was conducted with further lower concentrations of 0.00025 (T7), 0.0005 (T8) and 0.001 (T9) mg/mL of culture media. In the absence of S9 mix, the mean mitotic index observed was 10.03 (NC), 9.95 (VC), 8.69 (T7), 6.54 (T8), 5.79 (T9) and 8.54 (PC). In the presence of S9 mix, the mean mitotic index observed was 10.05 (NC), 9.94 (VC), 8.84 (T7), 6.55 (T8), 5.74 (T9) and 8.55 (PC).

In the cytotoxicity experiment III the highest test concentration 0.001 (T9) mg/ mL of culture media show 41.8 % reduction in absence of metabolic activation and 42.18% in the presence of metabolic activation indicates slight cytotoxicity of test item. Hence 0.001 was selected as highest concentaration for main study considering the selection of test concentrations upto cytotoxicity. The mitotic index when compared to the respective vehicle control both in the presence or absence of metabolic activation.

Hence the concentrations selected for the main study are 0.00025, 0.0005 and 0.001 mg/mL. The main study was performed in two independent phases;

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: No data

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: No data
- Indication whether binucleate or mononucleate where appropriate: No data

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: No data
- Negative (solvent/vehicle) historical control data: Please refer table remarks section

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: No data
- Other observations when applicable: No data
Remarks on result:
other: No mutagenic potential

Cytotoxicity results:

                

Before conducting the chromosomal aberration study, the test chemical was evaluated for cytotoxicity both in the absence and presence of metabolic activation system (1%). Cytotoxicity was assessed at the concentrations of 0.016 (T1), 0.0312 (T2) and 0.0625 (T3) mg/mL at initial cytotoxicity experiment (cytotxicityexperiment I). All the tested concentrations atintialcytotoxicity experiment were cytotoxic. A second cytotoxicity experiment (cytotoxicity experiment II) was conducted with 0.002 (T4), 0.004 (T5) and 0.008 (T6) mg/mL of culture media. In second cytotoxicity experiment all tested concentrations were cytotoxic.

Hence one more cytotoxicity experiment (cytotoxic experiment III) was conducted with further lower concentrations of 0.00025 (T7), 0.0005 (T8) and 0.001 (T9) mg/mL of culture media. In the absence of S9 mix, the mean mitotic index observed was 10.03 (NC), 9.95 (VC), 8.69 (T7), 6.54 (T8), 5.79 (T9) and 8.54 (PC). In the presence of S9 mix, the mean mitotic index observed was 10.05 (NC), 9.94 (VC), 8.84 (T7), 6.55 (T8), 5.74 (T9) and 8.55 (PC).

In the cytotoxicity experiment III the highest test concentration 0.001 (T9) mg/ mL of culture media show 41.8 % reduction in absence of metabolic activation and 42.18% in the presence of metabolic activation indicates slight cytotoxicity of test item. Hence0.001 mg/mL was selected as highest concentaration for main study considering the selection of test concentrations upto cytotoxicity. The mitotic index when compared to the respective vehicle control both in the presence or absence of metabolic activation.

Hence the concentrations selected for the main study are 0.00025, 0.0005 and 0.001 mg/mL. The main study was performed in two independent phases;

Phase 1 results:           

In the experiment, the cultures were exposed to Methyl-2-napthyl ether (CAS no. 93-04-9) for a short period of time (4 h) both in the absence and in the presence of metabolic activation system (1%). The mean percentage of aberrant cells was 0.333 (NC), 0.667 (VC), 0.667 (T1), 0.667 (T2), 0.667 (T3) and 10.333 (PC) in the absence of metabolic activation and 0.667 (NC), 0.667 (VC), 0.667 (T1), 0.6676 (T2), 0.667 (T3) and 10.000 (PC) in the presence of metabolic activation at the concentration of 0.00 (NC), 0.00 (VC), 0.00025 (T1), 0.0005 (T2) and 0.001 (T3) mg/mL and positive controls, respectively.

Treatment with Ethyl methanesulfonate at the concentration of 600 µg/mL in the absence of metabolic activation and Cyclophosphamidemonohydrate at the concentration of30 µg/mL in the presence of metabolic activation (1%) causedsignificant increase in percent aberrant cells.Even though the analysis did not reveal any statistical significance, the increase was biologically significant.

During thetreatment with test item in the absence and presence of S9 mix, there was noreduction in mitotic index observed at the tested concentrations. The observed mean mitotic indexin the absence of metabolic activation were 10.02, 9.93, 8.58, 6.69, 5.39 and 8.48 andin the presence ofmetabolic activation were 10.02, 10.03, 8.73, 6.33, 5.68 and 8.62 for NC, VC, T1, T2, T3 and PC concentrations respectively.

