Naphthalene-2,6-dicarboxylic acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item revealed no mutagenic or genotoxicological potential in bacterial reverse mutation and chromosomal aberration tests.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

The study start date was January 9, 2015 and the study end date was March 23, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Expiration date of the lot/batch: No expiration date, but test material was reanalyzed and determined to be stable during the test period
- Purity test date: Before the study 99.7 %, retesting after the study 99.9 %

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigeration between 4.1 and 9.4 °C (measured)
- Stability under test conditions: analytically confirmed during the test period
- Solubility and stability of the test substance in the solvent/vehicle: Made fresh in DMSO
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: All dosing solutions were used within 2 hours of preparation. No heat generation, foaming or discoloration was observed in any of the prepared solutions.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9. Purchased from Kikkoman bio Chemifa Co., Ltd. Lot no. RAA201410A

Test concentrations with justification for top dose:

5, 15, 50, 150, 500, 1500, and 5000 µg/plate. Top dose was limit dose. No precipitation or significant cytotoxicity was obsered in a pre-test with concentrations up to 5000 µg/plate.

Vehicle / solvent:

As a result of confirming preparation of the test substance at the test facility, it did not dissolve in Japanese Pharmacopoeia Injection Water at 50 mg/mL and did not even suspend. Acetone was uniformly suspended at 220 mg/mL and 50 mg/mL, but precipitation occurred immediately. On the other hand, it was uniformly suspended in DMSO at 220 mg/mL and dissolved at 50 mg/mL. No heat generation, foaming or discoloration was observed in any of the prepared solutions. Therefore, DMSO was selected as the vehicle for the test substance and the negative control substance.

Details on test system and experimental conditions:

Pre-culture medium: Nutrient broth medium (Oxoid nutrient Broth No. 2, Oxoid Ltd. Lot number: 876774) was adjusted to 25 g/L by using water for injection (lot number 3 K 87, Otsuka Pharmaceutical Factory Co., Ltd.). To the medium of Salmonella typhimurium TA 98 and TA 100, ampicillin sodium (Lot No. M 3 B 7468, Nacalai Tesque, Inc.) was added at 25 μg/mL at the time of use.

Test medium (minimum glucose agar: hereinafter referred to as plate): Vital Medium AMT-O Medium (Far East Pharmaceutical Industry Co., Ltd. Lot number: DZLFAV02)

Medium for stratification: (A) soft agar and (B) amino acid solution of the composition of the table on the next page were prepared using water for injection (lot number 3K 87, Otsuka Pharmaceutical Factory Co., Ltd.) = 10: 1. An amino acid solution of L-histidine and D-biotin was used for Salmonella typhimurium, and an amino acid solution of L-tryptophan was used for Escherichia coli. These multilayer culture media were kept at 47 °C until use.

S9 mix: Kikkoman Bio Chemifa Co., Ltd., Lot number: RAA201410A. It was prepared from liver homogenate of S1c: SD rat (male, 7 weeks old) which was induced by intraperitoneal administration of phenobarbital and 5,6-benzoflavone. Freezing storage at -80 ºC or below after purchase


3.7.1 Dose setting test
Each strain was tested in direct method (in the absence of S9 metabolic activation system) and metabolic activation method (in the presence of S9 metabolic activation system).
For both the direct method and the metabolic activation method, the maximum dose of the test substance was set at 5000 μg / plate.

Each strain was tested by direct method and metabolic activation method. As a result of the dose setting test, the average value of the number of revertive mutant colonies of the test substance group was less than twice the average value of the negative control group in both the direct method of each strain and the test series of the metabolic activation method, there was also no increase in the number of revertant mutant colonies with increasing dose of substance. Growth inhibition by treatment with the test substance and precipitation of the test substance were not observed either.

Negative control group (dimethyl sulfoxide) and positive control group of the following table were set for each test series in both the dose setting test and this test.

