Methyl 2-naphthyl ether

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation test in vitro:

Bacterial reverse mutation test:

The registered substance, 2-methoxynapthalene (CAS No. 93-04-9), was tested non-mutagenic (negative) in a bacterial reverse mutation test both in the presence and absence of S9 metabolic activation system. The test was performed according to OECD TG 471 and GLP.

In vitro mammalian chromosome aberration test:

The registered substance, i.e., 2-methoxynaphthalene (CAS: 93-04-9), tested non-clastogenic (negative) in primary cultures of human peripheral blood lymphocytes in the presence and absence of S9 metabolic activation system. The test was performed according to OECD TG 473 and GLP.

In vitro mammalian gene mutation test: 

An in vitro gene mutataion test with the registered substance (CAS 93-04-9) according to OECD TG 476 and GLP is ongoing. This results will be submitted at a later timepoint.

 

 

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Data waiving:

other justification

Justification for data waiving:

other:

Reference 2

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Version / remarks:

29. July 2016

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Purity: 99.06%
Molecular Weight:158.2 g/mol
Molecular Formula: C11H10O
Manufacture Date: June 29, 2018
Expiry Date: June 29, 2023

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

Human lymphocytes were collected from the blood. Blood samples were obtained from a healthy donor in the range of 25-29 years of age.

Metabolic activation:

with and without

Metabolic activation system:

Cofactor-supplemented liver S9 microsomal fraction was used. The S9 fraction was obtained from Aroclor 1254-induced rat.
An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution, which resulted in a final concentration in the S9 mix of approximately 1 % (v/v) and 2 % (v/v) for Phase I and Phase II, respectively.
The cofactor solution contained the following quantity of chemicals in 500 mL of Reverse Osmosis (RO) water:

D-glucose-6-phosphate: 0.80 g
MgCl2: 1.00 g
KCl:1.35 g
Na2HPO4: 6.40 g
NaH2PO4.H2O: 1.40 g
β-NADP: 1.75 g

Test concentrations with justification for top dose:

Test concentrations: 0.0 (NC: distilled water), 0.0 (VC: DMSO), 0.0006 mg/ml, 0.0012 mg/ml, 0.0024 mg/ml

Justifications:
Based on the solubility, precipitation, and pH tests, three test concentrations, 0.125, 0.063 and 0.031 mg/ml in culture media, were selected for evaluation. Cytotoxicity was determined by a reduction in the mitotic index in comparison with vehicle control. The test concentration 0.0024 mg/mL produced a 58.59 % and 56.08 % decrease in the mitotic index compared to VC in the absence (-S9) and presence of metabolic activation (+S9), respectively.

Vehicle / solvent:

Dimethyl sulfoxide was used as vehicle.

Untreated negative controls:

yes

Remarks:

Distilled water

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): Duplicates were used.
- Number of independent experiments: Two (Phase I-II)

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): NA
- Test substance added in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk: In medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: NA
- Exposure duration/duration of treatment: 4 hrs (Phase I), 24 hrs (Phase II)
- Harvest time after the end of treatment (sampling/recovery times): 24 hrs (Phase I-II)

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): indicate the identity of mitotic spindle inhibitor used (e.g., colchicine), its concentration and, duration and period of cell exposure.:Three hours before cell harvesting, 240 µL of colcemid® (10 µg/mL) (final concentration: 0.3 µg/mL) was added to each culture tube/flask and continued incubation at 37 °C.

- If cytokinesis blocked method was used for micronucleus assay: indicate the identity of cytokinesis blocking substance (e.g. cytoB), its concentration, and duration and period of cell exposure. NA
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): Microscopic slides with the mitotic metaphase spreads were prepared by dropping the cell suspension onto a clean pre-chilled microscope slide. Two slides were made from each sample. The cells were stained with 5 % fresh Giemsa stain in phosphate buffer and mounted using DPX.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): Three hundred well-spread metaphases per concentration and control were scored for cytogenetic damage on coded slides.
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification): NA
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): NA
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): Evaluation of the slides was performed using microscopes with 100 x oil immersion objectives. Chromosomal and chromatid gaps, breaks, acentric, fragments, exchanges, 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.
- Determination of polyploidy: Yes
- Determination of endoreplication: Yes

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition; mitotic index (MI); relative population doubling (RPD); relative increase in cell count (RICC); replication index; cytokinesis-block proliferation index; cloning efficiency; relative total growth (RTG); relative survival (RS); other: Cytotoxicity was demonstrated by calculating the Mitotic Index (MI).
- Any supplementary information relevant to cytotoxicity:

Evaluation criteria:

A test item is 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-dependent when evaluated with the appropriate trend test,
- any of the results are outside the historical vehicle control range.
A test item is classified as non-clastogenic if:
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent vehicle control.
- if there is no dose-dependent increase.
- all results are inside the historical vehicle control range.

Statistics:

Statistical significance was confirmed using the non-parametric Mann-Whitney test. However, both biological and statistical significance were considered together.

Key result

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In Phase I, the concentration of 0.0024 mg/ml produced 55.74% and 52.16% without and with S9 mix, respectively. In Phase II, cytotoxicity was 54.67% and 41.22% at 0.0024 mg/ml without and with S9 mix, respectively.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: The pH of the culture medium (with the test item added) was measured following 0 and 4 hours of exposure at 37°C. No significant changes in pH were observed at 0 or 4 hours compared to the vehicle.
- Data on osmolality:NA
- Possibility of evaporation from medium: NA
- Water solubility: The test substance was insoluble in water.
- Precipitation and time of the determination: Precipitation was observed at concentrations of 2.0, 1.0, 0.5, and 0.25 mg/ml at 0th hour. Slight precipitation was observed at 0.125 mg/mL test concentration at the 0th hour.
- Definition of acceptable cells for analysis:
- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES (if applicable):

STUDY RESULTS
- Concurrent vehicle negative and positive control data: See in section “Any other information on results incl. tables”

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible
- Statistical analysis; p-value if any: See in section “Any other information on results incl. tables”

Chromosome aberration test (CA) in mammalian cells:
- Results from cytotoxicity measurements: See in section “Any other information on results incl. tables”

o For lymphocytres in primary cultures: mitotic index (MI):
o For cell lines: relative population doubling (RPD), relative Increase in cell count (RICC), number of cells treated and cells harvested for each culture, information on cell cycle length, doubling time or proliferation index.
- Genotoxicity results (for both cell lines and lymphocytes)
o Definition for chromosome aberrations, including gaps: Chromosomal and chromatid gaps, breaks, acentric, fragments, exchanges, 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.
o Number of cells scored for each culture and concentration, number of cells with chromosomal aberrations and type given separately for each treated and control culture, including and excludling gaps: See in section “Any other information on results incl. tables”

o Changes in ploidy (polyploidy cells and cells with endoreduplicated chromosomes) if seen: See in section “Any other information on results incl. tables”

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: Yes. See in section “Any other information on results incl. tables”

- Negative (solvent/vehicle) historical control data: Yes. See in section “Any other information on results incl. tables”

Remarks on result:

other: Non-clastogenic (negative)

Table 1: Mitotix index - Cytotoxicity experiment III

Treatment	R	Mitotic Index (%)
		Absence of Metabolic Activation (-S9)				Presence of Metabolic Activation (1% S9)
		Mitotic Index	Mean	SD	Percent Reduction vs. NC	Mitotic Index	Mean	SD	Percent Reduction vs. NC
NC (0.0 mg/mL)	R1	9.89	9.73	0.23	-	9.85	9.55	0.43	-
	R2	9.57				9.24
VC (0.0 mg/mL)	R1	9.66	9.62	0.06	-	9.75	9.55	0.28	-
	R2	9.58				9.35
T7 (0.0006 mg/mL)	R1	6.69	6.43	0.36	33.13	6.29	6.53	0.34	31.67
	R2	6.18				6.77
T8 (0.0012 mg/mL)	R1	5.38	5.58	0.28	42.00	5.18	5.33	0.22	44.16
	R2	5.78				5.49
T9 (0.0024 mg/mL)	R1	3.89	3.98	0.14	58.59	4.30	4.20	0.14	56.08
	R2	4.08				4.10
PC	R1	7.99	8.14	0.21	15.37	8.48	8.24	0.35	13.76
	R2	8.29				7.99

 

