Naphthalene-2,6-dicarboxylic acid

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From Feb 12, 2015 (treatment started) to March 23, 2015 (study end date)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

"On the method of teting related to new chemicl substances" (March 31, 2011 Department of Medicine Diet 0331 No. 7. Heisei 23. 03-29. Bureau of manufacture No. 5 Personal Protection NO. 110331009. Welfare Ministry of Health, Labor and Welfare Director, Ministry of Economy, Trade and Industry Director of Manufacturing Industry bureau. Director of Environmental Policy Bureau Directorate of the Ministry of the Environment)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Naphthalene-2,6-dicarboxylic acid

EC Number:

214-527-0

EC Name:

Naphthalene-2,6-dicarboxylic acid

Cas Number:

1141-38-4

Molecular formula:

C12H8O4

IUPAC Name:

naphthalene-2,6-dicarboxylic acid

Test material form:

solid

Specific details on test material used for the study:

Solubility: insoluble in water, sparingly soluble in alcohols, soluble in N, N-dimethylformamide

In the test facility, preparation was confirmed using water, dimethylsulfoxide and acetone.

Lot number: J82SE
Purity: 99.7% (GC), 99.6% (neutralization titration)
Manufacturer: Kept confidential by Japan MHLW
Date of acquisition: October 20, 2014
Acquisition volume:100 g (shared with related tests)
Stability: After completion of the test operation including the related test, a part of the test substance was sent to Tokyo Chemical Industry Co., Ltd. for analysis. As a result of the analysis, the obtained purities were 99.9% (GC) and 100.3% (neutralization titration) (report, disposition No. Z0062, February 21, 2015), the test substance was stable during the test period was analytically confirmed

Storage conditions: refrigeration [1 to 10 ºC: measurement range 5.0 to 9.4 ºC (test substance storage room), 3.9 to 7.5 ºC (mutagenicity testing room)], dark place, airtight

Storage location and storage period: Test substance storage room [Obtained October 20, 2014 (January 13, 2015)] and Mutagenicity Laboratory [January 13, 2015 to February 12, 2015 test substance treatment]]

Method

Species / strain

Species / strain / cell type:

mammalian cell line, other:

Details on mammalian cell type (if applicable):

CHL / IU: Chinese hamster (lung of newborn Chinese hamster (female))

Obtained from: DS Pharma Biomedical Co., Ltd.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 Source: Kikkoman Bio Chemifa Co., Ltd. Lot number: CAM 201412A Manufacturing date: December 19, 2014

Test concentrations with justification for top dose:

As a result of preliminary test, cell proliferation rate decreased to about 60% of the control values at the highest dose of 2200 μg / mL, but no cell proliferation suppression of 50% or more was observed at any dose. Based on this result, the doses for the main chromosome aberration test were set at 275, 550, 1100, 1650 and 2200 μg / mL.

Vehicle / solvent:

In this test, 1100 mg of the test substance was weighed. Appropriate amount of DMSO was added and suspended by stirring with a touch mixer, then dissolved in 5 mL to prepare a maximum concentration preparation solution (220 mg / mL). A portion of the maximum concentration preparation solution was diluted with DMSO at the following stage to prepare 165, 110, 55 and 27.5 mg / mL.
Preparation Frequency: The test substance preparation solution was prepared at the time of use, and used within 40 minutes after preparation in the preliminary test, within 50 minutes after preparation in the definitive test. Addition volume of preparation solution: 1 vol% to the solution in the plate was added.

Controls

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

mitomycin C

Details on test system and experimental conditions:

Short time treatment - S9 treatment, short time treatment + S9 treatment and continuous treatment method 24 - hour treatments were conducted.

As a result of preliminary test, cell proliferation rate decreased to about 60% of control values at the highest dose of 2200 μg / mL in any of the test series cultures, but no cell proliferation suppression of 50% or more was observed at any dose. Based on this result, the dose of chromosome aberration test was set at the dose of 275, 550, 1100, 1650 and 2200 μg / mL with the highest dose of 2200 μg / mL.

A satellite group for confirming the influence of the test substance on cell proliferation was set for each dose except for the positive control group.

Four plates (2 satellite groups) were used for each group except for the positive control group and two plates were used for the positive control group. The identification number was specified on each plate.

Continuous treatment and 24-hour treatment: Confirmation of precipitation of test substanc and confirmed whether there is an influence on pH of culture solution by test substance preparation solution with pH test paper (BTB, lot number; 10712001, Toyo Roshi Kaisha) because the change in color tone was observed in the culture liquid color at the dose of 550 μg / mL or more.

