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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
supporting 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 J-check

Data source

Reference
Reference Type:
review article or handbook
Title:
In vitro mammalian chromosome aberration test for CAS no 2173-57-1
Author:
National Institute of Technology and Evaluation
Year:
2017
Bibliographic source:
Japan Chemicals Collaborative Knowledge Database, 2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Principles of method if other than guideline:
In vitro mammalian chromosome aberration test was performed to determine the mutagenic nature of 2-(2-methylpropoxy)naphthalene
GLP compliance:
not specified
Type of assay:
other: In vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material: 2-(2-Methylpropoxy)Naphthalene
- Molecular formula: C14H16O
- Molecular weight: 200.279 g/mol
- Substance type: Organic
- Physical state: No data
- Purity: No data
- Impurities (identity and concentrations): No data
Specific details on test material used for the study:
- Name of test material: 2-(2-Methylpropoxy)Naphthalene
- Molecular formula: C14H16O
- Molecular weight: 200.279 g/mol
- Substance type: Organic
- Physical state: No data
- Purity: No data
- Impurities (identity and concentrations): No data

Method

Target gene:
No data
Species / strainopen allclose all
Species / strain / cell type:
other: Chinese hamster lung (CHL/IU) cells
Remarks:
Short term treatment
Details on mammalian cell type (if applicable):
No data
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
other: Chinese hamster lung (CHL/IU) cells
Remarks:
Continuous treatmnet
Details on mammalian cell type (if applicable):
No data
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
No data
Test concentrations with justification for top dose:
Continuous treatment: 0, 61.4, 76.8, 96.0 and 120 µg/mL
Short term treatment: 0, 76.8, 96.0 and 120 µg/mL without S9 mix and at 0, 16.1, 20.1 and 25.2 µg/mL with S9 mix
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO
Controls
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 3 days
- Exposure duration:
Short term treatment: 6 hrs
Continuous treatment: 24 hrs
- Expression time (cells in growth medium):
Short term treatment: 6 hrs
Continuous treatment: 24 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: No data

NUMBER OF CELLS EVALUATED: 200 metaphase cells/dose (100/plate) for structural aberrations (i.e. gap, ctb, csb, cte, cse and other abnormalities) and for polyploid

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: yes, cytotoxicity was measured as inhibition of cell growth

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: No data
- Other: No data

OTHER: No data
Rationale for test conditions:
No data
Evaluation criteria:
The final result of each test was decided for structural aberrations or polyploid cells, separately as follows:negative (-): if the frequency of aberrant cells was less than 5%,inconclusive (±): in greater than 5% but less than 10%,and positive (+): if 10% or more.
Statistics:
No data

Results and discussion

Test resultsopen allclose all
Species / strain:
other: Chinese hamster lung (CHL/IU) cells
Remarks:
Short term treatment
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
cytotoxicity (more than 50% inhibition of cell growth) was shown at 120 µg/mL and above under the absence of metabolic activation and at 25.2 µg/mL and above under the presence of metabolic activation.
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
other: Chinese hamster lung (CHL/IU) cells
Remarks:
Long term treatment
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
cytotoxicity (more than 50% inhibition of cell growth) was shown at 120 µg/mL and above under the absence of metabolic activation and at 25.2 µg/mL and above under the presence of metabolic activation.
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
No data
Remarks on result:
other: No mutagenic potential

Any other information on results incl. tables

Table: Historical control data

Historical control values of structural aberrations

Group

Test system

N

Incidence of structural aberration (%) [Mean±SD]

Range

Lower

Upper

Negative control

Short term treatment (-S9)

157

0.6±0.8

0.0

2.9*

Short term treatment (+S9)

166

0.4±0.6

0.0

2.3*

Continuous treatment (24 hrs)

135

0.6±0.7

0.0

2.8*

Positive control

Short term treatment (-S9)

157

39.0±9.7

10.0

68.2*

Short term treatment (+S9)

164

29.2±10.5

10.0

60.8*

Continuous treatment (24 hrs)

133

30.3±8.2

10.0

54.9*

 

Historical control values of polyploidy cells

Group

Test system

N

Incidence of structural aberration (%) [Mean±SD]

Range

Lower

Upper

Negative control

Short term treatment (-S9)

157

0.3±0.5

0.0

1.9*

Short term treatment (+S9)

166

0.2±0.5

0.0

1.7*

Continuous treatment (24 hrs)

135

0.4±0.7

0.0

2.4*

 

* The range is calculated by Mean±3 X SD

 

Table: Results of growth inhibition test (Short term treatment)

Compound

Dose (µg/mL)

Relative cell growth (%)

Mean

-S9

+S9

-S9

+S9

DMSO

0

100.0

100.0

100.0

100.0

100.0

100.0

2- Napthylisobutyl ether

15.6

100.0

103.9

100.5

101.0

100.8

98.0

31.3

91.1

31.4

86.5

21.7

97.2

20.4

62.6

89.7

22.9

86.5

21.7

83.3

20.4

125

28.3

15.9

22.7

16.0

17.1

16.0

250d

7.5

6.6

6.3

10.1

5.1

13.6

501d

6.0

6.1

6.7

6.1

7.3

6.1

1002d

12.9

11.1

12.5

11.0

12.0

10.8

2003d

6.6

10.7

5.5

9.2

4.4

7.6

d: visible precipitation was noted

Table 2. Chromosome aberration test

[Short-term treatment : -S9]