Table 2: Summary of mitotic index

Mitotic Index (%)
Phase I
Treatment	Absence of Metabolic Activation (-S9)			Treatment	Presence of Metabolic Activation (+S9) (1%)
	Mean	SD	Percentage reduction vs. VC		Mean	SD	Percentage reduction vs. VC
NC	9.87	0.13	-	NC	9.53	0.20	-
VC	9.66	0.28	2.12	VC	8.66	0.56	9.17
T1 (0.0006 mg/mL)	6.87	0.14	28.94	T1 (0.0006 mg/mL)	5.59	0.00	35.41
T2 (0.0012 mg/mL)	5.57	0.14	42.35	T2 (0.0012 mg/mL)	4.92	0.36	43.13
T3 (0.0024 mg/mL)	4.28	0.13	55.74	T3 (0.0024 mg/mL)	4.14	0.08	52.16
PC	8.37	0.58	13.40	PC	7.69	0.56	11.19

Mitotic Index (%)
Phase II
Treatment	Absence of Metabolic Activation (-S9)			Treatment	Presence of Metabolic Activation (+S9) (2%)
	Mean	SD	Percentage reduction vs. VC		Mean	SD	Percentage reduction vs. VC
NC	9.35	0.15	-	NC	8.71	0.06	-
VC	9.12	0.35	2.42	VC	8.31	0.06	4.52
T1 (0.0006 mg/mL)	6.38	0.15	30.05	T1 (0.0006 mg/mL)	7.12	0.35	14.29
T2 (0.0012 mg/mL)	5.82	0.07	36.16	T2 (0.0012 mg/mL)	5.38	0.29	35.26
T3 (0.0024 mg/mL)	4.14	0.07	54.67	T3 (0.0024 mg/mL)	4.89	0.44	41.22
PC	8.12	0.1	10.97	PC	7.62	0.21	8.34

 

Table 3: Summary of Percent aberrant cells

Percent Aberrant Cells
Phase I
Treatment	Absence of Metabolic Activation (-S9)		Treatment	Presence of Metabolic Activation (+S9) (1%)
	Mean	SD		Mean	SD
NC	0.333	0.471	NC	0.333	0.471
VC	0.333	0.471	VC	0.333	0.471
T1 (0.0006 mg/mL)	0.667	0.943	T1 (0.0006 mg/mL)	0.000	0.000
T2 (0.0012 mg/mL)	0.667	0.000	T2  (0.0012 mg/mL)	0.333	0.471
T3 (0.0024 mg/mL)	0.667	0.000	T3  (0.0024 mg/mL)	0.667	0.000
PC	11.000	1.414	PC	10.333	1.414

Percent Aberrant Cells
Phase II
Treatment	Absence of Metabolic Activation (-S9)		Treatment	Presence of Metabolic Activation (+S9) (2%)
	Mean	SD		Mean	SD
NC	0.333	0.471	NC	0.667	0.000
VC	0.333	0.471	VC	0.333	0.471
T1 (0.0006 mg/mL)	0.667	0.943	T1 (0.0006 mg/mL)	0.000	0.000
T2 (0.0012 mg/mL)	0.333	0.471	T2 (0.0012 mg/mL)	1.000	0.471
T3 (0.0024 mg/mL)	0.333	0.471	T3 (0.0024 mg/mL)	0.333	0.471
PC	10.667	0.943	PC	11.000	1.414

 

Table 4: Induvidual observations for slides for mitotic index and chromosme aberrations

Phase I [In the Presence of Metabolic Activation, (1% S9)]

Treatment	Culture No.	Mitotic Index	Frequencies of Aberration	Total No. of Aberration	Number of aberrated cells	Percentage of Aberrated Cells
NC	R1	9.67	1 cse	1	1	0.67
	R2	9.39	-	0	0	0.00
VC	R1	8.26	-	0	0	0.00
	R2	9.05	1 cte, 1 csb	2	1	0.67
T1 (0.0006 mg/mL)	R1	5.59	-	0	0	0.00
	R2	5.59	-	0	0	0.00
T2  (0.0012 mg/mL)	R1	4.67	1 cse	1	1	0.67
	R2	5.17	-	0	0	0.00
T3  (0.0024 mg/mL)	R1	4.20	1 ctb, 1 cte	2	1	0.67
	R2	4.09	1 cse	1	1	0.67
PC	R1	7.29	5 ctb, 5 cte, 2 ctg, 5 csb, 5 cse,  2 AC, 04 fragments	26	17	11.33
	R2	8.08	4 ctb, 4 cte, 3 ctg, 4 csb, 3 cse, 2 csg, 2  DC, 2 AC, 06 fragments	25	14	9.33

Phase I [In the Absence of Metabolic Activation, (-S9)]

Treatment	Culture No.	Mitotic Index	Frequencies of Aberration	Total No. of Aberration	Number of aberrated cells	Percentage of Aberrated Cells
NC	R1	9.96	-	0	0	0.00
	R2	9.78	1 cse	1	1	0.67
VC	R1	9.46	1 csb	1	1	0.67
	R2	9.86	-	0	0	0.00
T1 (0.0006 mg/mL)	R1	6.97	-	0	0	0.00
	R2	6.77	1 ctb, 1cse	2	2	1.33
T2  (0.0012 mg/mL)	R1	5.47	1 cse	1	1	0.67
	R2	5.67	1 csb	1	1	0.67
T3  (0.0024 mg/mL)	R1	4.18	1 cse	1	1	0.67
	R2	4.37	1 cte, 1 csb	2	1	0.67
PC	R1	8.77	6 ctb, 8 cte,  4 csb, 4 cse, 1 csg, 3 AC, 01 fragments	26	18	12.00
	R2	7.96	4 ctb, 5 cte,  3 csb, 3 cse, 3 csg, 3 AC, 04 fragments	22	15	10.00

Phase II [In the Absence of Metabolic Activation (-S9)]

Treatment	Culture No.	Mitotic Index	Frequencies of Aberration	Total No. of Aberration	Number of aberrated cells	Percentage of Aberrated Cells
NC	R1	9.24	1cse	1	1	0.67
	R2	9.45	-	0	0	0.00
VC	R1	8.87	-	0	0	0.00
	R2	9.37	1 cte	1	1	0.67
T1 (0.0006 mg/mL)	R1	6.27	-	0	0	0.00
	R2	6.49	1 ctb, 1 cse	2	2	1.33
T2  (0.0012 mg/mL)	R1	5.77	1 cse	1	1	0.67
	R2	5.88	-	0	0	0.00
T3  (0.0024 mg/mL)	R1	4.19	-	0	0	0.00
	R2	4.08	1 cte	1	1	0.67
PC	R1	8.19	5 ctb, 6 cte, 2 ctg, 5 csb, 4 cse, 1DC, 3 AC, 04 fragments	28	17	11.33
	R2	8.05	4 ctb, 5 cte, 4 csb, 3 cse, 1 csg, 1 DC, 3 AC, 02 fragments	22	15	10.00

Phase II [In the Presence of Metabolic Activation (2% S9)]

Treatment	Culture No.	Mitotic Index	Frequencies of Aberration	Total No. of Aberration	Number of aberrated cells	Percentage of Aberrated Cells
NC	R1	8.75	1 csb	1	1	0.67
	R2	8.67	1 cse	1	1	0.67
VC	R1	8.36	-	0	0	0.00
	R2	8.27	1 csb	1	1	0.67
T1 (0.0006 mg/mL)	R1	6.88	-	0	0	0.00
	R2	7.37	-	0	0	0.00
T2  (0.0012 mg/mL)	R1	5.59	1 cse	1	1	0.67
	R2	5.17	1 cte, 1 csb	2	2	1.33
T3  (0.0024 mg/mL)	R1	4.58	1 cte	1	1	0.67
	R2	5.19	-	0	0	0.00
PC	R1	7.77	6 ctb, 4 cte, 3 ctg, 4 csb, 3 cse, 2 csg, 3 AC, 09 fragments	29	18	12.00
	R2	7.47	5 ctb, 4 cte, 2 ctg, 5 csb, 4 cse, 1 csg , 2 AC, 06 fragments	26	15	10.00

Key: MI = Mitotic Index, ctg = Chromatid gap, ctb = Chromatid break, csg = Chromosomal gap, csb = Chromosomal break, DC = Dicentric, AC = Acentric, cte = Chromatid exchange, cse = Chromosome exchange, NC = Negative Control, VC = Vehicle Control,  PC = Positive Control.