Preparation of chromosome specimen: Two hours before termination of culture, colestimide (lot number; 1393179, GIBCO) at a final concentration of 0.2 μg / mL was added to each plate. At the end of the culture, the plates were collected in a centrifuge tube, and each plate was placed in a 0.02% EDTA-0.25% trypsin (0.5 M EDTA: lot number; 1045847, GIBCO, 2.5% trypsin: lot number, 1511084, GIBCO), And the cells were detached. The resulting cell suspension was collected in the same centrifuge tube and centrifuged at 1000 rpm for 5 minutes. The supernatant was removed and 0.075 mol / L potassium chloride (lot number; 310 U 1825, Kanto Kagaku Co., Ltd.) was added, and the cells were gently pipetted and the cells were bloated for 15 minutes at 37 ° C. The cells were semi-fixed by adding ice-cold Carnoy's fixative (methanol: acetic acid = 3: 1, methanol: lot number; 609 B 1143, Kanto Kagaku Co., Ltd., acetate: lot number: KPM 0110, Wako Pure Chemical Industries, Ltd.), Centrifuged at 1000 rpm for 5 minutes to remove the supernatant, and fixed with a fresh Carnoy's fixative. After repeating the fixing operation of the cells three times, the cell suspension was dropped onto the slide glass and air dried. From each plate, two chromosome specimens were prepared. Each slide was stained with 3% Giemsa solution [Giemsa solution: lot number; AJ 257, Wako Pure Chemical Industries, Ltd., instant phosphate buffer solution (pH 6.8): lot number; T 403, LSI Mediences Inc.] for 20 minutes, after washing with water and air drying, it was sealed with an encapsulant (Marinol, lot number: 1300901, Muto Chemical Co., Ltd.).

The three highest consecutive doses (1100, 1650 and 2200) were evaluated, since the cell proliferation inhibition exceeding 50% was not observed in any test series. In addition, specimens of the control group were evaluated. It was confirmed that about 50 or more metaphase cells were obtained per specimen. For specimens of the selected observation dose, one specimen per plate was selected and coded.

Mid-metaphase images of 100 (200 per dose) per plate were selected and observed with a microscope (BX 51 TF; Olympus Corporation) with a total magnification of 1000 ×, and chromosome abnormality was judged according to the following classification.
For structural abnormalities, those with chromosome of 25 ± 2 were taken as observation targets: structural anomaly (structural aberration) and chromatid break (ctb). A distinct discontinuous part (cut part) of the chromosome, where the discontinuous part is the chromatin. When the width was greater than or equal to the width, or when the cut piece was out of the longitudinal axis of the chromatid, it was judged as a chromatid cut. Chromatid exchange (cte: exchange): those in which two or more cleavage sites of the chromatid were mutually exchanged (binding reaction) were judged as chromatid exchanges. Chromosome break (csb: chromosome break) was judged when cutting occurred at the same position of both chromosomes. The criterion for cutting was similar to that of chromosome splitting. Chromosome exchange (cse: chromosome exchange): when exchanges occurred in the same direction at the same position of both chromosomes, chromosome exchange was judged. Other (others): other structural abnormalities include fragmentation. It was judged as fragmentation when cutting and gaps appeared on almost all chromosomes of one mid-metaphase image and no exchange type abnormality was included. Gap: when the width of the non-stained part was narrower than the width of the chromatid part at the non-stained part (part where dyeability was not observed at all) generated on the chromatid or chromosome. Numerical aberration: polyploid (poly: polyploid) when the number of chromosomes (25 ± 2) was doubled and the number of chromosomes was 36 or more (triploid, tetraploid, etc.) was judged as a polyploid. Others: other numerical abnormalities are intranuclear doubling. When doubled chromosomes did not separate but were lined up in parallel, it was judged to be endoreduplication and it was distinguished from the polyploid and counted.