Phase 2 results:

            

The phase II experiment was performed to confirm the negative results obtained in the absence and in the presence of metabolic activation in Phase I. In the Phase II, test item concentrations used were 0.00025 (T1), 0.0005 (T2) and 0.001 (T3) mg/mL culture both in presence and in absence of metabolic activation (2%). The duration of exposure to the test item in presence of metabolic activation system was 4 hours and in absence of metabolic activation the duration of exposure was 24 hours. The mean percent aberrant cells were 0.667 (NC), 0.667 (VC), 0.667 (T1), 0.667 (T2), 0.667 (T3) and 9.667 (PC) in the absence of metabolic activation and 0.667 (NC), 0.333 (VC), 0.333 (T1), 0.667 (T2), 0.667 (T3) and 10.000 (PC) in the presence of metabolic activation at the concentration of 0.00 (NC), 0.00 (VC), 0.00025 (T1), 0.0005 (T2) and 0.001 (T3) mg/mL of culture and positive control, respectively.

Treatment with Ethyl methanesulfonate at the concentration of 600 µg/mL in the absence of metabolic activation and Cyclophosphamidemonohydrate at the concentration of30 µg/mL in the presence of metabolic activation (2%) causedsignificant increase in percent aberrant cells.Though the analysis did not reveal any statistical significance, the increase was biologically significant.

The increased frequency of aberrations observed in the concurrent positive control groups (Phase I and II) demonstrated the sensitivity of the test system, suitability of the methods and conditions employed in the experiment.

Treatment with test item in the absence and presence of S9 mix, there was no reduction in mitotic index was observed at the tested concentrations. The observed mean mitotic indexin the absence of metabolic activation were 9.99, 10.01, 8.12, 6.78, 5.59 and 8.48 andin the presence ofmetabolic activation were 9.97, 9.94, 8.67, 6.68, 5.54 and 8.62 for NC, VC, T1, T2, T3 and PC concentrations respectively.

Conclusions:
Under the specified conditions, Methyl 2-napthyl ether is non-clastogenic up to the concentration of 0.001 mg/ml in human peripheral blood lymphocytes both in the presence (1 % and 2 %) and in the absence of metabolic activation and hence, it is not likely to classify as mutagenic substance according to the criteria mentioned in CLP regulations.
Executive summary:

An in vitro mammalian chromosome aberration test (OECD TG 473) was conducted to determine the chromosomal aberration induction potential ofMethyl 2-napthyl etherin human peripheral blood lymphocyte cultures. Blood was drawn from a healthy volunteer, by venous puncture using heparinised syringe. The experiment was performed both in the presence and in the absence of metabolic activation system after 48 hours mitogenic stimulation. The test chemical was dissolved in DMSO and used at dose level of 0, 0.00025, 0.0005 and 0.001 mg/ml, in the presence and absence of S9 metabolic activation system in Trial 1 and Trial 2. Trial I was performed by short term treatment method both in the presence and absence of metabolic activation system (1 %, S9 mix). Phase II was performed either by short-term treatment (4 hours) as well as long-term treatment (24 hours) methods. Long term treatment was performed in absence of metabolic activation to confirm the negative results obtained in the absence of metabolic activation in Phase I. Short term treatment method was performed with increased metabolic activation (2%) condition to confirm the negative results obtained in the presence of metabolic activation in Phase I. The doses for the main study were based on the cytotoxicity study conducted both in the presence and absence of metabolic activation system. Three test concentrations (0.5, 1 and 2mg/mL of culture media) based on the solubility, precipitation and pH test of the test item were used. Minimum 300 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides andthe percent aberrant cells was calculated.Before conducting the chromosome aberration study,Methyl 2-napthyl etherwas evaluated for cytotoxicity both in the absence and presence of metabolic activation system (S9 mix, 1 %). A minimum of 1000 cells were counted in different fields of slide per culture and the number of metaphases were recorded for mitotic index (MI) calculation.In the cytotoxicity experiment, exposure to 0.001 mg/ml of test substance induced slight cytotoxicity and the mitotic index showed 41.8 % reduction in the absence of metabolic activation and 42.18 % in the presence of metabolic activation. In the mutagenicity test,no structural abnormality of chromosome and induction of ploidy cells were observed at any dose tested in both confirmatory trials. The percent aberrant cells were comparable in control and treated groups, both in the presence and in the absence of exogenous metabolic activation. Similarly, there was no reduction in mitotic index after exposure to the test substance at any concentration tested, in both trials.In conclusion, under the experimental conditions reported,Methyl 2-napthyl etherdid not induce structural and numerical chromosome aberration and, consequently it is not clastogenic up to the concentration of 0.001 mg/ml in human peripheral blood lymphocytes both in the presence (1-2 %) and absence of metabolic activation.

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from study report
Qualifier:
according to
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 the test chemical 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:
no
Type of assay:
mammalian cell gene mutation assay
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 / 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.
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
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
Remarks on result:
other: No mutagenic potential

Table 1A.Effect of the test chemical 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

2a/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

1/4b

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/Aa

N/A

0.25 mM

N/A

N/A

0.5 mM

N/A

N/Ab

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.