HISTORICAL DATA

HISTORICAL DATA FOR IN VITRO MAMMALIAN CHROMOSOMAL ABERRATION TEST (CA-H) RCC LABORATORIES INDIA PVT LTD, HYDERABAD, INDIA.
S.No.	Study No.	Vehicle	Phase I				Phase II
			Absence of S9		Presence of S9		Absence of S9		Presence of S9
			Vehicle Control	Positive Control	Vehicle Control	Positive Control	Vehicle Control	Positive Control	Vehicle Control	Positive Control
1	1151	DMSO	0.000	9.000	0.000	10.500	0.000	9.000	0.000	8.000
2	1333	DMSO	0.000	8.000	0.000	7.500	0.500	8.500	0.500	9.000
3	2060	DMSO	0.500	8.000	0.000	7.000	1.500	6.500	0.000	9.000
4	2450	DMSO	0.000	10.000	0.000	10.500	0.000	11.500	0.000	12.000
5	2452	DMSO	0.000	10.000	0.000	8.500	0.000	9.500	0.000	8.500
6	3000	PBS	0.000	7.500	0.000	8.500	0.000	11.000	0.000	10.000
7	3313	DMSO	0.000	8.000	0.000	10.500	0.500	9.500	0.000	9.500
8	3422	DMSO	0.000	9.000	0.500	10.000	1.000	9.500	1.000	8.500
9	3665	RPMI	0.500	8.500	0.000	7.500	0.000	8.500	0.500	8.000
10	3801	Sodium Phosphate Buffer	1.500	9.500	1.000	9.000	1.000	9.500	0.500	9.500
11	3862	DMSO	1.500	9.500	1.000	9.000	1.000	9.500	0.500	9.500
12	4792	PBS	0.500	7.500	0.500	8.500	0.500	8.500	0.000	8.000
13	4938	DMSO	0.500	8.500	1.000	8.500	0.500	8.000	1.000	8.000
14	5123	DMSO	0.333	9.000	0.667	8.667	0.333	9.667	0.333	9.000
15	5739	Dimethyl sulfoxide	0.333	10.333	0.333	10.000	0.333	9.667	0.333	10.667
16	5824	PBS	0.333	10.000	0.333	11.000	0.333	9.333	0.333	10.000
17	6461	PBS	0.333	10.000	0.333	9.667	0.333	9.000	0.333	10.000
18	6196	RPMI	0.333	11.000	0.333	10.000	0.333	10.667	0.333	10.000
19	6121	DMSO	0.667	8.667	0.667	9.667	0.667	9.667	0.667	9.333
20	6678	DMSO	0.333	10.333	0.333	10.000	0.333	9.667	0.333	10.667
21	6687	DMSO	0.333	11.333	0.333	11.333	0.333	12.333	0.333	12.000
22	6221	DMSO	0.333	9.667	0.333	10.667	0.333	9.667	0.333	10.333
23	6834	DMSO	0.333	10.333	0.333	11.333	0.333	11.333	0.333	10.667
24	6759	PBS	0.667	10.667	0.000	10.000	0.333	12.000	0.333	11.333
25	6430	DMSO	0.333	9.000	0.333	10.000	0.667	9.667	0.667	9.667
26	7703	DMSO	0.333	10.000	0.333	10.000	0.333	10.333	0.333	10.333
27	7576	RPMI	0.333	10.000	0.333	10.667	0.333	10.333	0.333	10.333
28	7572	DMSO	0.667	10.333	0.667	10.000	0.667	9.667	0.333	10.000
29	7574	Dimethyl sulfoxide	0.333	10.333	0.333	10.333	0.333	10.000	0.333	10.333
30	7434	DMSO	0.667	10.000	0.333	10.667	0.333	9.667	0.333	11.000
31	7708	DMSO	0.333	9.667	0.333	10.333	0.333	9.333	0.333	9.667
32	7263	DMSO	0.333	10.667	0.333	10.333	0.333	10.667	0.333	9.667
33	8072	DMSO	0.333	10.333	0.333	10.000	0.333	10.333	0.333	10.333

HISTORICAL DATA FOR IN VITRO MAMMALIAN CHROMOSOMAL ABERRATION TEST (CA-H) RCC LABORATORIES INDIA PVT LTD, HYDERABAD, INDIA.
S.No.	Study No.	Vehicle	Phase I				Phase II
			Absence of S9		Presence of S9		Absence of S9		Presence of S9
			Vehicle Control	Positive Control	Vehicle Control	Positive Control	Vehicle Control	Positive Control	Vehicle Control	Positive Control
34	4825	DMSO	0.667	9.000	0.667	9.333	0.333	10.000	0.667	9.000
35	8112	DMSO	0.333	9.667	0.333	10.000	0.333	10.667	0.333	10.333
36	8142	DMSO	0.333	10.000	0.333	10.333	0.333	10.000	0.333	10.333
37	8091	DMSO	0.333	10.333	0.333	10.333	0.333	10.000	0.333	10.000
38	8174	RPMI	0.333	11.000	0.333	10.000	0.333	9.667	0.333	10.667
39	7657	DMSO	0.333	10.333	0.333	11.333	0.333	10.000	0.333	11.000
40	8176	DMSO	0.333	11.333	0.333	8.667	0.333	10.667	0.333	10.000
41	8541	DMSO	0.667	9.667	0.333	10.000	0.667	9.667	0.333	10.000
42	8064	DMSO	0.333	10.667	0.667	9.667	0.333	10.333	0.333	10.000
43	8486	DMSO	0.333	10.000	0.333	10.333	0.333	10.333	0.667	11.333
44	8660	RPMI	0.333	11.000	0.333	10.000	0.333	10.333	0.333	10.000
45	8722	DMSO	0.667	10.000	0.667	10.667	0.667	9.333	0.667	10.666
46	8670	DMSO	0.333	10.000	0.333	11.000	0.333	10.667	0.667	10.000
47	8680	DMSO	0.333	10.333	0.667	10.333	0.333	10.000	0.333	10.000
48	8658	DMSO	0.333	10.000	0.333	11.000	0.333	10.667	0.667	10.000
49	9845	DMSO	0.333	10.000	0.333	11.000	0.333	10.667	0.333	10.667
50	9861	DMSO	0.333	10.667	0.333	10.333	0.333	10.333	0.667	10.000
51	9862	DMSO	0.667	10.000	0.333	9.000	0.333	10.000	0.333	10.667
52	9911	DMSO	0.333	10.667	0.333	11.333	0.333	9.333	0.333	10.000
53	9925	DMSO	0.333	11.333	0.333	11.000	0.333	11.333	0.333	11.000
54	10049	DMSO	0.333	10.000	0.333	11.000	0.333	9.667	0.667	11.000
55	9939	DMSO	0.333	9.667	0.333	11.000	0.333	8.000	0.333	9.000
56	10679	DMSO	0.333	11.667	0.333	10.667	0.333	13.333	0.333	15.000
57	10807	DMSO	0.333	10.333	0.667	11.000	0.333	9.000	0.333	10.667
58	10858	RPMI	0.667	10.333	0.667	11.000	0.333	11.000	0.333	10.667
59	10859	DMSO	0.333	10.667	0.667	10.000	0.667	10.333	0.333	10.000
Mean			0.395	9.887	0.384	10.0012	0.411	9.955	0.384	10.082
SD			0.275	0.938	0.242	0.998	0.254	1.071	0.213	1.114
Mean + 2SD			0.945	11.764	0.868	12.005	0.919	12.096	0.810	12.310
Mean - 2SD			-0.155	8.010	-0.100	8.012	-0.098	7.813	-0.043	7.854

Conclusions:

The registered substance, i.e., 2-methoxynaphthalene (CAS: 93-04-9), tested non-clastogenic (negative) in primary cultures of human peripheral blood lymphocytes in the presence and absence of S9 metabolic activation system. The test was performed according to OECD TG 473 and GLP.

Executive summary:

The clastogenic potential of the registered substance, i.e.,  2-methoxynaphthalene (CAS: 93-04-9), was assessed in human peripheral blood lymphocytes in the presence and absence of an exogenous metabolic activation system in an OECD 473 study. Cofactor-supplemented liver S9 microsomal fraction was used as an exogenous metabolic activation system. The S9 fraction was derived from the Aroclor 1254-induced rats. The test concentrations were selected based on the solubility, precipitation and pH checks and a preliminary cytotoxicity test. The test substance was soluble in Dimethyl Sulfoxide (DMSO) up to 200 mg/ml to give the final treatment concentration of 2 mg/ml. No significant changes in pH were observed at 0 or 4 hours when compared with the vehicle control. Precipitation was observed at concentrations of 2.0, 1.0, 0.5, and 0.25 mg/ml. Slight precipitation was observed at 0.125 mg/m test concentration at the 0^th hour, which was judged not to interfere with the conduct of the test. Hence, the concentration of  0.125 mg/ml was selected as the high dose for the cytotoxicity experiment.