Test strain Positive substance (dosage: μg / plate)
Direct method Metabolic activation method
Salmonella typhimurium TA100 AF-2 (0.01) 2-AA (1)
Salmonella typhimurium TA1535 NaN3 (0.5) 2-AA (2)
Escherichia coli WP2uvrA AF-2 (0.01) 2-AA (10)
Salmonella typhimurium TA98 AF-2 (0.1) 2-AA (0.5)
Salmonella typhimurium TA1537 9-AA (80) 2-AA (2)

AF-2: 2- (2-furyl) -3- (5-nitro-2-furyl) acrylamide
NaN 3: sodium azide
9-AA: 9-aminoacridine hydrochloride hydrate
2-AA: 2-aminoanthracene

The number of plates was set to 3 for each of the negative control group, the test substance treated group and the positive control group. The number of plates for sterility test was set to 2 for each of the maximum concentration preparation solution and S9-mix. Labels were affixed to the plate to identify the test number and test group.

Test method: Preculture of test strain
12 mL of preculture medium (Nutrient Broth medium) was placed in an L-shaped tube having a capacity of about 40 mL, and 12 μL of the thawed preserved bacteria was inoculated, and the temperature was set at 37 °C., the amplitude was 40 mm, and the shaking speed was set to 100 rpm Reciprocal shaking culture was carried out for 10 hours with a shaking incubator (Personal-11 · EX, Taitec Corporation). After the inoculation of the strain, the L-shaped tube was cooled (ice-cooled) until the start of the shaking culture, 6 hours and 20 minutes in the dose setting test and 6 hours and 35 minutes in the present test, respectively. At the end of the culture, the OD 660 nm of the resulting bacterial culture was measured with a turbidity meter (visible photometer CO 7500: Funakoshi Co., Ltd.), and the viable cell count was calculated from the viable cell count - OD 660 nm correlation formula of each strain. Cultures in which the viable cell count was confirmed to be greater than 1E+9 cells / mL were used for the test.
The viable cell count (calculated value) of each culture broth was as shown in the following table.

Test strain Number of bacteria (calculated value) (1E9 cells / mL)
Dose setting test This exam
Salmonella typhimurium TA100 2.61 2.54
Salmonella typhimurium TA1535 2.69 2.57
Escherichia coli WP2uvrA 5.66 5.3
Salmonella typhimurium TA98 4.13 3.97
Salmonella typhimurium TA1537 1.03 1.02

Treatment of test substance and control substance preparation solution was performed by preincubation method. That is, 0.1 mL/L Na-phosphate buffer solution (pH 7.4) 0.5 mL for the direct method is added to 0.1 mL of the test substance or control substance preparation solution using a polyethylene tube (5 mL capacity) In the case of the metabolic activation method, 0.5 mL of S9-mix was added and mixed, further 0.1 mL of the bacterial culture solution was added, and the shaking incubator set at 37 °C., amplitude 40 mm, shaking speed 100 times / min Personal-11 · EX, Taitec Corp.) for 20 minutes with shaking (preincubation). After the pre-incubation, Salmonella typhimurium contains 2 mL of a multilayer medium containing 0.05 mmol/L L-histidine and 0.05 mmol/L D-biotin, Escherichia coli contains 0.05 mmol / L L-tryptophan for stratification medium 2 mL, and the mixture was overlaid on a plate. Plate the plate in an incubator (MIR-262, Sanyo BioMedica Co., Ltd.) set at 37 ± 0.5 ºC after solidifying the multilayer medium in a flat place and incubate for 49 hours (dose setting test) or 49 hours and 20 minutes (this test) was carried out stationally. For each test, a sterility test was conducted on the maximum concentration preparation solution of the test substance to be used for the test and S9 mix, and the presence or absence of contaminating bacteria was confirmed.

With respect to the negative control group, the test substance treated group and the positive control group of each test system, the presence or absence of growth inhibition of the strain in the plate was confirmed with a stereoscopic microscope (SZ6045 TR, Olympus Optical Co., Ltd.) With or without precipitation of the test substance was visually confirmed. Next, for the negative control group, the test substance treated group and the positive control group of each test system, the number of revertive mutant colonies was measured using a colony analyzer (CA-11D, System Science Co., Ltd.). Since there was no plate thought that precipitation of the test substance or inhibition of growth affected the colony analyzer counting, visual counting of the number of revertive mutant colonies using a stereomicroscope was not carried out.