Conclusions:
In an in vitro mammalian cell gene mutation study, Chinese Hamster Ovary (CHO) cells were exposed to the test chemical in the concentration of 0, 0.01, 0.25, 0.5 or 1 mM in the absence of 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 absence of exogenous 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 when administered to Chinese Hamster Ovary (CHO) cells. The test substance was administered to CHO cells for 3 hours at the dose levels of 0, 0.1, 0.25, 0.5 or 1.0 mM in the absence or presence of exogenous metabolic activation (S9 mix). CHO cells representing the negative controls were exposed to the vehicle. Positive controls, such asN-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.No mutant colonies were detected following the exposure to positive control of7,12-dimethylbenz(a) anthracene and, therefore no conclusion could be drawn from experiments performed in the presence of exogenous metabolic activation. No clear indication of gene mutation was seen when CHO cells were treated with methyl 2-naphthyl etherat concentration of0, 0.1, 0.25, 0.5 or 1.0 mM for 3 hoursin the absence of S9 mix. One very diffuse colony was seen in one well out of four at the concentration 0.5 mM, but this colony was a small cluster of apoptotic cells as it was only mildly coloured by crystal violet and, consequently it was considered irrelevant tothepresenttest.This was 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.In addition, no cytotoxic effects were observed when CHO cells were exposed to the test substance at any concentration used. In conclusion,methyl 2-napthyl etherdoes not give rise to gene mutations when CHO cells are exposed to the test chemical at 0, 0.1, 0.25, 0.5 or 1.0 mM for 3 hours in the absence of exogenous metabolic activation (-S9 mix). In addition,methyl 2-napthyl etherdoes not induce cytotoxic effects at concentrations ≤ 10.0 mM. 

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In vivo gene mutation tests:

Available data in the literature on the test chemical, Methyl 2-naphthyl ether (Cas No. 93-04-9) and its read across analogues were reviewed to determine the toxic nature of the test chemical. The studies are as mentioned below:

#1

A Sex-Linked Recessive Lethal (SLRL) mutagenicity test was carried out in fruit fly, Drosophila melanogaster to assess the genotoxic nature of2-Ethoxynaphthalene (Cas No. 93-18-5) in vivo. Wild-type males were fed the solution of test substance, prepared in 5 % of saccharose at final concentration of 25 mM. Treated males were mated to appropriate females. The offspring of these females were scored for lethal effects corresponding to the effects on mature sperm, mid or late-stage spermatids, early spermatids, spermatocytes and spermatogonia at the time of treatment. Absence of wild type males in F2 generation indicated that a sex-linked recessive lethal mutation has occurred in a germ cell of the P1 male. As seen by the results, no significant increase in percentage of sex-linked recessive lethal mutations in the germ line of insects were detected comparing treated and control groups. Hence,2-ethoxynaphthalenwas non mutagenic SLRL test in vivo performed in Drosophila melanogaster.

#2

A micronucleus (MN) test was performed using male and female NMRI mice to determine the mutagenic nature of 2-ethoxynaphthalen in vivo. The test substance was administered in olive oil at the final concentration of 0, 344, 603 and 861 mg/kg via a single intraperitoneal injection. Then the mice were sacrificed, and bone-marrow smears were prepared 30 hours after treatment. The smears were stained according to the method of Schmid (1976) and were observed for cytogenetic effects in polychromated erthrocytes. As seen by the results, a single exposure to2-ethoxynaphthalen did not induce and increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow samples as compared with the concurrent control. Hence, in vivo micronucleus test performed in male and female NMRI mice did not indicate mutagenic potential of 2-ethoxynaphthalen.

#3

A Sex-Linked Recessive Lethal (SLRL) mutagenicity test was employed to determine the mutagenic nature of 2-(2-Methylpropoxy)Naphthalene (Cas No. 2173-57-1) in Drosophila melanogaster. The test substance was dissolved in 5 % saccharose solution and administered to wild type male flies at final concentration of 25 mM. There was a significant increase in sex-linked recessive lethal mutation (SRL) in single tests (Brood II) of treated groups as compared to control, but repeated tests did not confirm the mutagenic activity of 2-(2-Methylpropoxy)Naphthalene. Thus, this apparent effect that was observed only in single experiment probably occurred spontaneously. In conclusion, 2-(2-Methylpropoxy)Naphthalene was non mutagenic in SLRL test in vivo performed in Drosophila melanogaster.

#4

An in vivo micronucleus test was performed in male and female NMRI mice to evaluate the mutagenic potential of 2-(2-Methylpropoxy)Naphthalene (Cas No. 2173-57-1).The test substance was administered in olive oil at the final concentration of 0, 800, 1400 and 2000 mg/kg via a single intraperitoneal injection. Then the mice were sacrificed, and bone-marrow smears were prepared 30 hours after treatment. A single exposure to2-(2-Methylpropoxy)Naphthalene did not increase the frequency of micronucleated polychromatic erythrocytes in the bone marrow of treated mice as compared with the concurrent control. Hence, in vivo micronucleus test performed in male and female NMRI mice did not indicate mutagenic potential of 2-(2-Methylpropoxy)Naphthalene.