In the cytotoxicity test, cells were exposed to test item concentrations of  0.0 (NC: distilled water), 0.0 (VC: DMSO), 0.125, 0.063 and 0.031 mg/ml in culture media in the presence and absence of S9 metabolic activation. Cytotoxicity was determined by a reduction in the mitotic index in comparison with vehicle control data. All the tested concentrations at the initial cytotoxicity experiment were cytotoxic both in the presence and absence of the S9 metabolic activation system. Next, the cytotoxicity was assessed at test item concentrations of 0.0 (NC), 0.0 (VC), 0.0006, 0.0012 and 0.015 mg/ml of culture media in a second cytotoxicity experiment (Experiment II). In cytotoxicity experiment (II), all tested concentrations were cytotoxic both in the presence and absence of the S9 metabolic activation system. Cytotoxicity was then assessed at test item concentrations of 0.0 (NC), 0.0 (VC), 0.0006, 0.0012 and 0.0024 mg/ml in culture media in the third cytotoxicity experiment (Experiment III). The test concentration of 0.0024 mg/ml produced a 58.59 % and 56.08 % decrease in the mitotic index compared to VC in the absence (-S9) and the presence of metabolic activation (+S9), respectively. The dose level that results in approximately 55 ± 5 % reduction in the mitotic index was selected as the highest test concentration in the main test. Hence, 0.0024 mg/ml was chosen as the highest test item concentration for the main study, both in the presence and absence of metabolic activation. In the CA test, proliferating peripheral blood cells (whole blood) were used for the test, and the treatment of cultures with the test item was conducted in two independent phases. In the Phase I experiment, cells were exposed to test item concentrations of 0.0 (NC), 0.0 (VC), 0.0006, 0.0012 and 0.0024 mg/ml along with positive controls (EMS: 600 µg/ml without S9 mix and CPA: 30 600 µg/ml with S9 mix) for 4 hours both in the presence and absence of S9 metabolic activation system (1 v/v %) using duplicates. In the Phase II experiment, cells in duplicate cultures were treated with 0.0 (NC), 0.0 (VC), 0.0006, 0.0012 and 0.0024 mg/ml for 4 hours in the presence of the S9 metabolic activation system (2 v/v%) or 24 hours in the absence of S9 metabolic activation. Cells were harvested 24 hrs after treatments in both experiements. A minimum of 1000 cells was counted in different fields of slide per culture, and the number of metaphases was recorded for Mitotic Index (MI) calculation. Three hundred well-spread metaphases per concentration and control were scored for cytogenetic damage on coded slides. Chromosomal and chromatid gaps, breaks, acentric, fragments, exchanges, 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. Results: In the cytotoxicity test (Experiment III), the test concentration 0.0024 mg/ml produced a 58.59 % and 56.08 % decrease in the mitotic index compared to VC in the absence (-S9) and the presence of metabolic activation (+S9), respectively. The test concentration 0.0012 mg/ml produced 42.00 % and 44.16 % decreases in the mitotic index compared to VC in the absence (-S9) and presence (+S9) of metabolic activation, respectively. The test concentration 0.0006 mg/ml produced a 33.13 % and 31.67 % decrease in the mitotic index (cytotoxicity) compared to VC in the absence (-S9) and presence (+S9) of metabolic activation, respectively.

In Phase I experiment, the cultures were exposed to the test substance for 4 hours both in the presence (+S9) and absence (-S9) of the metabolic activation system (1%). The mean percentages of aberrant cells were 0.333 (NC), 0.333 (VC), 0.667 ( at 0.0006 mg/ml), 0.667 (at 0.0012 mg/ml), 0.667 (at 0.0024 mg/ml) and 11.000 (PC: 600 µg/mL EMS) in the absence of metabolic activation. In the presence of metabolic activation (1 v/v%), the mean percentage of aberrant cells were 0.333 (NC), 0.333 (VC), 0.000 (at 0.0006 mg/ml), 0.333 (at 0.0012 mg/ml), 0.667 (at 0.0024 mg/ml) and 10.333 (PC: 30 µg/ml CPA). Treatment with positive control substances caused significant increases in the percent of aberrant cells. Even though the analysis did not reveal any statistical significance, the increases were deemed biologically significant. A 55.74% and 52.16 % of reduction in the mitotic index, i.e., cytotoxicity, was observed at the tested concentration of 0.0024 mg/ml compared to vehicle control (VC) in the absence (-S9) and presence (+S9) of metabolic activation system, respectively. The observed mean mitotic index in the absence of metabolic activation were 9.87 (NC), 9.66 (VC), 6.87 (at 0.0006 mg/ml), 5.57 (at 0.0012 mg/ml), 4.28 (at 0.0024 mg/ml) and 8.37 (PC: 600 µg/mL EMS). In the presence of metabolic activation (1 v/v%), the mean mitotic index was 9.53 (NC), 8.66 (VC), 5.59 (at 0.0006 mg/ml), 4.92 (at 0.0012 mg/ml), 4.14 (at 0.0024 mg/ml) and 7.69 (PC: 30 µg/mL CPA). The Phase II experiment was performed to confirm the negative results obtained in Phase I in the presence and absence of S9 metabolic activation. In the absence of metabolic activation, the mean percent aberrant cells were 0.333 (NC), 0.333 (VC), 0.667 (at 0.0006 mg/ml), 0.333 (at 0.0012 mg/ml), 0.333 (at 0.0024 mg/ml) and 10.667 (PC: 600 µg/mL EMS). In the presence of the metabolic activation, the mean percent aberrant cells were 0.667 (NC), 0.333 (VC), 0.000 (at 0.0006 mg/ml), 1.000 (at 0.0012 mg/ml ), 0.333 (at 0.0024 mg/ml) and 11.000 (PC: 30 µg/mL CPA). Treatment with positive control substances in the absence and presence of metabolic activation caused a significant increase in percent aberrant cells. Even though the analysis did not reveal any statistical significance, the increases were deemed biologically significant. A reduction of 54.67 and 41.22 in the mitotic index was observed at the tested concentration of 0.0024 mg/ml compared to the vehicle control (VC) in the absence (-S9) and presence (+S9) of the  metabolic activation system, respectively. The observed mean mitotic index in the absence (-S9) of metabolic activation were 9.35 (NC), 9.12 (VC), 6.38 (at 0.0006 mg/ml), 5.82 (at 0.0012 mg/ml), 4.14 (at 0.0024 mg/ml) and 8.12 (PC: 600 µg/mL EMS). In the presence (+S9) of metabolic activation, the mean mitotic index was 8.71 (NC), 8.31 (VC), 7.12 (at 0.0006 mg/ml), 5.38 (at 0.0012 mg/ml), 4.89 (at 0.0024 mg/ml) and 7.62 (PC: 30 µg/mL CPA). No significant increase in the percent of aberrant cells was observed in the vehicle control groups  (Dimethyl sulfoxide) in Phase I and Phase II when compared to the negative control (distilled water). The increased frequency of aberrant cells 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. Conclusion: The registered substance, i.e., 2-methoxynaphthalene (CAS: 93-04-9),  is non-clastogenic in human peripheral lymphocytes both in the presence (1% and 2%) and in the absence of metabolic activation.

Reference 3

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Adopted on July 21st 1997, corrected June 26th 2020

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Purity: 99.06%
Manufacture Date: June 29, 2018
Expiry Date: June 29, 2023

Target gene:

Histidine gene

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Cofactor-supplemented liver S9 microsomal fraction was used as an exogenous metabolic activation system. The S9 fraction derived from Aroclor 1254-injected rats.
An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to get a final concentration of approximately 10 % v/v in the S9 mix.

Cofactor solution contains the following quantity of chemicals in 500 mL of distilled water.
D-glucose-6-phosphate: 0.8 g
β-NADP: 1.75 g
MgCl2: 1.0 g
KCl: 1.35 g
Na2HPO4.H2O: 6.4 g
NaH2PO4.H2O: 1.4 g

Test concentrations with justification for top dose:

Test concentrations: 0.0 (NC, Distilled water), 0.0 (VC, DMSO), 0.039, 0.078, 0.156, 0.313 and 0.625 ug/plate

Justification:
Test concentrations were selected based on a preliminary cytotoxicity test. In this pre-test, bacterial cells were exposed to test item concentrations of 0.0 (NC), 0.0 (VC), 0.020, 0.039, 0.078, 0.156, 0.313, 0.625, 1.25 and 2.5 µg/plate together with positive control substances. Complete inhibition of the bacterial background lawn was observed at ≥1.25 µg/plate, while at 0.625 µg/plate, moderate inhibition of the background lawn was noted in TA98 and TA100 testers both in the presence and absence of S9 metabolic activation.

Vehicle / solvent:

Dimethyl sulphoxide was used as a vehicle.