The number of cells was determined for each plate and the total value was calculated for each test group. Furthermore, the appearance rate (%) is found for each structural abnormality (counted when the number of cells having structural abnormality is 1 even if there is a plurality of structural abnormality in one cell) and the total of cells having numerical abnormality. The appearance rate (%) was calculated as a percentage of the number of occurrences with respect to the observed cell number (the number of metaphase images).
a) Structural anomaly
• Ctb: number of cells with chromatid cleavage
• Cte: number of cells with chromatid exchanges
• Csb: Number of cells with chromosome breakage
• Cse: Number of cells with chromosome exchange
• Others: number of cells with other structural abnormalities
• Total: the number of cells having some structural abnormality
b) About gap
• gap: Number of cells with gaps
c) About numerical anomalies
• Poly: number of cells in polyploid
• Others: number of other numerically abnormal cells
• Total: the number of cells with some numerical abnormality

Evaluation criteria:

The following two conditions were set (and met) as conditions under which the test result was correctly evaluated.
a) The occurrence rates of structural abnormality and numerical abnormality of chromosomal abnormality in the negative control group of each test series are both less than 5%.
b) The occurrence rate of structural abnormality of chromosomal abnormality in the positive control group of each test series is 10% or more.

Evaluation of test results
If the appearance rate (%) of chromosomal structural abnormality and numerical abnormality are all less than 5%, it is negative. It is equivocal if either occurrence rate or both appearance rates are 5% or more and less than 10%. In cases where reproducibility was observed and either occurrence rate or both occurrence rates of 10% or more, and cases where increase with reproducibility or increase in dose were observed was considered positive. No statistical method was used.

Retest
In any of the test series, if the quality of the chromosome specimen is poor, or the occurrence rate of the chromosomal abnormality of the negative control group or the positive control group does not satisfy the establishment condition of the test, the corresponding test series or all the tests then conduct a retest on the series. The relevant matter did not occur in the test and no retest was conducted.

Results and discussion

Test results

Key result

Species / strain:

mammalian cell line, other: Chinese Hamster Lung Derived Cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Also precipitate at highest dose tested of 2200 ug/ml

Vehicle controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

No precipitation of test substance was observed at 275 ug/ml. At both 550 and 1100 μg / mL, precipitation of test substances was observed at the beginning of treatment, but not at the end of treatment. Precipitation was observed at both the start and end of treatment at the two highes doses of 1650 and 2200 μg / mL.

At 275 μg/ml there was no change in the pH of the culture medium. At 550 ug/ml the pH of the culture solution was found to decrease at the beginning of treatment, but not the end of treatment. At 1100, 1650 and 2200 μg / mL precipitation was observed at both the beginning and end of treatment.

The cell proliferation rate was evaluated at the same time as the chromosomal aberration. Cell proliferation was dose-dependently lower at 1100, 1650, and 2200 ug/ml with growth rates of 94, 95.5, and 67% control values, respectively, none of the cultures demonstrated growth inhibiiton of >50%.

The frequency of structural and numerical chromosome abnormalities was less than 5% in all of the short-time treatment without S 9 mix, the short-time treatment method with S 9 mix, and the continuous treatment method 24-hour treatment cultures.

The occurrence rate of chromosome structural abnormality in the positive control group was 41.0% in the short-time treatment method without S 9, 40.0% in the short-time treatment method with S 9, and 46.0% in the continuous treatment method.

Applicant's summary and conclusion

Conclusions:

2,6-naphthalene dicarboxylic acid, when tested up to a liimit dose of 2200 ug/ml in a chromosomal abberation test using mammalian cultured cells did not show any evidence of a potential to induce chromosomal aberrations.

Executive summary:

Using Chinese hamster lung-derived cells (CHL / IU) and the test substance 2,6 -naphthalene dicarboxylic acid, the presence or absence of chromosomal abnormalities was examined. The tests were carried out in three test series: Short Time Processing - S 9 Processing, Short Time Processing Process + S 9, and Processing and Continuous Processing Method 24 Hours Processing.

As a result of the preliminary test (cytostatic test: dose of the test substance: 17.2, 34.4, 68.8, 138, 275, 550, 1100 and 2200 μg / mL), in the main test (chromosome aberration test), doses of 275, 550, 1100, 1650 and 2200 μg / mL were set .

Based on the measurement results of the cell proliferation rate, 1100, 1650 and 2200 μg / mL were evaluated in detail. As a result, the occurrence rate of structural abnormality and numerical abnormality of chromosome was less than 5% at any dose of each test series, and the result was negative. In addition, the reproducibility was confirmed because the influence on cell proliferation of each test series, precipitation by test substance preparation treatment and pH on the culture solution were consistent with the results of the preliminary test. The appearance rate of chromosome structural abnormality in the positive control group showed clear positive values in each test series and it was confirmed that this test system had appropriate sensitivity.

From the above, it was judged that 2,6-naphthalene dicarboxylic acid does not induce chromosomal abnormality in mammalian cultured cells under the test conditions.