Based on the observations madeit can be concluded that, thetest chemical ofMethyl 2-naphthyl ether (Cas No. 93-04-9) did not inducegene and chromosome mutations, structural and numerical chromosome aberrationsin vivo and hence, it is not likely to classify as mutagenic substance in vivo. 

Link to relevant study records
Reference
Endpoint:
genetic toxicity in vivo, other
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Remarks:
Experimental data from various test chemicals
Justification for type of information:
Data for the target chemical is based on data from various test chemicals
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
other: Sex linked recessive lethal mutation assay (Wuergler et al., 1977)
Version / remarks:
1
Principles of method if other than guideline:
WoE derived based on the experimental data from various test chemicals
GLP compliance:
not specified
Type of assay:
Drosophila SLRL assay
Species:
other: 1. Drosophila melanogaster, 2. Mouse, 3. Drosophila melanogaster, 4. Mouse
Strain:
other: 1. No data, 2. NMR1, 3. No data, 4. NMR1
Details on species / strain selection:
No data
Sex:
male/female
Details on test animals and environmental conditions:
1. TEST ANIMALS
- Source: No data
- Age at study initiation: Mature sperm
- Weight at study initiation: No data
- Assigned to test groups randomly: [no/yes, under following basis: ] No data
- Fasting period before study: No data
- Housing: No data
- Diet (e.g. ad libitum): No data
- Water (e.g. ad libitum): No data
- Acclimation period: No data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): No data
- Humidity (%): No data
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): No data

IN-LIFE DATES: From: To: No data

2. TEST ANIMALS
- Source: lvanovas GmbH, Kisslegg
- Age at study initiation: 10 to 14 week old
- Weight at study initiation: No data
- Assigned to test groups randomly: [no/yes, under following basis: ] No data
- Fasting period before study: No data
- Housing: No data
- Diet (e.g. ad libitum): standard chow (Altromin) ad libitum
- Water (e.g. ad libitum): Water ad libitum
- Acclimation period: No data

3. TEST ANIMALS
- Source: No data
- Age at study initiation: Mature sperm
- Weight at study initiation: No data
- Assigned to test groups randomly: [no/yes, under following basis: ] No data
- Fasting period before study: No data
- Housing: No data
- Diet (e.g. ad libitum): No data
- Water (e.g. ad libitum): No data
- Acclimation period: No data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): No data
- Humidity (%): No data
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): No data

IN-LIFE DATES: From: To: No data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): No data
- Humidity (%):No data
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): No data

IN-LIFE DATES: From: To: No dataTEST ANIMALS
- Source: lvanovas GmbH, Kisslegg
- Age at study initiation: 10 to 14 week old
- Weight at study initiation: No data
- Assigned to test groups randomly: [no/yes, under following basis: ] No data
- Fasting period before study: No data
- Housing: No data
- Diet (e.g. ad libitum): standard chow (Altromin) ad libitum
- Water (e.g. ad libitum): Water ad libitum
- Acclimation period: No data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): No data
- Humidity (%):No data
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): No data

IN-LIFE DATES: From: To: No data

4. TEST ANIMALS
- Source: lvanovas GmbH, Kisslegg
- Age at study initiation: 10 to 14 week old
- Weight at study initiation: No data
- Assigned to test groups randomly: No data
- Fasting period before study: No data
- Housing: No data
- Diet (e.g. ad libitum): standard chow (Altromin) ad libitum
- Water (e.g. ad libitum): Water ad libitum
- Acclimation period: No data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): No data
- Humidity (%):No data
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): No data

IN-LIFE DATES: From: To: No data
Route of administration:
oral: feed
Vehicle:
1. - Vehicle(s)/solvent(s) used: Solutions or emulsions of test substances to be fed to the flies were prepared in 5% saccharose, with the addition of 2% ethanol and 2% Tween 80 for compounds that were poorly soluble in water.
- Justification for choice of solvent/vehicle: No data
- Concentration of test material in vehicle: 25mM
- Amount of vehicle (if gavage or dermal): No data
- Type and concentration of dispersant aid (if powder): No data
- Lot/batch no. (if required): No data
- Purity: No data

2. - Vehicle(s)/solvent(s) used: Olive oil
- Justification for choice of solvent/vehicle: The chemical was soluble in olive oil
- Concentration of test material in vehicle: 0, 1 x 344, 1 x 603, 1 x 861 mg/Kg
- Amount of vehicle (if gavage or dermal): No data
- Type and concentration of dispersant aid (if powder): No data
- Lot/batch no. (if required): No data
- Purity: No data

3. - Vehicle(s)/solvent(s) used: Solutions or emulsions of test substances to be fed to the flies were prepared in 5% saccharose, with the addition of 2% ethanol and 2% Tween 80 for compounds that were poorly soluble in water.
- Justification for choice of solvent/vehicle: No data
- Concentration of test material in vehicle: 25mM
- Amount of vehicle (if gavage or dermal): No data
- Type and concentration of dispersant aid (if powder): No data
- Lot/batch no. (if required): No data
- Purity: No data