Untreated negative controls:

yes

Remarks:

Distilled water

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-Nitro-o-phenylenediamine, 2-Aminoanthracene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): Triplicates were used.
- Number of independent experiments: Two (Trial I-II)

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 1-2 x 109 cells/mL
- Test substance added in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk: Trial I: plate incorporation, Trial II: preincubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 60 min
- Exposure duration/duration of treatment: 48 hrs
- Harvest time after the end of treatment (sampling/recovery times): 48 hrs

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition; mitotic index (MI); relative population doubling (RPD); relative increase in cell count (RICC); replication index; cytokinesis-block proliferation index; cloning efficiency; relative total growth (RTG); relative survival (RS); other: Cytotoxicity was determined by reduction in the number of revertant counts and/or inhibition of the bacterial background lawn growth.
- Any supplementary information relevant to cytotoxicity:
A pre-experiment was performed with strains TA98 and TA100. Eight concentrations (i.e. 0.0 (negative control; NC), 0.0 (vehicle control; VC), 0.001, 0.003, 0.008, 0.025, 0.079, 0.250, 0.791 and 2.5 mg/plate) with half-log intervals (√10) were tested for toxicity and mutation induction with 3 plates each (triplicates). The experimental conditions in the pre-experiment were the same as described below for Trial I.

Evaluation criteria:

The Substance is considered as a mutagen, if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA98, TA100 and TA102) or thrice (strains TA1535 and TA1537) the colony count of the corresponding vehicle/solvent control is observed.
A dose-dependent increase is considered biologically relevant if the threshold of biological significance is exceeded at more than one concentration. An increase exceeding the threshold of biological significance at only one concentration was judged as biologically relevant if it is reproduced in an independent second experiment.
A dose-dependent increase in the number of revertant colonies below the threshold of biological significance is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remained within the historical range of negative and vehicle control, the increase is not considered biologically relevant.

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Inhibition of the bacterial background lawn is noted at 0.625 ug/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Inhibition of the bacterial background lawn is noted at 0.625 ug/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Inhibition of the bacterial background lawn is noted at 0.625 ug/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Inhibition of the bacterial background lawn is noted at 0.625 ug/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Inhibition of the bacterial background lawn is noted at 0.625 ug/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: No data
- Data on osmolality: No data
- Possibility of evaporation from medium:NA
- Water solubility: Insoluble
- Precipitation and time of the determination: Precipitation was observed at 5 and 3.75 mg/plate concentrations, which was considered to interfere with the colony count. At 2.5 mg/plate concentration, slight precipitation was observed, which was considered to be non-interfering with colony count.
- Definition of acceptable cells for analysis: Regular background growth in the negative and solvent (vehicle) control.
- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES (if applicable):

STUDY RESULTS
- Concurrent vehicle negative and positive control data: Raw data is shown in section “Any other information on results incl, tables”.
For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible
- Statistical analysis; p-value if any: No data
- Any other criteria: e.g. GEF for MLA

Ames test:
- Signs of toxicity: Toxicity was noted as inhibition of the bacterial background lawn.
- Individual plate counts: Raw data is shown in section “Any other information on results incl, tables”.
- Mean number of revertant colonies per plate and standard deviation: Raw data is shown in section “Any other information on results incl, tables”.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: Available. Raw data is shown in section “Any other information on results incl, tables”.

- Negative (solvent/vehicle) historical control data: Available . Raw data is shown in section “Any other information on results incl, tables”.

Remarks on result:

other: Non-mutagenic

Table 1: Revertant counts in the preliminary cytotoxicity test

Dose (mg/plate)	R	Absence of Metabolic Activation (-S9)		Presence of Metabolic Activation (+S9)
		TA100	TA 98	TA100	TA 98
NC (0.00)	R1	106	21	120	20
	R2	112	18	106	22
	R3	114	20	108	20
VC (0.00)	R1	118	24	116	26
	R2	126	23	120	24
	R3	112	25	124	25
T1 (0.020)	R1	110	24	120	22
	R2	118	21	114	25
	R3	112	22	116	23
T2 (0.039)	R1	120	23	112	25
	R2	106	22	118	21
	R3	118	24	108	22
T3 (0.078)	R1	108	24	112	24
	R2	118	20	110	23
	R3	114	21	120	20
T4 (0.156)	R1	116	22	108	25
	R2	104	19	110	23
	R3	118	25	118	24
T5 (0.313)	R1	116	24	120	22
	R2	112	26	110	20
	R3	114	20	116	24
T6 (0.625)	R1	108 (+++)	22 (+++)	112 (+++)	23 (+++)
	R2	114 (+++)	23 (+++)	122 (+++)	25 (+++)
	R3	112 (+++)	18 (+++)	110 (+++)	25 (+++)
T7 (1.25)	R1	116 (++)	24 (++)	112 (++)	22 (++)
	R2	112 (++)	21 (++)	120 (++)	23 (++)
	R3	110 (++)	23 (++)	116 (++)	24 (++)
T8 (2.5)	R1	118 (++)	23 (++)	112 (++)	24 (++)
	R2	106 (++)	20 (++)	118 (++)	23 (++)
	R3	112 (++)	21 (++)	110 (++)	24 (++)
PC	R1	1280	816	1328	1288
	R2	1160	912	1240	1456
	R3	1304	848	1376	1360

NC=Negative control;  VC = Vehicle Control;  PC=Positive control

R = Replicate, T= Test concentration (T8: Highest, T1: Lowest)

Background Lawn: (+++) = Moderate Inhibition; (++) = Complete Inhibition

4-Nitro-o-phenylenediamine [10μg/plate]: TA 98, Sodium azide [10μg/plate]: TA 100,

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

 

Table 2: Revertant counts in Trial I (Plate incorporation assay)

Dose (mg/plate)	R	Presence of Metabolic Activation (+S9)
		TA 1537	TA 1535	TA 98	TA 100	TA 102
NC (0.00)	R1	5	12	20	120	240
	R2	4	11	22	106	232
	R3	5	15	20	108	244
VC. (0.00)	R1	6	14	26	116	264
	R2	6	17	24	120	252
	R3	7	16	25	124	256
T1 (0.039)	R1	5	15	25	112	256
	R2	6	16	21	118	240
	R3	4	13	22	108	236
T2 (0.078)	R1	6	14	24	112	232
	R2	7	15	23	110	256
	R3	5	14	20	120	248
T3 (0.156)	R1	5	13	25	108	236
	R2	6	12	23	110	256
	R3	5	16	24	118	232
T4 (0.313)	R1	4	13	22	120	244
	R2	7	11	20	110	232
	R3	6	15	24	116	252
T5 (0.625)	R1	5	13	23	112	232
	R2	3	13	25	122	252
	R3	6	14	25	110	236
PC	R1	164	368	1288	1328	1440
	R2	172	328	1456	1240	1560
	R3	156	448	1360	1376	1392

Dose (mg/plate)	R	Absence of Metabolic Activation (-S9)
		TA 1537	TA 1535	TA 98	TA 100	TA 102
NC (0.00)	R1	4	14	21	106	232
	R2	4	10	18	112	224
	R3	3	12	20	114	248
VC. (0.00)	R1	5	15	24	118	256
	R2	7	13	23	126	260
	R3	5	16	25	112	240
T1 (0.039)	R1	5	15	23	120	240
	R2	5	12	22	106	236
	R3	4	13	24	118	252
T2 (0.078)	R1	5	13	24	108	232
	R2	5	15	20	118	248
	R3	6	11	21	114	236
T3 (0.156)	R1	5	14	22	116	244
	R2	4	15	19	104	236
	R3	4	12	25	118	232
T4 (0.313)	R1	6	12	24	116	248
	R2	4	13	26	112	240
	R3	5	12	20	114	236
T5 (0.625)	R1	5	14	22	108	232
	R2	3	13	23	114	248
	R3	4	11	18	112	228
PC	R1	184	1260	816	1280	1536
	R2	160	1140	912	1160	1404
	R3	176	1284	848	1304	1608

NC = Negative Control, VC = Vehicle Control, T = Test concentration (T5: Highest, T1: Lowest), R = Replicate, PC = Positive control       

2-Aminoanthracene [2.5μg/plate]: TA 1537, TA1535, 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.

