2(1H)-Naphthalenone, 3,4-dihydro-5-methoxy-

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 2012 - December 2012

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:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

22 July 2012

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

457-080-9

EC Name:

-

Cas Number:

32940-15-1

Molecular formula:

C11H12O2

IUPAC Name:

5-methoxy-1,2,3,4-tetrahydronaphthalen-2-one

Test material form:

solid

Details on test material:

- Appearance: Ochre-yellow solid
- Storage condition of test material: In refrigerator (2-8°C) in the dark

Method

Species / strain

Species / strain / cell type:

lymphocytes: human peripheral blood

Details on mammalian cell type (if applicable):

Type and identity of media:
Blood samples
Blood samples were collected by venapuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started.

- Culture medium
Culture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 µg/mL respectively) and 30 U/mL heparin.

- Lymphocyte cultures
Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 ml (9 mg/mL) phytohaemagglutinin was added.

The Average Generation Time (AGT) of the cells and the age of the donor at the time the AGT was determined:
Dose range finding study: age 33, AGT = 15.8 h
First cytogenetic assay: age 28, AGT = 14.1 h
Cytogenetic assay 1A: age 31, AGT = 13.4 h
Second cytogenetic assay: age 29, AGT = 15.3 h

Cytokinesis block (if used):

Cytochalasine B (5 μg/ml)

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Dose range finding test:
With and without S9-mix, 3hr exposure; 24 hr fixation: 10, 33, 100, 333 and 1000 µg/mL
With S9-mix, 27hr exposure: 3, 10, 33, 100, 333, 1000 and 1762 µg/mL

First cytogenetic test:
Test concentrations:
Without S9-mix, 3hr exposure; 24 hr fixation : 10, 30, 50, 60, 70, 80 and 90 μg/ml culture medium
With S9-mix, 3hr exposure; 24 hr fixation : 10, 30, 100, 150, 200, 250 and 300 μg/ml culture medium
Concentrations selected for scoring of micronuclei:
Without S9-mix, 3hr exposure; 24 hr fixation: 10, 30 and 50 µg/mL
With S9-mix, 3hr exposure; 24 hr fixation: 10, 30 and 100 µg/mL
Since in the presence of S9-mix not enough cells could be scored for the presence of micronuclei to draw an appropriate conclusion, this part of the experiment was repeated.
Cytogenetic test 1A:
Test concentrations:
With S9-mix, 3 hr exposure; 24 hr fixation: 10, 30, 60, 100, 125 and 150 µg/mL
Concentrations selected for scoring of micronuclei:
With S9-mix, 3 hr exposure; 24 hr fixation: 10, 60, 100 and 125 µg/mL

Second cytogenetic test:
Test concentrations:
Without S9-mix, 24hr exposure: 3, 10, 30, 50, 70 and 90 µg/mL
Concentrations selected for scoring of micronuclei:
Without S9-mix, 24hr exposure: 3, 10 and 30 µg/mL

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of vehicle:
Substance could be dissolved in DMSO and DMSO is accepted and approved by authorities and international guidelines

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9, 0.25 and 0.38 µg/mL for a 3 hours exposure period and 0.15 and 0.23 µg/mL for a 24 hours exposure period

Controls 2

Positive control substance:

other: colchicine

Remarks:

without S9, 0.1 µg/mL for a 3 hours exposure period and 0.05 µg/mL for a 24 hours exposure period.

Controls 3

Positive control substance:

cyclophosphamide

Remarks:

with S9, 10 or 15 µg/mL for a 3 hours exposure period

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 46 ± 2 hr
- Exposure duration:
Short-term treatment
Without and with S9-mix: 3 hr treatment, 24 hr recovery/harvest time
Continuous treatment
Without S9-mix: 24 hr treatment/harvest time

ARREST OF CELL DIVISION: 5 µg/mL Cytochalasine B
STAIN: Giemsa

NUMBER OF REPLICATIONS: duplicates

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
To harvest the cells, cell cultures were centrifuged (5 min, 365 g) and the supernatant was removed. Cells in the remaining cell pellet were swollen by a 5 min treatment with hypotonic 0.56% (w/v) potassium chloride solution at 37°C. Cells were collected by centrifugation (8 min, 135 g) and fixed with 3 changes of methanol : acetic acid fixative (3:1 v/v).
Fixed cells were dropped onto cleaned slides and stained for 10 - 30 min with 5% (v/v) Giemsa solution in tap water, rinsed in tap-water and allowed to dry. The dry slides were automatically embedded in a 1:10 mixture of xylene/pertex and mounted with a coverslip in an automated coverslipper.