4. - Vehicle(s)/solvent(s) used: Olive oil
- Justification for choice of solvent/vehicle: The chemical was soluble in olive oil
- Concentration of test material in vehicle: 0, 800, 1400, 2000 mg/Kg
- Amount of vehicle (if gavage or dermal): No data
- Type and concentration of dispersant aid (if powder): No data
- Lot/batch no. (if required): No data
- Purity: No data
Details on exposure:
1. No data
2. No data
3. No data
4. No data
Duration of treatment / exposure:
1. No data
2. 0 and 24 hrs
3. No data
4. 0 and 24 hrs
Frequency of treatment:
1. No data
2. Once
3. No data
4. Once
Post exposure period:
1. No data
2. 6 hrs
3. No data
4. 6 hrs
Remarks:
25.0 mM/ 1
Remarks:
0, 1 x 344, 1 x 603 or 1 x 861 mg/Kg / 2
Remarks:
25.0 mM/ 3
Remarks:
0, 800, 1400 or 2000 mg/Kg / 4
No. of animals per sex per dose:
1. No data

2. Total: 16
0 mg/Kg: 4
344 mg/Kg: 4
603 mg/Kg: 4
861 mg/Kg: 4

3. No data

4. Total: 16
0 mg/Kg: 4
800 mg/Kg: 4
1400 mg/Kg: 4
2000 mg/Kg: 4
Control animals:
not specified
Positive control(s):
No data
Tissues and cell types examined:
1. Sex linked recessive lethal chromosomes
2. Bone marrow micronucleus were examined
3. Sex linked recessive lethal chromosomes
4. Bone marrow micronucleus were examined
Details of tissue and slide preparation:
1. No data
2. CRITERIA FOR DOSE SELECTION: No data

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): 30 hrs

DETAILS OF SLIDE PREPARATION: The smears were stained according to the method of Schmid (1976)

METHOD OF ANALYSIS: increase in the number of micronucleus

OTHER: No data

3. No data

4. CRITERIA FOR DOSE SELECTION: No data

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): 30 hrs

DETAILS OF SLIDE PREPARATION: The smears were stained according to the method of Schmid (1976)

METHOD OF ANALYSIS: increase in the number of micronucleus

OTHER: No data
Evaluation criteria:
1. Increase in the number of recessive lethal chromosomes
2. The smears were noted for micronucleated polychromatic erythrocytes
3. Increase in the number of recessive lethal chromosomes
4. The smears were noted for micronucleated polychromatic erythrocytes
Statistics:
1. No data
2. No data
3. No data
4. No data
Sex:
male
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
not specified
Negative controls validity:
not specified
Positive controls validity:
not specified
Remarks on result:
other: No mutagenic effects were observed.
Remarks:
1
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
not valid
Negative controls validity:
not specified
Positive controls validity:
not specified
Remarks on result:
other: No mutagenic effects were observed
Sex:
male
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
not specified
Negative controls validity:
not specified
Positive controls validity:
not specified
Remarks on result:
other: No mutagenic effects were observed
Remarks:
3
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
not specified
Negative controls validity:
not specified
Positive controls validity:
not specified
Remarks on result:
other: No mutagenic effects were observed
Additional information on results:
1. No data
2. RESULTS OF RANGE-FINDING STUDY
- Dose range: No data
- Solubility: No data
- Clinical signs of toxicity in test animals: No data
- Evidence of cytotoxicity in tissue analyzed: No data
- Rationale for exposure: No data
- Harvest times: No data
- High dose with and without activation: No data
- Other: No data


RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): No data
- Induction of micronuclei (for Micronucleus assay):Yes
- Ratio of PCE/NCE (for Micronucleus assay):
Micronucleus formation:
Dose(mg/kg) Surviving/ treated mice Mean no. of micronucleated
PE/1000 PE
0 4/4 1.3
344 4/4 1.0
603 4/4 1.3
861 4/4 2.0
(PE =Polychromatic erythrocytes)
- Appropriateness of dose levels and route: Intraperitoneal
- Statistical evaluation:No data

3. No data

4. RESULTS OF RANGE-FINDING STUDY
- Dose range: No data
- Solubility: No data
- Clinical signs of toxicity in test animals: No data
- Evidence of cytotoxicity in tissue analyzed: No data
- Rationale for exposure: No data
- Harvest times: No data
- High dose with and without activation: No data
- Other: No data


RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): No data
- Induction of micronuclei (for Micronucleus assay):Yes
- Ratio of PCE/NCE (for Micronucleus assay):
Micronucleus formation:

Dose(mg/kg) Surviving/ treated mice Mean no. of micronucleated
PE/1000 PE
0 4/4 1.5
800 4/4 1.8
1400 4/4 1.0
200 4/4 2.5

(PE =Polychromatic erythrocytes)
- Appropriateness of dose levels and route: Intraperitoneal
- Statistical evaluation:No data
Conclusions:
The test chemical, Methyl 2-naphthyl ether (Cas No. 93-04-9) did not induce gene and chromosome mutations, structural and numerical chromosome aberrations in vivo and hence, it is not likely to classify as mutagenic substance in vivo.
Executive summary:

Available data in the literature on the test chemical, Methyl 2-naphthyl ether (Cas No. 93-04-9) and its read across analogues were reviewed to determine the toxic nature of the test chemical. The studies are as mentioned below:

#1

A Sex-Linked Recessive Lethal (SLRL) mutagenicity test was carried out in fruit fly, Drosophila melanogaster to assess the genotoxic nature of2-Ethoxynaphthalene (Cas No. 93-18-5) in vivo. Wild-type males were fed the solution of test substance, prepared in 5 % of saccharose at final concentration of 25 mM. Treated males were mated to appropriate females. The offspring of these females were scored for lethal effects corresponding to the effects on mature sperm, mid or late-stage spermatids, early spermatids, spermatocytes and spermatogonia at the time of treatment. Absence of wild type males in F2 generation indicated that a sex-linked recessive lethal mutation has occurred in a germ cell of the P1 male. As seen by the results, no significant increase in percentage of sex-linked recessive lethal mutations in the germ line of insects were detected comparing treated and control groups. Hence,2-ethoxynaphthalenwas non mutagenic SLRL test in vivo performed in Drosophila melanogaster.

#2

A micronucleus (MN) test was performed using male and female NMRI mice to determine the mutagenic nature of 2-ethoxynaphthalen in vivo. The test substance was administered in olive oil at the final concentration of 0, 344, 603 and 861 mg/kg via a single intraperitoneal injection. Then the mice were sacrificed, and bone-marrow smears were prepared 30 hours after treatment. The smears were stained according to the method of Schmid (1976) and were observed for cytogenetic effects in polychromated erthrocytes. As seen by the results, a single exposure to2-ethoxynaphthalen did not induce and increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow samples as compared with the concurrent control. Hence, in vivo micronucleus test performed in male and female NMRI mice did not indicate mutagenic potential of 2-ethoxynaphthalen.

#3

A Sex-Linked Recessive Lethal (SLRL) mutagenicity test was employed to determine the mutagenic nature of 2-(2-Methylpropoxy)Naphthalene (Cas No. 2173-57-1) in Drosophila melanogaster. The test substance was dissolved in 5 % saccharose solution and administered to wild type male flies at final concentration of 25 mM. There was a significant increase in sex-linked recessive lethal mutation (SRL) in single tests (Brood II) of treated groups as compared to control, but repeated tests did not confirm the mutagenic activity of 2-(2-Methylpropoxy)Naphthalene. Thus, this apparent effect that was observed only in single experiment probably occurred spontaneously. In conclusion, 2-(2-Methylpropoxy)Naphthalene was non mutagenic in SLRL test in vivo performed in Drosophila melanogaster.

#4

An in vivo micronucleus test was performed in male and female NMRI mice to evaluate the mutagenic potential of 2-(2-Methylpropoxy)Naphthalene (Cas No. 2173-57-1).The test substance was administered in olive oil at the final concentration of 0, 800, 1400 and 2000 mg/kg via a single intraperitoneal injection. Then the mice were sacrificed, and bone-marrow smears were prepared 30 hours after treatment. A single exposure to2-(2-Methylpropoxy)Naphthalene did not increase the frequency of micronucleated polychromatic erythrocytes in the bone marrow of treated mice as compared with the concurrent control. Hence, in vivo micronucleus test performed in male and female NMRI mice did not indicate mutagenic potential of 2-(2-Methylpropoxy)Naphthalene. 

.

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Genetic Toxicity (In Vitro):

Available data in the literature on the test chemical, Methyl 2-naphthyl ether (Cas No. 93-04-9) was reviewed to determine the mutagenic nature of the test chemical. The studies are as mentioned below:

Ames test:

Study 1:

Ames assay was performed to investigate the potential of Methyl-2-napthyl ether (CAS no. 93-04-9) to induce gene mutations in comparison to vehicle control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102. The assay was performed in two independent experiments both with and without liver microsomal activation(S9 mix). Each concentration, including the negative, vehicle and positive controls was tested in triplicate. Based on the pre-experiment results, the test item was tested with the following concentrations 0, 0.003, 0.008, 0.025, 0.079 and 0.250 mg/plate for main study, both in the presence of metabolic activation (+S9) and in the absence of metabolic activation (-S9). No substantial increase in revertant colony numbers in any of the tester strains were observed following treatment with Methyl-2-napthyl ether at any dose level in both the confirmatory trials, neither in the presence nor in the absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.The spontaneous reversion rates in the negative, vehicle and positive controlswerewithin the range of historical data. The positive controls used for various strains showed a distinct increase in induced revertant colonies in both the methods i.e. Plate incorporation method and Pre-incubation method. In conclusion, it is stated that during the described mutagenicity test and under the experimental conditions reported, Methyl-2-napthyl ether did not induce gene mutations by base pair changes or frame shifts in the genome of Salmonella typhimurium strains used.