The presence or absence of growth inhibition was judged according to the following criteria, and one or more was defined as having growth inhibition.

0: Growth inhibition is not observed.
A fine background loan (observable at a magnification of about 50 times) is observed on the entire surface of the culture medium, and when there is no difference from the background loan of the negative control group.
1: Slight growth inhibition is observed when the background loan is reduced and the size of each individual colony is larger than in the negative control group.
2: Moderate growth inhibition is observed when raised large reverse mutation colony and flat and small background loan coexist.
3: Strong growth inhibition is observed when the background loan grows to the same size as the reversion mutant colony and it is difficult to discriminate between them.
4: No surviving bacteria are observed.

Evaluation criteria:

Sensitivity check of test system: The average value of the number of reverse mutation colonies of the negative control group of each test system was within the control value based on the background data of the test facility and the average value of the number of reversion mutation colonies of the positive control group of each test system was negative when it was more than twice the average value of the control group, it was judged that the test system had adequate sensitivity.

Criteria for judging test results: In at least one test system, the average value of the number of reversion mutant colonies of the test substance group is more than twice the average value of the negative control group, and the increase in the number of revertive mutant colonies accompanying the increase in the dose of the test substance was reproducible It was decided to be positive when it was acknowledged with. In addition, when reproducibility is not recognized in the test result, confirmation test etc. are carried out to confirm reproducibility. No statistical method was used for judging the test results.

Since no positive result was obtained in the test, the mutagens specific activity was not calculated.

Statistics:

Only for mean and standard deviation for results run in triplicate.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Reverse mutation test using naphthalenedicarboxylic acid bacteria (this test) (SR14250)

S9 mix (±)	Test substance (µg/plate)	Colony count / plate (mean ± SD)
		Base pair substitution type			Frame shift type
		TA100	TA1535	WP2uvrA	TA98	TA1537
S9 mix (-)	Negative control	111 145 111 (122 ± 20)	14 11 8 (11 ± 3)	24 22 18 (21 ± 3)	18 23 16 (19 ± 4)	10 10 8 (9 ± 1)
	313	97 97 98 (97 ± 1)	12 20 17 (16 ± 4)	19 17 20 (19 ± 2)	21 6 8 (12 ± 8)	14 7 7 (9 ± 4)
	625	92 114 112 (106 ± 12)	7 19 14 (13 ± 6)	21 26 19 (22 ± 4)	13 13 14 (13 ± 1)	6 11 7 (8 ± 3)
	1250	98 109 124 (110 ± 13)	12 16 17 (15 ± 3)	20 20 16 (19 ± 2)	21 13 19 (18 ± 4)	12 7 7 (9 ± 3)
	2500	115 105 102 (107 ± 7)	7 9 12 (9 ± 3)	21 22 18 (20 ± 2)	23 20 15 (19 ± 4)	8 7 6 (7 ± 1)
	5000	91 108 92 (97 ± 10)	13 8 3 (8 ± 5)	26 18 19 (21 ± 4)	10 10 8 (9 ± 1)	11 7 6 (8 ± 3)
S9 mix (+)	Negative control	120 146 116 (127 ± 16)	12 18 13 (14 ± 3)	23 22 27 (24 ± 3)	35 32 24 (30 ± 6)	17 16 11 (15 ± 3)
	313	131 122 129 (127 ± 5)	13 7 14 (11 ± 4)	24 18 17 (20 ± 4)	24 27 23 (25 ± 2)	22 7 8 (12 ± 8)
	625	123 111 135 (123 ± 12)	11 10 16 (12 ± 3)	26 17 21 (21 ± 5)	25 22 28 (25 ± 3)	15 9 8 (11 ± 4)
	1250	132 145 140 (139 ± 7)	16 12 10 (13 ± 3)	21 24 27 (24 ± 3)	29 31 27 (29 ± 2)	10 17 11 (13 ± 4)
	2500	122 135 122 (126 ± 8)	14 12 7 (11 ± 4)	25 26 23 (25 ± 2)	46 26 28 (33 ± 11)	13 15 8 (12 ± 4)
	5000	93 103 120 (105 ± 14)	15 6 20 (14 ± 7)	27 19 13 (20 ± 7)	32 22 40 (31 ± 9)	16 13 7 (12 ± 5)
Positive controlS9 mix (-)	Name of substance	AF-2 a)	NaN3 b)	AF-2	AF-2	9-AA c)
	(μg/plate)	0.01	0.5	0.01	0.1	80
	Number of colonies / plate	744 702 732 (726 ± 22)	208 260 244 (237 ± 27)	64 87 68 (73 ± 12)	331 327 311 (323 ± 11)	126 145 186 (152 ± 31)
Positive controlS9 mix (+)	Name of substance	2-AA d)	2-AA	2-AA	2-AA	2-AA
	(μg/plate)	1	2	10	0.5	2
	Number of colonies / plate	1622 1786 2070 (1826 ± 227)	444 537 447 (476 ± 53)	1103 1034 1122 (1086 ± 46)	336 392 355 (361 ± 28)	421 377 361 ( 386 ± 31)