NUMBER OF CELLS EVALUATED: 1000/culture (mono- and binucleated cells)

DETERMINATION OF CYTOTOXICITY
- The cytostasis/cytotoxicity was determined using the cytokinesis-block proliferation index (CPBI index)

ACCEPTABILITY OF ASSAY
An in vitro micronucleus test was considered acceptable if it meets the following criteria:
a) The number of mono and binucleated cells with micronuclei observed in 2000 cells of the solvent control should be less than 10.
b) The positive control substance colchicine produced at least a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number mononucleated cell with micronuclei and the positive control substances MMC-C and CP produced at least a statistically significant increase in the number of binucleated cells with micronuclei.
c) A homogeneous response between the duplicate cultures is observed.

Evaluation criteria:

A test substance was considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if:
a) It induces a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of mono or binucleated cells with micronuclei and/or
b) a statistically significant and biologically relevant increase is observed in the number of mono or binucleated cells with micronuclei in the absence of a clear dose-response relationship.

A test substance was considered negative (not clastogenic or aneugenic) in the in vitro micronucleus test if:
a) none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of mono and binucleated cells with micronuclei.
b) The number of mono and binucleated cells with micronuclei was within the laboratory historical control data range.

Statistics:

The incidence of micronucleated cells (cells with one or more micronuclei) for each exposure group was compared to that of the solvent control using Chi-square statistics.

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: human peripheral blood

Metabolic activation:

with and without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the first cytogenetic assay, both in the absence and presence of S9-mix (cytogenetic assay 1A), The substance induced a dose dependent, increase in the number of binucleated cells with micronuclei. The increase was statistically significant at the highest concentration tested (50 and 125 μg/ml in the absence and presence of S9-mix, respectively). In addition in the presence of S9-mix, a statistically significant dose dependent increase in the number of mononucleated cells with micronuclei was observed.
The increase in the number of binucleated cells with micronuclei in the presence of S9-mix was only observed at a high toxic concentration (64% cytotoxicity). However, the increase in the number of mononucleated cells was already observed at a mid-dose of 60 μg/ml which did not show severe cytotoxicity (27% cytotoxicity).
In the second cytogenetic assay at the 24 hours continuous exposure time, the substance induced a dose dependent increase in the number of binucleated cells with micronuclei. The increase was statistically significant at the highest concentration tested (30 μg/ml).

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation in the exposure medium was observed at dose levels of 1000 µg/ml and above

RANGE-FINDING/SCREENING STUDIES:
- Toxicity was observed at dose levels of 33 µg/ml and above in the absence of S9, 3 hr treatment/24 hr fixation, 27 hr continuous treatment and in the presence of S9, 3 hours treatment, 24 hours fixation

CYTOKINESIS BLOCK
- Distribution of mono-, bi- and multi-nucleated cells:
First cytogenetic assay
without S9-mix, 3 hours exposure time, 24 hours fixation time
Concentration Culture Number of cells with ….nuclei
µg/ml 1 2 3 or more
0 A 91 276 136
B 91 291 125
10 A 105 309 86
B 78 353 74
30 A 192 272 36
B 135 321 45
50 A 235 254 12
B 259 228 15
60 A 308 193 0
B 344 152 4
70 A 400 99 1
B 369 127 4
80 A 460 40 0
B 398 103 2
90 A 434 68 0
B 480 20 0
0.25 MMC-C A 162 319 23
B 158 311 31
0.38 MMC-C A 239 255 6
B 194 295 15
0.1 Colch A 425 66 9
B 451 44 5

+S9-mix, 3 hours exposure time, 24 hours fixation time
Concentration Culture Number of cells with ….nuclei
µg/ml 1 2 3 or more
0 A 95 283 122
B 78 289 133
10 A 109 302 89
B 109 296 95
30 A 125 293 83
B 95 320 85
100 A 252 240 9
B 293 194 13
150 A 377 121 3
B 309 185 6
200 A 495 25 0
B 421 74 7
250 A 453 62 0
B 493 7 0
300 A 496 8 0
B 483 17 0
15 CP A 348 153 2
B 320 188 3

cytogenetic assay 1A
+S9-mix, 3 hours exposure time, 24 hours fixation time
Concentration Culture Number of cells with ….nuclei
µg/ml 1 2 3 or more
0 A 112 302 97
B 88 286 129
10 A 149 277 97
B 99 304 97
30 A 150 302 63
B 129 313 71
60 A 191 261 54
B 170 276 56
100 A 237 249 17
B 187 300 14
125 A 312 188 3
B 331 174 1
150 A 395 105 3
B 406 100 3
10 CP A 312 182 9
B 295 202 4

