Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Data for test chemicals was reviewed to determine the mutagenic nature of 2-ethoxynaphthalene (93-18-5). The studies are as mentioned below:

AMES Assay

Ames assay was performed to investigate the potential of test substance 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. Each concentration, including the negative, vehicle and positive controls was tested in triplicate. Based on the solubility and precipitation test results eight different concentrations viz. 0, 0.001, 0.003, 0.008, 0.025, 0.079, 0.250, 0.791 and 2.5 mg/plate were selected for pre-experiment. Based on the pre-experiment results, the test item was tested with the following concentrations 0, 0.008, 0.025, 0.079, 0.250 and 0.791 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 test substance 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 controls are within the range of our 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, the test item did not induce gene mutations by base pair changes or frame shifts in the genome of the strains used.

In vitro Chromosomal abbreviation study in mammalian cell

Test substance is non-clastogenic at the highest tested concentration  both in the presence and in the absence of metabolic activation under the specified conditions and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
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
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 test substance 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.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Name of test material: 2-Ethoxynaphthalene (β-naphthyl ethyl ether)
- Molecular formula: C12H12O
- Molecular weight: 172.226 g/mol
- Subsatnce type: Organic
- Physical state: No data
- Purity: No data
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, 0.008, 0.025, 0.079, 0.250 and 0.791 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:
yes
Remarks:
RO water
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, 0.008, 0.025, 0.079, 0.250 and 0.791 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

              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)

6.00

2.00

11.67

1.15

16.00

1.73

93.67

8.02

229.33

12.22

VC

(0.00)

7.67

1.15

13.00

1.73

18.67

0.58

106.67

9.07

289.33

29.14

T1

(0.008)

5.33

2.31

9.67

0.58

21.33

4.16

90.67

11.02

232.67

5.03

T2

(0.025)

5.67

2.52

13.67

2.52

18.33

1.53

119.67

8.08

242.67

9.45

T3

(0.079)

7.33

1.53

13.00

2.65

17.00

2.00

111.00

8.54

258.67

17.24

T4

(0.250)

5.33

1.53

10.67

2.08

18.67

3.06

124.00

6.08

233.33

9.45

T5

(0.791)

6.33

2.89

13.33

1.53

16.67

1.53

93.00

4.58

294.00

12.00

PC

158.00

12.00

361.33

29.96

1014.67

45.49

1229.33

59.91

1740.67

101.87

 

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

1.00

9.00

2.65

16.00

1.00

82.00

7.55

246.67

6.11

VC

(0.00)

7.33

1.53

9.33

0.58

15.67

2.89

100.33

1.53

307.33

8.33

T1

(0.008)

4.33

0.58

13.33

2.08

24.67

6.11

114.33

8.50

281.33

15.53

T2

(0.025)

6.33

1.15

11.67

1.53

19.67

2.08

113.33

14.36

298.67

4.16

T3

(0.079)

4.33

1.53

14.67

1.15

23.00

3.61

107.00

4.58

300.00

14.42

T4

(0.250)

7.33

2.08

12.67

2.08

22.33

4.04

121.67

6.03

293.33

16.04

T5

(0.791)

5.33

1.15

12.33

2.08

17.33

1.53

79.33

3.21

302.00

4.00

PC

199.33

18.15

1182.67

151.66

788.00

136.18

1458.00

71.36

2007.33

18.15

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-INCUBATIONMETHOD
(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)

4.00

1.00

9.00

1.00

19.33

2.08

101.00

2.65

274.67

6.11

VC

(0.00)

5.33

2.08

11.00

3.00

22.00

2.65

110.00

7.00

294.00

11.14

T1

(0.008)

6.33

2.08

11.33

2.08

19.33

1.15

87.33

8.50

271.33

13.01

T2

(0.025)

5.00

1.73

12.33

1.53

26.00

2.00

102.33

4.51

285.33

8.33

T3

(0.079)

4.00

1.00

8.67

1.15

22.67

2.08

112.00

9.54

268.00

12.00

T4

(0.250)

5.33

1.53

10.67

1.53

21.00

2.65

95.00

16.52

303.33

7.57

T5

(0.791)

6.33

2.89

11.67

1.53

22.00

4.00

106.33

7.09

286.00

8.00

PC

176.67

11.72

380.00

48.04

1051.33

97.04

1013.33

82.81

1748.67

80.41

 

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)