Table 3: Revertant counts in Trial II (Preincubation assay)

Dose (mg/plate)	R	In the Presence of Metabolic Activation (+S9)
		TA 1537	TA 1535	TA 98	TA 100	TA 102
NC (0.00)	R1	4	12	24	106	240
	R2	5	14	21	116	236
	R3	4	11	22	112	248
VC. (0.00)	R1	7	15	27	124	272
	R2	6	13	28	118	256
	R3	6	17	24	120	260
T1 (0.039)	R1	5	13	26	110	256
	R2	7	15	24	120	240
	R3	5	14	23	116	248
T2 (0.078)	R1	6	13	25	108	252
	R2	4	16	21	116	260
	R3	6	12	24	114	244
T3 (0.156)	R1	5	15	24	114	256
	R2	4	13	25	110	240
	R3	6	16	23	120	248
T4 (0.313)	R1	5	13	27	114	236
	R2	5	16	23	108	252
	R3	4	14	24	118	244
T5 (0.625)	R1	4	13	22	110	248
	R2	5	14	24	114	256
	R3	4	12	23	118	236
PC	R1	180	424	1296	1452	1344
	R2	204	320	1392	1512	1488
	R3	168	368	1356	1332	1416

Dose (mg/plate)	R	In the Absence of Metabolic Activation (-S9)
		TA 1537	TA 1535	TA 98	TA 100	TA 102
NC (0.00)	R1	5	13	19	110	240
	R2	3	13	22	102	228
	R3	4	12	21	108	232
VC. (0.00)	R1	6	16	23	116	260
	R2	7	13	26	122	244
	R3	5	15	25	120	252
T1 (0.039)	R1	5	14	22	110	248
	R2	5	15	26	116	240
	R3	5	12	24	114	252
T2 (0.078)	R1	7	16	23	106	236
	R2	5	12	21	112	240
	R3	5	14	25	118	244
T3 (0.156)	R1	5	14	21	116	244
	R2	4	13	24	108	232
	R3	4	16	23	114	248
T4 (0.313)	R1	4	15	24	112	236
	R2	3	11	22	106	248
	R3	5	13	27	114	252
T5 (0.625)	R1	4	15	22	114	248
	R2	6	12	25	108	240
	R3	4	13	23	112	236
PC	R1	156	1152	852	1380	1404
	R2	184	1380	708	1284	1608
	R3	172	1284	972	1308	1452

NC = Negative Control, VC = Vehicle Control, T = Test concentration (T5: Highest, T1: Lowest), R= Replicate
PC = Positive control 2-Aminoanthracene [2.5μg/plate]: TA 1537, TA1535, TA98, TA100,
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.

Table 4: The mean revertant counts in Trial I (Plate incorporation assay)

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)	4.67	0.58	12.67	2.08	20.67	1.15	111.33	7.57	238.67	6.11
VC (0.00)	6.33	0.58	15.67	1.53	25.00	1.00	120.00	4.00	257.33	6.11
T1 (0.039)	5.00	1.00	14.67	1.53	22.67	2.08	112.67	5.03	244.00	10.58
T2 (0.078)	6.00	1.00	14.33	0.58	22.33	2.08	114.00	5.29	245.33	12.22
T3 (0.156)	5.33	0.58	13.67	2.08	24.00	1.00	112.00	5.29	241.33	12.86
T4 (0.313)	5.67	1.53	13.00	2.00	22.00	2.00	115.33	5.03	242.67	10.07
T5 (0.625)	4.67	1.53	13.33	0.58	24.33	1.15	114.67	6.43	240.00	10.58
PC	164.00	8.00	381.33	61.10	1368.00	84.29	1314.67	68.97	1464.00	86.53

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)	3.67	0.58	12.00	2.00	19.67	1.53	110.67	4.16	234.67	12.22
VC (0.00)	5.67	1.15	14.67	1.53	24.00	1.00	118.67	7.02	252.00	10.58
T1 (0.039)	4.67	0.58	13.33	1.53	23.00	1.00	114.67	7.57	242.67	8.33
T2 (0.078)	5.33	0.58	13.00	2.00	21.67	2.08	113.33	5.03	238.67	8.33
T3 (0.156)	4.33	0.58	13.67	1.53	22.00	3.00	112.67	7.57	237.33	6.11
T4 (0.313)	5.00	1.00	12.33	0.58	23.33	3.06	114.00	2.00	241.33	6.11
T5 (0.625)	4.00	1.00	12.67	1.53	21.00	2.65	111.33	3.06	236.00	10.58
PC	173.33	12.22	1228.00	77.15	858.67	48.88	1248.00	77.15	1516.00	103.46

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: The mean revertant counts in Trial II (preincubation assay)

Dose (mg/plate)	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)	4.33	0.58	12.33	1.53	22.33	1.53	111.33	5.03	241.33	6.11
VC (0.00)	6.33	0.58	15.00	2.00	26.33	2.08	120.67	3.06	262.67	8.33
T1 (0.039)	5.67	1.15	14.00	1.00	24.33	1.53	115.33	5.03	248.00	8.00
T2 (0.078)	5.33	1.15	13.67	2.08	23.33	2.08	112.67	4.16	252.00	8.00
T3 (0.156)	5.00	1.00	14.67	1.53	24.00	1.00	114.67	5.03	248.00	8.00
T4 (0.313)	4.67	0.58	14.33	1.53	24.67	2.08	113.33	5.03	244.00	8.00
T5 (0.625)	4.33	0.58	13.00	1.00	23.00	1.00	114.00	4.00	246.67	10.07
PC	184.00	18.33	370.67	52.05	1348.00	48.50	1432.00	91.65	1416.00	72.00

Dose (mg/plate)	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)	4.00	1.00	12.67	0.58	20.67	1.53	106.67	4.16	233.33	6.11
VC (0.00)	6.00	1.00	14.67	1.53	24.67	1.53	119.33	3.06	252.00	8.00
T1 (0.039)	5.00	0.00	13.67	1.53	24.00	2.00	113.33	3.06	246.67	6.11
T2 (0.078)	5.67	1.15	14.00	2.00	23.00	2.00	112.00	6.00	240.00	4.00
T3 (0.156)	4.33	0.58	14.33	1.53	22.67	1.53	112.67	4.16	241.33	8.33
T4 (0.313)	4.00	1.00	13.00	2.00	24.33	2.52	110.67	4.16	245.33	8.33
T5 (0.625)	4.67	1.15	13.33	1.53	23.33	1.53	111.33	3.06	241.33	6.11
PC	170.67	14.05	1272.00	114.47	844.00	132.18	1324.00	49.96	1488.00	106.66

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

Table 6: Historical control data

Trial I (Plate Incorporation Method)
Strains	Metabolic Activation	Treatment	Mean	SD	Max	Min
TA 1537	S9 +	Negative control	6	2	10	2
	S9 -		6	2	10	2
	S9 +	Solvent control	6	2	10	2
	S9 -		6	2	10	2
	S9 +	Positive control	168	38	245	92
	S9 -		175	43	261	89
TA 1535	S9 +	Negative control	12	3	18	7
	S9 -		12	3	18	7
	S9 +	Solvent control	13	3	18	7
	S9 -		13	3	18	7
	S9 +	Positive control	336	211	757	86
	S9 -		1200	263	1726	674
TA 98	S9 +	Negative control	24	6	36	11
	S9 -		23	6	35	11
	S9 +	Solvent control	25	6	37	13
	S9 -		23	5	33	13
	S9 +	Positive control	1099	312	1722	476
	S9 -		815	284	1383	248
TA 100	S9 +	Negative control	117	28	173	61
	S9 -		114	26	166	62
	S9 +	Solvent control	116	28	172	60
	S9 -		113	26	165	61
	S9 +	Positive control	1488	390	2268	709
	S9 -		1311	298	1906	715
TA 102	S9 +	Negative control	274	42	358	190
	S9 -		271	55	382	161
	S9 +	Solvent control	279	65	409	150
	S9 -		277	82	442	112
	S9 +	Positive control	1648	305	2258	1037
	S9 -		1896	364	2624	1168

Trial II (Pre-Incubation Method)
Strains	Metabolic Activation	Treatment	Mean	SD	Max	Min
TA 1537	S9 +	Negative control	6	2	10	2
	S9 -		6	2	10	2
	S9 +	Solvent control	6	2	10	3
	S9 -		6	2	10	2
	S9 +	Positive control	170	39	249	91
	S9 -		182	43	268	96
TA 1535	S9 +	Negative control	13	3	18	7
	S9 -		12	3	18	7
	S9 +	Solvent control	13	3	18	8
	S9 -		13	3	18	7
	S9 +	Positive control	299	197	694	145
	S9 -		1244	260	1765	724
TA 98	S9 +	Negative control	24	6	35	13
	S9 -		23	5	33	13
	S9 +	Solvent control	24	5	35	14
	S9 -		23	5	32	14
	S9 +	Positive control	1269	275	1819	719
	S9 -		740	210	1160	320
TA 100	S9 +	Negative control	117	25	166	67
	S9 -		113	23	159	66
	S9 +	Solvent control	116	22	159	73
	S9 -		112	20	151	73
	S9 +	Positive control	1469	347	2163	775
	S9 -		1352	263	1878	827
TA 102	S9 +	Negative control	281	32	345	218
	S9 -		276	28	331	220
	S9 +	Solvent control	281	34	350	212
	S9 -		276	34	344	207
	S9 +	Positive control	1595	287	2168	1022
	S9 -		1753	248	2248	1258

Conclusions:

The registered substance, 2-methoxynapthalene (CAS No. 93-04-9), was tested non-mutagenic (negative) in a bacterial reverse mutation test both in the presence and absence of S9 metabolic activation system. The test was performed according to OECD TG 471 and GLP.