Compound Dose

Time of exposure

Relative cell growth

No. of cells analyzed

No. of cells with structural aberrations

No. of cells with aberrations

No. of cells analyzed for polyploid

No. of polyploid cells

(μg/mL)

(h)

(%)

gap

ctb

cte

csb

cse

oth

-gap(%)

(%)

DMSO a) 0

6

100

200

0

1

0

0

0

0

1 ( 0.5)

200

0 ( 0.0)

2-Naphthylisobutyl ether

76.8

6

77.2

200

0

0

0

0

0

0

0 ( 0.0)

200

0 ( 0.0)

96

6

53.5

200

0

2

1

0

0

0

3 ( 1.5)

200

0 ( 0.0)

120

6

24.1

200

0

2

4

0

0

0

5 ( 2.5)

200

1 ( 0.5)

150

6

11

NA

MMC b) 0.1

6

85.6

200

2

30

69

0

1

0

88 (44.0)

200

0 ( 0.0)

Abbreviation: ctb; chromatid break, cte: chromatid exchange, csb: chromosome break, cse: chromosome exchange, oth: others

gap: total No. of cells with aberrations except gap

NA:Not analyzed

a): Negative control (Dimethyl sulfoxide, 10 μL/mL)

b): Positive control:Mitomycin C

[Short-term treatment : +S9]

Compound Dose

Time of exposure

Relative cell growth

No. of cells analyzed

No. of cells with structural aberrations

No. of cells with aberrations

No. of cells analyzed for polyploid

No. of polyploid cells

(μg/mL)

(h)

(%)

gap

ctb

cte

csb

cse

oth

-gap(%)

(%)

DMSO a) 0

6

100

200

0

0

0

0

0

0

0 ( 0.0)

200

1 ( 0.5)

2-Naphthylisobutyl ether

16.1

6

64.3

200

0

0

0

0

0

0

0 ( 0.0)

200

1 ( 0.5)

20.1

6

56.2

200

0

0

0

0

0

0

0 ( 0.0)

200

0 ( 0.0)

25.2

6

40.2

200

0

1

2

0

0

0

3 ( 1.5)

200

2 ( 1.0)

31.5

6

14.6

NA

CP b) 12.5

6

88

200

3

8

28

0

0

0

34 (17.0)

200

0 ( 0.0)

Abbreviation: ctb; chromatid break, cte: chromatid exchange, csb: chromosome break, cse: chromosome exchange, oth: others

gap: total No. of cells with aberrations except gap

NA:Not analyzed

a): Negative control (Dimethyl sulfoxide, 10 μL/mL)

b): Positive control: Cyclophosphamide

[Continuous treatment : 24 h]

Compound Dose

Time of exposure

Relative cell growth

No. of cells analyzed

No. of cells with structural aberrations

No. of cells with aberrations

No. of cells analyzed for polyploid

No. of polyploid cells

(μg/mL)

(h)

(%)

gap

ctb

cte

csb

cse

oth

-gap(%)

(%)

DMSO a) 0

24

100

200

1

1

0

0

0

0

1 ( 0.5)

200

1 ( 0.5)

2-Naphthylisobutyl ether

61.4

24

72.5

200

0

2

0

0

0

0

2 ( 1.0)

200

0 ( 0.0)

76.8

24

83

200

1

1

0

0

0

0

1 ( 0.5)

200

0 ( 0.0)

96

24

66.5

200

1

2

0

0

0

0

2 ( 1.0)

200

0 ( 0.0)

120

24

40.3

182

2

3

1

0

0

0

4 ( 2.2)

200

0 ( 0.0)

150

24

26.3

Toxic

MMC b) 0.05

24

123.1

200

5

33

68

0

0

0

84 (42.0)

200

1 ( 0.5)

Abbreviation: ctb; chromatid break, cte: chromatid exchange, csb: chromosome break, cse: chromosome exchange, oth: others

gap: total No. of cells with aberrations except gap

a): Negative control (Dimethyl sulfoxide, 10 μL/mL)

b): Positive control: Mitomycin C

Applicant's summary and conclusion

Conclusions:
2-(2-Methylpropoxy)Naphthalene did not induce chromosome aberration in Chinese hamster lung (CHL/IU) cells in the short term treatment (with and without) and continuous treatment (without) and hence it is not likely to classify as a gene mutant in vitro.
Executive summary:

In vitro mammalian chromosome aberration test was performed to determine the mutagenic nature of the test chemical using Chinese hamster lung cells (CHL/IU). The test chemical was dissolved in DMSO and used at dose levels of 0, 76.8, 96.0 and 120µg/mL without S9 mix and at 0, 16.1, 20.1 and 25.2µg/mL with S9 mix for the short-term treatment, and at 0, 61.4, 76.8, 96.0 and 120µg/mL for continuous treatment. In either test condition, no increase in structural aberrations or polyploidy was observed, although cytotoxicity (more than 50% inhibition of cell growth) was shown at 120µg/mL and above under the absence of metabolic activation and at 25.2µg/mL and above under the presence of metabolic activation. The positive controls were effective for induction of chromosome aberrations. The test chemical did not induce chromosome aberration in Chinese hamster lung (CHL/IU) cells in the short term treatment (with and without) and continuous treatment (without) and hence it is not likely to classify as a gene mutant in vitro.