4.33

1.53

10.33

1.53

18.33

2.08

105.33

9.45

270.00

15.62

VC

(0.00)

5.00

2.00

11.33

1.53

20.67

2.52

86.33

12.10

267.33

3.06

T1

(0.008)

3.33

0.58

11.33

3.51

21.00

5.57

98.33

10.79

282.00

14.42

T2

(0.025)

5.33

1.53

11.33

2.08

21.67

2.08

85.33

9.45

292.67

16.65

T3

(0.079)

7.00

1.00

11.00

2.65

22.67

4.16

118.67

5.13

277.33

11.37

T4

(0.250)

4.67

1.53

11.00

3.00

24.33

3.06

109.33

5.03

296.00

9.17

T5

(0.791)

5.33

2.52

9.67

2.08

22.67

3.51

113.00

9.85

300.67

9.02

PC

199.33

14.74

1112.00

41.90

969.33

72.04

1121.33

85.19

1792.00

51.73

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:
Test substance 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 mutant as per the criteria mentioned in CLP regulation.
Executive summary:

Ames assay was performed to investigate the potential of test substance 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. Each concentration, including the negative, vehicle and positive controls was tested in triplicate. Based on the solubility and precipitation test results eight different concentrations viz. 0, 0.001, 0.003, 0.008, 0.025, 0.079, 0.250, 0.791 and 2.5 mg/plate were selected for pre-experiment.

 

Based on the pre-experiment results, the test item was tested with the following concentrations 0, 0.008, 0.025, 0.079, 0.250 and 0.791 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 test substance 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 controls are within the range of our 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.

 

Conclusion

 

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

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from handbook or collection of data.
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
other: As given below
Principles of method if other than guideline:
Weight of evidence prepared from various publication discribed below
1,This in vitro assay was performed to assess the potential of test substance 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.
2,Chromosomal aberration study was performed to determine the mutagenic nature of the test chemical
GLP compliance:
not specified
Type of assay:
other: In vitro mammalian chromosome aberration assay
Specific details on test material used for the study:
- Name of test material: 2-Ethoxynaphthalene (β-naphthyl ethyl ether)
- Molecular formula: C12H12O
- Molecular weight: 172.226 g/mol
- Subsatnce type: Organic
- Physical state: No data
- Purity: No data
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
Species / strain / cell type:
other: Chinese hamster fibroblast cell line CHL
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:
1,0.00 (NC), 0.00025 (T1), 0.0005 (T2) and 0.001 (T3) mg/mL
2,At three different doses with 0.5 mg/mL being the maximum dose concentration
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
Untreated negative controls:
yes
Remarks:
Untreated cells served as negative control
Negative solvent / vehicle controls:
yes
Remarks:
Medium
True negative controls:
not specified
Positive controls:
not specified
Positive control substance:
not specified
Details on test system and experimental conditions:
1,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

2,METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: No data
- Exposure duration: 24 and 48 hrs
- Expression time (cells in growth medium): 24 and 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): Giemsa solution (1.5%, pH 6.8)
SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: No data

NUMBER OF CELLS EVALUATED: 100 well spread metaphases

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: No data
- Other: 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
Species / strain:
other: Chinese hamster fibroblast cell line CHL
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
not specified
Additional information on results:
1,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
Conclusions:
Test substance is non-clastogenic at the highest tested concentration both in the presence and in the absence of metabolic activation under the specified conditions and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.
Executive summary:

Data for the various test chemicals was reviewed to determine the mutagenic nature of 2-ethoxynaphthalene (93-18-5). The studies are as mentioned below:

This study was conducted to determine the chromosomal aberration induction potential of Methyl-2-napthyl ether (CAS no. 93-04-9) in human peripheral blood lymphocyte cultures. The methods followed were as per OECD guideline No. 473, adopted on 29thJuly 2016 “ In Vitro Mammalian Chromosome Aberration Test. The experiment was conducted using human peripheral blood lymphocytes. 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 h 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 phase 1 and phase 2. Phase I of experiment was performed by short term treatment method both in the presence and absence of metabolic activation system(1%). Phase II of experiment was performed by short term treatment as well as long term treatment method. 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. 3 test concentrations (0.5, 1 and 2mg/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 negative control. The medium of the proliferatingblood culture was removed by centrifugation at 1500 rpm for 10 minutes. The cells were suspended in plain medium (medium without serum) mixed with S9 mix (Phase I - 1 % and Phase II - 2 % v/v) and in complete media mixed with phosphate buffer for the treatment in presence and in absence of metabolic activation system respectively. A volume of 7.92 mL of proliferating culture was dispensed to individual sterile culture tubes/flasks. Each tube/flask according to treatment groups was identified. Negative control tubes were treated with 80 µL of RPMI media and treatment group were treated with 80 µL of respective test item stock solution. The cultures were incubated at 37 ± 2 °C for duration (exposure period). 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 slides were dried over a slide warmer and labelled. At least two slide was made from each sample. The cells were stained with 5 % fresh Giemsa stain in phosphate buffer and mounted using DPX mountant. Evaluation of the slides was performed using microscopes with 100 x oil immersion objectives. 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.300 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides. Evaluation of the slides was performed using microscopes with 100 x oil immersion objectives. Chromosomal and chromatid breaks, acentric fragments, deletions, exchanges, pluverization, polyploidy (including endoreduplication) and disintegrations were recorded as structural chromosomal aberrations. Gaps were recorded as well, but they were not included in the calculation of the aberration rates. Only metaphases with 46± 2 centromere regions were included in the analysis. Methyl-2-napthyl ether (CAS no. 93-04-9) is non-clastogenic at the highest tested concentration of 0.001mg/ml both in the presence (1% and 2%) and in the absence of metabolic activation under the specified conditions and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

 Chromosomal aberration study was performed to determine the mutagenic nature of the test chemical. The cells were exposed to the test material at three different doses with 0.5 mg/mL being the maximum concentration for 48hr. Colcemid (final concn 0.2µg/ml) was added to the culture 2 hr before cell harvesting. The cells were then trypsinized and suspended in a hypotonic KCI solution (0.075 M) for 13 min at room temperature. After centrifugation the cells were fixed with acetic acid-methanol (1:3, v/v) and spread on clean glass slides. After air-drying, the slides were stained with Giemsa solution for 12-15 min. A hundred well-spread metaphases were observed under the microscope. In the present studies, no metabolic activation systems were applied. The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate. Untreated cells and solvent-treated cells served as negative controls, in which the incidence of aberrations was usually less than 3.0%. The results were considered to be negative if the incidence was less than 4.9%, equivocal if it was between 5.0 and 9.9%, and positive if it was more than 10.0%. The incidence of polyploid cells for 48hr after treatment was 0.0%. The incidence of cells with structural chromosomal aberrations at 0.4mg/ml for 48hr after treatment was 47.0%. Also positive at 0.3 mg/ml at 24hr (11.0 %) and at 48hr (30.0%). D20 was 0.25 mg/ml (the dose at which structural aberrations were detected in 20 % of theMetaphases observed). TR value (the frequency of cells with exchange-type aberrations per unit dose(mg/ml) ) was 0.35. The test chemical did not induce chromosomal aberration in Chinese hamster fibroblast cell line CHL in the absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant.

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

Genetic toxicity in vivo

Description of key information

Gene mutation toxicity study was performed to determine the mutagenic nature of 2-Ethoxynaphthalene using Drosophila SLRL test in vivo. 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. 2-Ethoxynaphthalene was used at dose levels of 25mM. Sex linked recessive lethal mutation were noted in the chromosomes. 2-Ethoxynaphthalene gave negative gene mutation results in the Drosophila SLRL test performed using male Drosophila melanogaster species.

Link to relevant study records
Reference
Endpoint:
genetic toxicity in vivo, other
Remarks:
Drosophila SLRL assay
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from peer reviwed publication
Qualifier:
according to
Guideline:
other: Sex linked recessive lethal mutation assay (Wuergler et al., 1977)
Principles of method if other than guideline:
Drosophila SLRL test test was performed to determine the mutagenic nature of 2-Ethoxynaphthalene
GLP compliance:
not specified
Type of assay:
Drosophila SLRL assay
Specific details on test material used for the study:
- Name of test material: 2-Ethoxynaphthalene
- Molecular formula: C12H12O
- Molecular weight: 172.226 g/mol
- Subsatnce type: Organic
- Physical state: Solid
- Purity: No data
Species:
Drosophila melanogaster
Strain:
not specified
Details on species / strain selection:
No data
Sex:
male
Details on test animals and environmental conditions:
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
Route of administration:
oral: feed
Vehicle:
- 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
Details on exposure:
No data
Duration of treatment / exposure:
No data
Frequency of treatment:
No data
Post exposure period:
No data
Dose / conc.:
25 other: mM
No. of animals per sex per dose:
No data
Control animals:
not specified
Positive control(s):
No data
Tissues and cell types examined:
Sex linked recessive lethal chromosomes
Details of tissue and slide preparation:
No data
Evaluation criteria:
Increase in the number of recessive lethal chromosomes
Statistics:
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 effect were observed.
Additional information on results:
No data