Executive summary:

The mutagenic potential of the registered substance, i.e., 2-methoxynapthalene (CAS No. 93-04-9), was tested according to OECD TG 471 and GLP using Salmonella Typhimurium strains TA98, TA100, TA1535, TA1537 and TA102. The test was performed in the presence and absence of an exogenous metabolic activation system. Cofactor-supplemented liver S9 microsomal fraction was used, and the S9 fraction was obtained from Aroclor 1254-induced rats. Test concentrations were selected based on the solubility, precipitation, and preliminary cytotoxicity tests. The Substance was insoluble in water and was soluble in Dimethyl sulphoxide up to 50 mg/ml. Insolubility was assessed as precipitation in the final mixture under the actual test conditions and was evident to the unaided eye. The test item was dissolved in DMSO at 50 mg/mL concentration and was checked for precipitation on agar. Precipitation was observed at 5 and 3.75 mg/plate concentrations, which was considered to interfere with the colony count. At 2.5 mg/plate concentration, slight precipitation was observed, which was considered non-interfering with colony count. Therefore 2.5 mg/plate was selected as the highest concentration for the pre-experiment. In the preliminary cytotoxicity test, bacterial cells of TA 98 and TA100 were exposed to test concentrations of 0.0 (NC, distilled water), 0.0 (VC, DMSO), 0.020, 0.039, 0.078, 0.156, 0.313, 0.625, 1.25 and 2.5 µg/plate in the presence and absence of S9 metabolic activation. Complete inhibition of the bacterial background lawn was observed at ≥1.25 µg/plate, while at  0.625 µg/plate, moderate inhibition of the background lawn was noted in TA98 and TA100 testers both in the presence and absence of S9 metabolic activation. Thus, the main test was performed with the flowing test item concentrations: 0.0 (VC), 0.0 (NC), 0.039, 0.078, 0.156, 0.313 and 0.625 µg/plate both in the presence and absence of S9 metabolic activation system using five Salmonella Typhimurium tester strains. For each strain and dose level, including negative, vehicle and positive controls, three plates (triplicate) were tested. The following positive control substances were used: sodium azide (TA 1535, TA100, without S9 mix), 4-Nitro-o-phenylenediamine (TA1537, TA98, without S9 mix), Methyl methanesulfonate (TA102 without S9), 2-Aminoanthracene (TA98, TA100, TA1535, TA1537 and TA102 wit S9 mix). Trial I was performed according to the plate incorporation method with five test concentrations along with the negative (Distilled water), vehicle (DMSO), and positive controls, and using three strains, i.e., TA1537, TA1535 and TA102.For TA98 and TA100 tester strains, the revertant colony counts were directly incorporated in Trial I from the pre-experiment up to the five concentrations (0.625 mg/plate to 0.039 mg/plate). Trial II was performed according to the preincubation method with all five tester strains along with the negative, vehicle, and positive controls. Results: No significant increase in the number of revertant colonies was observed following the treatment with the test substance up to the concentration of 0.625 µg/plate either in the presence or absence of S9 metabolic activation in all five tester strains in both trials (Trial I-II) when compared to the vehicle control. No trend of an increased number of revertant colonies with increased dosing of the test item was observed. Each strain-specific positive control showed a significant increase in the number of revertant colonies both in the presence and absence of metabolic activation, thus confirming the validity of the assay. Conclusion: The registered substance, i.e., 2-methoxynapthalene (CAS No. 93-04-9), did not induce gene mutation within the histidine operon by base-pair exchange or frameshift in Salmonella Typhimurium tester strains (TA98, TA100, TA1535, TA1537 and TA102) either in the presence or absence of S9 metabolic activation system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

 

 

 

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Genetic Toxicity / In Vitro:

Bacterial reverse mutation test:

The mutagenic potential of the registered substance, i.e., 2-methoxynapthalene (CAS No. 93-04-9), was tested according to OECD TG 471 and GLP using Salmonella Typhimurium strains TA98, TA100, TA1535, TA1537 and TA102. The test was performed in the presence and absence of an exogenous metabolic activation system. Cofactor-supplemented liver S9 microsomal fraction was used, and the S9 fraction was obtained from Aroclor 1254-induced rats. Test concentrations were selected based on the solubility, precipitation, and preliminary cytotoxicity tests. The Substance was insoluble in water and was soluble in Dimethyl sulphoxide up to 50 mg/ml. Insolubility was assessed as precipitation in the final mixture under the actual test conditions and was evident to the unaided eye. The test item was dissolved in DMSO at 50 mg/mL concentration and was checked for precipitation on agar. Precipitation was observed at 5 and 3.75 mg/plate concentrations, which was considered to interfere with the colony count. At 2.5 mg/plate concentration, slight precipitation was observed, which was considered non-interfering with colony count. Therefore 2.5 mg/plate was selected as the highest concentration for the pre-experiment. In the preliminary cytotoxicity test, bacterial cells of TA 98 and TA100 were exposed to test concentrations of 0.0 (NC, distilled water), 0.0 (VC, DMSO), 0.020, 0.039, 0.078, 0.156, 0.313, 0.625, 1.25 and 2.5 µg/plate in the presence and absence of S9 metabolic activation. Complete inhibition of the bacterial background lawn was observed at ≥1.25 µg/plate, while at  0.625 µg/plate, moderate inhibition of the background lawn was noted in TA98 and TA100 testers both in the presence and absence of S9 metabolic activation. Thus, the main test was performed with the flowing test item concentrations: 0.0 (VC), 0.0 (NC), 0.039, 0.078, 0.156, 0.313 and 0.625 µg/plate both in the presence and absence of S9 metabolic activation system using five Salmonella Typhimurium tester strains. For each strain and dose level, including negative, vehicle and positive controls, three plates (triplicate) were tested. The following positive control substances were used: sodium azide (TA 1535, TA100, without S9 mix), 4-Nitro-o-phenylenediamine (TA1537, TA98, without S9 mix), Methyl methanesulfonate (TA102 without S9), 2-Aminoanthracene (TA98, TA100, TA1535, TA1537 and TA102 wit S9 mix). Trial I was performed according to the plate incorporation method with five test concentrations along with the negative (Distilled water), vehicle (DMSO), and positive controls, and using three strains, i.e., TA1537, TA1535 and TA102.For TA98 and TA100 tester strains, the revertant colony counts were directly incorporated in Trial I from the pre-experiment up to the five concentrations (0.625 mg/plate to 0.039 mg/plate). Trial II was performed according to the preincubation method with all five tester strains along with the negative, vehicle, and positive controls. Results: No significant increase in the number of revertant colonies was observed following the treatment with the test substance up to the concentration of 0.625 µg/plate either in the presence or absence of S9 metabolic activation in all five tester strains in both trials (Trial I-II) when compared to the vehicle control. No trend of an increased number of revertant colonies with increased dosing of the test item was observed. Each strain-specific positive control showed a significant increase in the number of revertant colonies both in the presence and absence of metabolic activation, thus confirming the validity of the assay. Conclusion: The registered substance, i.e., 2-methoxynapthalene (CAS No. 93-04-9), did not induce gene mutation within the histidine operon by base-pair exchange or frameshift in Salmonella Typhimurium tester strains (TA98, TA100, TA1535, TA1537 and TA102) either in the presence or absence of S9 metabolic activation system.

 

In vitro mammalian chromosme aberration test:

The clastogenic potential of the registered substance, i.e.,  2-methoxynaphthalene (CAS: 93-04-9), was assessed in human peripheral blood lymphocytes in the presence and absence of an exogenous metabolic activation system in an OECD 473 study. Cofactor-supplemented liver S9 microsomal fraction was used as an exogenous metabolic activation system. The S9 fraction was derived from the Aroclor 1254-induced rats. The test concentrations were selected based on the solubility, precipitation and pH checks and a preliminary cytotoxicity test. The test substance was soluble in Dimethyl Sulfoxide (DMSO) up to 200 mg/ml to give the final treatment concentration of 2 mg/ml. No significant changes in pH were observed at 0 or 4 hours when compared with the vehicle control. Precipitation was observed at concentrations of 2.0, 1.0, 0.5, and 0.25 mg/ml. Slight precipitation was observed at 0.125 mg/m test concentration at the 0^th hour, which was judged not to interfere with the conduct of the test. Hence, the concentration of  0.125 mg/ml was selected as the high dose for the cytotoxicity experiment.