Negative control: Dimethylsulfoxide

a) AF-2 :2- (2-furyl) -3- (5-nitro-2-furyl) acrylamide

b) NaN3 :Sodium azide

c) 9-AA :9-aminoacridine hydrochloride hydrate

d) 2-AA :2-aminoanthracene

Conclusions:

2,6-naphthalene dicarboxic acid did not demonstrate mutagenic potential in the reverse mutation test using bacterial strains S. typhimurium strains TA98, TA100, TA1535, TA1537, and E. coli WPuvrA.

Executive summary:

The presence or absence of gene mutagenicity in 2,6 -naphthalene dicarboxylic acid bacteria was examined by a reversion mutation test using Salmonella typhimurium TA 100, TA 1535, TA 98, TA 1537 and Escherichia coli WP2uvrA. The test was carried out by a preincubation method in a direct method (in the absence of S9 metabolic activation system) and a metabolic activation method (in the presence of S9 metabolic activation system).

In the dose setting test, the maximum dose of the test substance was set at 5000 μg / plate for both the direct method and the metabolic activation method, and the total of 7 doses (5 to 5000 μg/plate) was set at 6 ratios with a common ratio of about 3. In the present study, from the results of the dose setting test, both the direct method and the metabolic activation method, the maximum dose of the test substance was set at 5000 μg/plate, and the total dose was set to 5 doses (156 to 5000 μg/plate).

As a result of the test, the average value of the number of revertant mutant colonies of the test substance-treated group in the test series of the direct setting method and the metabolic activation method of each strain in the dose setting test and the present test was the average value of the negative control group. It was less than 2-fold, and no increase in the number of revertant colonies with increasing dose was observed. Precipitation of the test substance and inhibition of growth by the test substance were not observed in any of the strains. In this way, reproducibility was confirmed in the dose setting test and the results of this test.

In both the dose setting test and the main test, the average value of the number of reversion mutant colonies of the negative control group of each strain was within the control value based on the background data of the test facility. The average value of the number of revertant colonies of the positive control group of each strain was clearly increased more than 2 times as compared with the average value of each negative control group. From these results, it was confirmed that each strain had adequate sensitivity to mutagens.

From the above, it was judged that 2,6-naphthalene dicarboxylic acid does not have mutagenic potential to the test strains employed under the test conditions described.

Reference 2

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

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

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]]

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.

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.

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

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.

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.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 1537

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Test concentrations with justification for top dose:

-Concentration range: 667-10 000 µg/plate with or without rat liver S9 fraction, prepared in solution of dimethylsulphoxide (DMSO)

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Conclusions:

The test item was not mutagenic in the test system with or without metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Based on the available mutagenicity and genotoxicity studies, no classification is triggered according to Regulation (EC) No 1272/2008.