Table: Results of Basc tests on Drosophila for sex-linked recessi',e lethal mutations

Concentration(mM)

No.of sex-linked recessive lethal chromosomes tested in

Brood I

Brood II

Brood III

25

 

8/2383

0.34

3/2379

0.13

5/2423

0.21

Conclusions:
2-Ethoxynaphthalene gave negative gene mutation results in the Drosophila SLRL test performed using male Drosophila melanogaster species.
Executive summary:

Gene mutation toxicity study was performed to determine the mutagenic nature of 2-Ethoxynaphthalene using Drosophila SLRL test in vivo. 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. 2-Ethoxynaphthalene was used at dose levels of 25mM. Sex linked recessive lethal mutation were noted in the chromosomes. 2-Ethoxynaphthalene gave negative gene mutation results in the Drosophila SLRL test performed using male Drosophila melanogaster species.

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

Additional information

Data for test chemicals was reviewed to determine the mutagenic nature of 2-ethoxynaphthalene (93-18-5). The studies are as mentioned below:

In Vitro Genetic Mutation study

AMES Assay

Ames assay was performed to investigate the potential of test substance 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. Each concentration, including the negative, vehicle and positive controls was tested in triplicate. Based on the solubility and precipitation test results eight different concentrations viz. 0, 0.001, 0.003, 0.008, 0.025, 0.079, 0.250, 0.791 and 2.5 mg/plate were selected for pre-experiment. Based on the pre-experiment results, the test item was tested with the following concentrations 0, 0.008, 0.025, 0.079, 0.250 and 0.791 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 test substance 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 controls are within the range of our 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, the test item did not induce gene mutations by base pair changes or frame shifts in the genome of the strains used.

Gene mutation toxicity study was performed to determine the mutagenic nature of the test compound . The test was performed as per the Spot test protocol. Test substance was dissolved in ethanol and applied at a concentration of 0 or 3 µmole/plate. In absence of a background lawn of bacteria on the plates (indicating toxicity) the test was repeated with a lower concentration of the substance. Test substance did not induce reversion of mutant strains in S. typhimurium LT-2 strain TA 98, TA 100, TA 1535 and TA 1537 in the presence and absence of S9 metabolic activation system and hence is not likely to classify as gene mutant in vitro.

Gene mutation toxicity study was performed to determine the mutagenic nature of ethyl 2-naphthyl ether. Ames assay was performed by the preincubation assay by dissolving the chemical in DMSO and used at a dosage level of 1.22- 5000 µg/plate both in the presence and absence of S9 metabolic activation system. Ethyl 2-naphthyl ether failed to induce mutation in Salmonella tyohimurium strains TA100, TA98, TA1537 and TA1535 in the presence and absence of S9 metabolic activation system and hence is not likely to classify for gene mutation in vitro.

Gene mutation toxicity was performed to determine the mutagenic nature of test substance .The test was performed by plate incorporation method using Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, TA1538 with and without S9 metabolic activation system at 5 different doses upto 3600 µg/plate and the plates were incubated for 48hrs. 2-Ethoxynaphthalene failed to induce mutation in Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, TA1538 in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

 

In vitro Chromosomal abbreviation study in mammalian cell

 