In the cytotoxicity test, cells were exposed to test item concentrations of  0.0 (NC: distilled water), 0.0 (VC: DMSO), 0.125, 0.063 and 0.031 mg/ml in culture media in the presence and absence of S9 metabolic activation. Cytotoxicity was determined by a reduction in the mitotic index in comparison with vehicle control data. All the tested concentrations at the initial cytotoxicity experiment were cytotoxic both in the presence and absence of the S9 metabolic activation system. Next, the cytotoxicity was assessed at test item concentrations of 0.0 (NC), 0.0 (VC), 0.0006, 0.0012 and 0.015 mg/ml of culture media in a second cytotoxicity experiment (Experiment II). In cytotoxicity experiment (II), all tested concentrations were cytotoxic both in the presence and absence of the S9 metabolic activation system. Cytotoxicity was then assessed at test item concentrations of 0.0 (NC), 0.0 (VC), 0.0006, 0.0012 and 0.0024 mg/ml in culture media in the third cytotoxicity experiment (Experiment III). The test concentration of 0.0024 mg/ml produced a 58.59 % and 56.08 % decrease in the mitotic index compared to VC in the absence (-S9) and the presence of metabolic activation (+S9), respectively. The dose level that results in approximately 55 ± 5 % reduction in the mitotic index was selected as the highest test concentration in the main test. Hence, 0.0024 mg/ml was chosen as the highest test item concentration for the main study, both in the presence and absence of metabolic activation. In the CA test, proliferating peripheral blood cells (whole blood) were used for the test, and the treatment of cultures with the test item was conducted in two independent phases. In the Phase I experiment, cells were exposed to test item concentrations of 0.0 (NC), 0.0 (VC), 0.0006, 0.0012 and 0.0024 mg/ml along with positive controls (EMS: 600 µg/ml without S9 mix and CPA: 30 600 µg/ml with S9 mix) for 4 hours both in the presence and absence of S9 metabolic activation system (1 v/v %) using duplicates. In the Phase II experiment, cells in duplicate cultures were treated with 0.0 (NC), 0.0 (VC), 0.0006, 0.0012 and 0.0024 mg/ml for 4 hours in the presence of the S9 metabolic activation system (2 v/v%) or 24 hours in the absence of S9 metabolic activation. Cells were harvested 24 hrs after treatments in both experiements. A minimum of 1000 cells was counted in different fields of slide per culture, and the number of metaphases was recorded for Mitotic Index (MI) calculation. Three hundred well-spread metaphases per concentration and control were scored for cytogenetic damage on coded slides. Chromosomal and chromatid gaps, breaks, acentric, fragments, exchanges, 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. Results: In the cytotoxicity test (Experiment III), the test concentration 0.0024 mg/ml produced a 58.59 % and 56.08 % decrease in the mitotic index compared to VC in the absence (-S9) and the presence of metabolic activation (+S9), respectively. The test concentration 0.0012 mg/ml produced 42.00 % and 44.16 % decreases in the mitotic index compared to VC in the absence (-S9) and presence (+S9) of metabolic activation, respectively. The test concentration 0.0006 mg/ml produced a 33.13 % and 31.67 % decrease in the mitotic index (cytotoxicity) compared to VC in the absence (-S9) and presence (+S9) of metabolic activation, respectively.

In Phase I experiment, the cultures were exposed to the test substance for 4 hours both in the presence (+S9) and absence (-S9) of the metabolic activation system (1%). The mean percentages of aberrant cells were 0.333 (NC), 0.333 (VC), 0.667 ( at 0.0006 mg/ml), 0.667 (at 0.0012 mg/ml), 0.667 (at 0.0024 mg/ml) and 11.000 (PC: 600 µg/mL EMS) in the absence of metabolic activation. In the presence of metabolic activation (1 v/v%), the mean percentage of aberrant cells were 0.333 (NC), 0.333 (VC), 0.000 (at 0.0006 mg/ml), 0.333 (at 0.0012 mg/ml), 0.667 (at 0.0024 mg/ml) and 10.333 (PC: 30 µg/ml CPA). Treatment with positive control substances caused significant increases in the percent of aberrant cells. Even though the analysis did not reveal any statistical significance, the increases were deemed biologically significant. A 55.74% and 52.16 % of reduction in the mitotic index, i.e., cytotoxicity, was observed at the tested concentration of 0.0024 mg/ml compared to vehicle control (VC) in the absence (-S9) and presence (+S9) of metabolic activation system, respectively. The observed mean mitotic index in the absence of metabolic activation were 9.87 (NC), 9.66 (VC), 6.87 (at 0.0006 mg/ml), 5.57 (at 0.0012 mg/ml), 4.28 (at 0.0024 mg/ml) and 8.37 (PC: 600 µg/mL EMS). In the presence of metabolic activation (1 v/v%), the mean mitotic index was 9.53 (NC), 8.66 (VC), 5.59 (at 0.0006 mg/ml), 4.92 (at 0.0012 mg/ml), 4.14 (at 0.0024 mg/ml) and 7.69 (PC: 30 µg/mL CPA). The Phase II experiment was performed to confirm the negative results obtained in Phase I in the presence and absence of S9 metabolic activation. In the absence of metabolic activation, the mean percent aberrant cells were 0.333 (NC), 0.333 (VC), 0.667 (at 0.0006 mg/ml), 0.333 (at 0.0012 mg/ml), 0.333 (at 0.0024 mg/ml) and 10.667 (PC: 600 µg/mL EMS). In the presence of the metabolic activation, the mean percent aberrant cells were 0.667 (NC), 0.333 (VC), 0.000 (at 0.0006 mg/ml), 1.000 (at 0.0012 mg/ml ), 0.333 (at 0.0024 mg/ml) and 11.000 (PC: 30 µg/mL CPA). Treatment with positive control substances in the absence and presence of metabolic activation caused a significant increase in percent aberrant cells. Even though the analysis did not reveal any statistical significance, the increases were deemed biologically significant. A reduction of 54.67 and 41.22 in the mitotic index was observed at the tested concentration of 0.0024 mg/ml compared to the vehicle control (VC) in the absence (-S9) and presence (+S9) of the  metabolic activation system, respectively. The observed mean mitotic index in the absence (-S9) of metabolic activation were 9.35 (NC), 9.12 (VC), 6.38 (at 0.0006 mg/ml), 5.82 (at 0.0012 mg/ml), 4.14 (at 0.0024 mg/ml) and 8.12 (PC: 600 µg/mL EMS). In the presence (+S9) of metabolic activation, the mean mitotic index was 8.71 (NC), 8.31 (VC), 7.12 (at 0.0006 mg/ml), 5.38 (at 0.0012 mg/ml), 4.89 (at 0.0024 mg/ml) and 7.62 (PC: 30 µg/mL CPA). No significant increase in the percent of aberrant cells was observed in the vehicle control groups  (Dimethyl sulfoxide) in Phase I and Phase II when compared to the negative control (distilled water). The increased frequency of aberrant cells 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. Conclusion: The registered substance, i.e., 2-methoxynaphthalene (CAS: 93-04-9),  is non-clastogenic in human peripheral lymphocytes both in the presence (1% and 2%) and in the absence of metabolic activation.

 

In vitro gene mutation in mammalian cells:

An in vitro gene mutation study in mammalian cells, according to OECD TG 476 with the registered substance (CAS: 93-04-9) has been initiated. This information will be submitted at a later time point.

Justification for classification or non-classification

A combination of in vitro genotoxicity tests was conducted with the registered substance, i.e., 2-methoxynaphthalene (CAS: 93-04-9), to assess its genotoxic properties. The substance tested non-mutagenic (negative) in Salmonella Typhimurium TA98, TA100, TA1535, TA1537 and TA102 according to OECD TG 471 and GLP both in the presence and absence of cofactor-supplemented liver S9 microsomal fraction. It has also been tested non-clastogenic (negative) in the primary culture of human peripheral blood cells according to OECD TG 473 and GLP both in the presence and absence of cofactor-supplemented liver S9 microsomal fraction. An in vitro gene mutation study with the Substance according to OECD TG 476 and GLP is ongoing. Justification for germ cell mutagenicity will be provided after receiving the data from the OECD 476 study.