Study 2:

The mutagenic activity of 2-methoxynaphthalene (Cas No. 93 -04 -9) was assessed by Ames test (spot test) using Salmonella typhimurium LT-2 strains TA 1535, TA 1537, TA 98 and TA 100. The test chemical was dissolved in ethanol as vehicle and used at dose levels of 0 or 3 µmole/plate (475 μg/ plate) in the presence and in the absence of S9 liver microsome fractions from Aroclor-induced rats. Positive and vehicle controls were also included in the test. As seen by the results, no substantial increase in revertant (His+) colony numbers in any of the tester strains were seen following treatment with 2-methoxynaphthalenen either in the presence nor in the absence of metabolic activation. Hence, 2-methoxynaphthalene is not mutagenic (negative) in Salmonella typhimurium strains used,under the described experimental conditions.  

In vitro mammalian chromosome aberration test:

An in vitro mammalian chromosome aberration test (OECD TG 473) was conducted to determine the chromosomal aberration induction potential of Methyl 2-napthyl ether in human peripheral blood lymphocyte cultures. Blood was drawn from a healthy volunteer, by venous puncture using heparinised syringe. The experiment was performed both in the presence and in the absence of metabolic activation system after 48 hours mitogenic stimulation. The test chemical was dissolved in DMSO and used at dose level of 0, 0.00025, 0.0005 and 0.001 mg/ml, in the presence and absence of S9 metabolic activation system in Trial 1 and Trial 2. Trial I was performed by short term treatment method both in the presence and absence of metabolic activation system (1 %, S9 mix). Phase II was performed either by short-term treatment (4 hours) as well as long-term treatment (24 hours) methods. Long term treatment was performed in absence of metabolic activation to confirm the negative results obtained in the absence of metabolic activation in Phase I. Short term treatment method was performed with increased metabolic activation (2%) condition to confirm the negative results obtained in the presence of metabolic activation in Phase I. The doses for the main study were based on the cytotoxicity study conducted both in the presence and absence of metabolic activation system. Three test concentrations (0.5, 1 and 2 mg/mL of culture media) based on the solubility, precipitation and pH test of the test item were used. Minimum 300 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides andthe percent aberrant cells was calculated. Before conducting the chromosome aberration study, Methyl 2-napthyl ether was evaluated for cytotoxicity both in the absence and presence of metabolic activation system (S9 mix, 1 %). A minimum of 1000 cells were counted in different fields of slide per culture and the number of metaphases were recorded for mitotic index (MI) calculation.In the cytotoxicity experiment, exposure to 0.001 mg/ml of test substance induced slight cytotoxicity and the mitotic index showed 41.8 % reduction in the absence of metabolic activation and 42.18 % in the presence of metabolic activation. In the mutagenicity test,no structural abnormality of chromosome and induction of ploidy cells were observed at any dose tested in both confirmatory trials. The percent aberrant cells were comparable in control and treated groups, both in the presence and in the absence of exogenous metabolic activation. Similarly, there was no reduction in mitotic index after exposure to the test substance at any concentration tested, in both trials.In conclusion, under the experimental conditions reported, Methyl 2 -napthyl ether did not induce structural and numerical chromosome aberration and, consequently it is not clastogenic up to the concentration of 0.001 mg/ml in human peripheral blood lymphocytes both in the presence (1-2 %) and absence of metabolic activation.

In vitro mammalian cell gene mutation test:

An in vitro mammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of methyl 2-napthyl ether when administered to Chinese Hamster Ovary (CHO) cells. The test substance was administered to CHO cells for 3 hours at the dose levels of 0, 0.1, 0.25, 0.5 or 1.0 mM in the absence or presence of exogenous metabolic activation (S9 mix). 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.No mutant colonies were detected following the exposure to positive control of 7,12-dimethylbenz(a) anthracene and, therefore no conclusion could be drawn from experiments performed in the presence of exogenous metabolic activation. No clear indication of gene mutation was seen when CHO cells were treated with methyl 2-naphthyl etherat concentration of0, 0.1, 0.25, 0.5 or 1.0 mM for 3 hoursin the absence of S9 mix. One very diffuse colony was seen in one well out of four at the concentration 0.5 mM, but this colony was a small cluster of apoptotic cells as it was only mildly coloured by crystal violet and, consequently it was considered irrelevant tothepresenttest.This was 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.In addition, no cytotoxic effects were observed when CHO cells were exposed to the test substance at any concentration used. In conclusion,methyl 2-napthyl etherdoes not give rise to gene mutations when CHO cells are exposed to the test chemical at 0, 0.1, 0.25, 0.5 or 1.0 mM for 3 hours in the absence of exogenous metabolic activation (-S9 mix). In addition,methyl 2-napthyl ether does not induce cytotoxic effects at concentrations ≤ 10.0 mM. 