This study was conducted to determine the chromosomal aberration induction potential of test chemical in human peripheral blood lymphocyte cultures. The methods followed were as per OECD guideline No. 473, adopted on 29thJuly 2016 “ In Vitro Mammalian Chromosome Aberration Test. The experiment was conducted using human peripheral blood lymphocytes. 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 h 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 phase 1 and phase 2. Phase I of experiment was performed by short term treatment method both in the presence and absence of metabolic activation system(1%). Phase II of experiment was performed by short term treatment as well as long term treatment method. 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. 3 test concentrations (0.5, 1 and 2mg/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 negative control. The medium of the proliferatingblood culture was removed by centrifugation at 1500 rpm for 10 minutes. The cells were suspended in plain medium (medium without serum) mixed with S9 mix (Phase I - 1 % and Phase II - 2 % v/v) and in complete media mixed with phosphate buffer for the treatment in presence and in absence of metabolic activation system respectively. A volume of 7.92 mL of proliferating culture was dispensed to individual sterile culture tubes/flasks. Each tube/flask according to treatment groups was identified. Negative control tubes were treated with 80 µL of RPMI media and treatment group were treated with 80 µL of respective test item stock solution. The cultures were incubated at 37 ± 2 °C for duration (exposure period). 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 slides were dried over a slide warmer and labelled. At least two slide was made from each sample. The cells were stained with 5 % fresh Giemsa stain in phosphate buffer and mounted using DPX mountant. Evaluation of the slides was performed using microscopes with 100 x oil immersion objectives. 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.300 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides. Evaluation of the slides was performed using microscopes with 100 x oil immersion objectives. Chromosomal and chromatid breaks, acentric fragments, deletions, exchanges, pluverization, polyploidy (including endoreduplication) and disintegrations were recorded as structural chromosomal aberrations. Gaps were recorded as well, but they were not included in the calculation of the aberration rates. Only metaphases with 46± 2 centromere regions were included in the analysis. Test chemical is non-clastogenic at the highest tested concentration of 0.001mg/ml both in the presence (1% and 2%) and in the absence of metabolic activation under the specified conditions and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

Chromosomal aberration study was performed to determine the mutagenic nature of the test chemical. The cells were exposed to the test material at three different doses with 0.5 mg/mL being the maximum concentration for 48hr. Colcemid (final concn 0.2µg/ml) was added to the culture 2 hr before cell harvesting. The cells were then trypsinized and suspended in a hypotonic KCI solution (0.075 M) for 13 min at room temperature. After centrifugation the cells were fixed with acetic acid-methanol (1:3, v/v) and spread on clean glass slides. After air-drying, the slides were stained with Giemsa solution for 12-15 min. A hundred well-spread metaphases were observed under the microscope. In the present studies, no metabolic activation systems were applied. The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate. Untreated cells and solvent-treated cells served as negative controls, in which the incidence of aberrations was usually less than 3.0%. The results were considered to be negative if the incidence was less than 4.9%, equivocal if it was between 5.0 and 9.9%, and positive if it was more than 10.0%. The incidence of polyploid cells for 48hr after treatment was 0.0%. The incidence of cells with structural chromosomal aberrations at 0.4mg/ml for 48hr after treatment was 47.0%. Also positive at 0.3 mg/ml at 24hr (11.0 %) and at 48hr (30.0%). D20 was 0.25 mg/ml (the dose at which structural aberrations were detected in 20 % of theMetaphases observed). TR value (the frequency of cells with exchange-type aberrations per unit dose(mg/ml) ) was 0.35. The test chemical did not induce chromosomal aberration in Chinese hamster fibroblast cell line CHL in the absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant.

In vivo genetic mutation assay

Gene mutation toxicity study was performed to determine the mutagenic nature of 2-Ethoxynaphthalene using Drosophila SLRL test in vivo. 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. 2-Ethoxynaphthalene was used at dose levels of 25mM. Sex linked recessive lethal mutation were noted in the chromosomes. 2-Ethoxynaphthalene gave negative gene mutation results in the Drosophila SLRL test performed using male Drosophila melanogaster species. 

Gene mutation toxicity was performed to determine the mutagenic nature of 2-Ethoxynaphthalene in vivo. Micronucleus assay was performed using bone marrow smears of male and female NMRI mice. 2-Ethoxynaphthalene was dissolved in olive oil and used at dose levels of 0, 1 x 344, 1 x 603, 1 x 861 mg/Kg. The chemical was given intraperitoneally during the 24 hrs study period. The mice were killed and bone-marrow smears were prepared 30 hr after treatment. The smears were stained according to the method of Schmid (1976) and were observed for genetic effects in polychromated erthrocytes. 2-Ethoxynaphthalene failed to produce genetic effects in the micronucleus assay performed and hence is not likely to classify as a gene mutant in vivo.

Based on the data summarized, 2-ethoxynaphthalene (93-18-5) is expected to not induce gene mutation both In Vitro and In vivo studies .Hence it is not likely to be mutagenic in vitro and vivo

Justification for classification or non-classification

Thus based on the above annotation and CLP criteria for target substance 2-ethoxynaphthalene (93-18-5)  does not exhibit gene mutation in vitro and vivo . Hence the test chemical is not likely to classify as a gene mutant in vitro and vivo.