Genetic Toxicity (In Vivo):

Available data in the literature on the read across analogues of the test chemical, Methyl 2-naphthyl ether (Cas No. 93-04-9) were reviewed to determine the mutagenic nature of the test chemical. The studies are as mentioned below:

Study 2:

A Sex-Linked Recessive Lethal (SLRL) mutagenicity test was carried out in fruit fly, Drosophila melanogaster to assess the genotoxic nature of2-Ethoxynaphthalene (Cas No. 93-18-5) in vivo. Wild-type males were fed the solution of test substance, prepared in 5 % of saccharose at final concentration of 25 mM. Treated males were mated to appropriate females. The offspring of these females were scored for lethal effects corresponding to the effects on mature sperm, mid or late-stage spermatids, early spermatids, spermatocytes and spermatogonia at the time of treatment. Absence of wild type males in F2 generation indicated that a sex-linked recessive lethal mutation has occurred in a germ cell of the P1 male. As seen by the results, no significant increase in percentage of sex-linked recessive lethal mutations in the germ line of insects were detected comparing treated and control groups. Hence,2-ethoxynaphthalenwas non mutagenic SLRL test in vivo performed in Drosophila melanogaster.

Study 3:

A micronucleus (MN) test was performed using male and female NMRI mice to determine the mutagenic nature of 2-ethoxynaphthalen in vivo. The test substance was administered in olive oil at the final concentration of 0, 344, 603 and 861 mg/kg via a single intraperitoneal injection. Then the mice were sacrificed, and bone-marrow smears were prepared 30 hours after treatment. The smears were stained according to the method of Schmid (1976) and were observed for cytogenetic effects in polychromated erthrocytes. As seen by the results, a single exposure to2-ethoxynaphthalen did not induce and increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow samples as compared with the concurrent control. Hence, in vivo micronucleus test performed in male and female NMRI mice did not indicate mutagenic potential of 2-ethoxynaphthalen.

Study 4:

A Sex-Linked Recessive Lethal (SLRL) mutagenicity test was employed to determine the mutagenic nature of 2-(2-Methylpropoxy)Naphthalene (Cas No. 2173-57-1) in Drosophila melanogaster. The test substance was dissolved in 5 % saccharose solution and administered to wild type male flies at final concentration of 25 mM. There was a significant increase in sex-linked recessive lethal mutation (SRL) in single tests (Brood II) of treated groups as compared to control, but repeated tests did not confirm the mutagenic activity of 2-(2-Methylpropoxy)Naphthalene. Thus, this apparent effect that was observed only in single experiment probably occurred spontaneously. In conclusion, 2-(2-Methylpropoxy)Naphthalene was non mutagenic in SLRL test in vivo performed in Drosophila melanogaster.

Study 5:

An in vivo micronucleus test was performed in male and female NMRI mice to evaluate the mutagenic potential of 2-(2-Methylpropoxy)Naphthalene (Cas No. 2173-57-1).The test substance was administered in olive oil at the final concentration of 0, 800, 1400 and 2000 mg/kg via a single intraperitoneal injection. Then the mice were sacrificed, and bone-marrow smears were prepared 30 hours after treatment. A single exposure to2-(2-Methylpropoxy)Naphthalene did not increase the frequency of micronucleated polychromatic erythrocytes in the bone marrow of treated mice as compared with the concurrent control. Hence, in vivo micronucleus test performed in male and female NMRI mice did not indicate mutagenic potential of 2-(2-Methylpropoxy)Naphthalene.

Based on the observations madeit can be concluded that, thetest chemical ofMethyl 2-naphthyl ether (Cas No. 93-04-9) did not inducegene and chromosome mutations, structural and numerical chromosome aberrationsin vivo and hence, it is not likely to classify as mutagenic substance in vivo. 

Considering all results gained from studies with the test substance it is demonstated that Methyl 2-napthyl ether (Cas No. 93-04-9) exhibits similar genotoxic effects as its structurally and functionally similar analogues both in vitro and vivo. Hence, the test substance of Methyl 2-naphthyl ether (Cas No. 93-04-9 is considered to be non-mutagenic in vitro and in vivo as the per the CLP regulations.

Justification for classification or non-classification

The mutagenic nature of methyl 2-naphthyl ether (Cas No. 93-04-9) has been evaluated by a combination of in vitro and in vivo genotoxicity tests: bacterial reverse mutation/Ames test, chromosome aberration and gene mutation tests of cultured mammalian cell, in vivo sex-linked recessive lethal mutation and micronucleus tests. The combination of these test approaches covers gene and chromosome mutations and clastogenic activity in somatic and germ cells in vitro and in vivo. As only limited toxicology data are available in the literature on methyl 2-naphthyl ether in silico evaluation was conducted by determining read across analogs for the target chemical. Based on structural similarity, reactivity, metabolism data and the physical-chemical properties, analogs of Isobutyl 2-naphthyl ether (Cas No. 2173-57-1) and Ethyl 2-naphthyl ether (Cas No. 93-18-5) were identified as read across of the target chemical for mutagenicity endpoint. Conclusive evidence from the studies discussed indicated, that methyl 2-naphthyl ether did not induce alterations in genetic material like gene and chromosome mutations, structural and numerical chromosome aberrations in vitro and in vivo neither in somatic nor in germ cells and, consequently methyl 2-naphthyl ether is "Not classified" for Mutagenicity according to the criteria of CLP regulations.