Second genetic assay
without S9-mix, 24 hours exposure time
Concentration Culture Number of cells with ….nuclei
µg/ml 1 2 3 or more
0 A 90 332 78
B 81 311 107
3 A 106 323 71
B 105 334 61
10 A 119 333 48
B 115 345 41
30 A 237 254 9
B 202 288 10
50 A 360 138 2
B 367 132 1
70 A 436 63 1
B 460 39 2
90 A 461 39 0
B 468 30 2
0.15 MMC-C A 190 304 6
B 163 328 9
0.23 MMC-C A 274 222 4
B 211 283 6
0.05 Colch A 473 26 2
B 473 28 3


NUMBER OF CELLS WITH MICRONUCLEI:
First cytogenetic assay
without S9-mix, 3 hours exposure time, 24 hours fixation time
Concentration (µg/ml) # mononucleated cells with micronuclei 1) # binucleated cells with micronuclei 1)
0 0 (1446) 2
10 2 (1128) 3 (1631)
30 1 (1933) 7
50 2 14**
0.25 MMC-C 0 76***
0.1 Colch 100*** (1704) 4 (288)

+S9-mix, 3 hours exposure time, 24 hours fixation time
Concentration (µg/ml) # mononucleated cells with micronuclei 1) # binucleated cells with micronuclei 1)
0 1 (841) 3
30 0 (507) 2
100 2 (740) 4 (881)
15 CP 1 (846) 23

cytogenetic assay 1A
+S9-mix, 3 hours exposure time, 24 hours fixation time
Concentration (µg/ml) # mononucleated cells with micronuclei 1) # binucleated cells with micronuclei 1)
0 4 (1164) 21
10 8 15
60 11 20
100 13 21
125 25** 66**
10 CP 10 114***

Second genetic assay
without S9-mix, 24 hours exposure time
Concentration (µg/ml) # mononucleated cells with micronuclei 1) # binucleated cells with micronuclei 1)
0 2 (643) 4 (1961)
3 1 (1007) 7
10 0 (1421) 9
30 6 (1930) 12*
0.15 MMC-C 0 (1516) 57*** (1815)
0.05 Colch 208*** 3 (93)

Significantly different from negative control group (Chi-square test), * P < 0.05, ** P < 0.01 or *** P < 0.001.
1) 2000 bi- and 2000 mononucleated cells scored for the presence of micronuclei (if less than 2000 cells were scored per culture the exact amount was reported between brackets).

- Indication whether binucleate or mononucleate where appropriate: In both cytogenetic assays in several cultures not enough mono- and/or binucleated cells could be scored for micronuclei in both cultures. In the first cytogenetic assay in the absence of S9-mix, the highest tested concentration did have enough cells to score for the presence of micronuclei. Therefore scoring additional cells in lower concentrations would not have changed the outcome of the study. In addition in the second cytogenetic assay, in the duplicate cultures of the highest tested concentrations 1000 cells could be scored for the presence of micronuclei. Scoring additional cells would therefore have limited additional information. Positive control cultures did show a statistically significant increase in the number of cells scored. Therefore scoring additional cells would have given limited additional information.


COMPARISON WITH HISTORICAL CONTROL DATA:
- The number of mono- and binucleated cells with micronuclei observed in 2000 cells found in the solvent and positive control cultures was within the laboratory historical range with the exception of cytogenetic assay 1A in which the number of micronucleuted binucleated cells was 10 to 11 per 1000 cells. Although this number was high, a clear positive response was observed at a concentration of 125 μg test item/mL and of the positive control group. Therefore the high background value does not influence the outcome of the study.
In both cytogenetic assays in several positive control cultures a low number of mono- and/or binucleated cells was present on the slides and therefore not enough cells could be examined for micronuclei from both cultures. Positive control cultures did show a statistically significant increase in the number of cells scored. Therefore scoring additional cells would have given limited additional information.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: CBPI
- Appropriate toxicity was reached at the dose levels selected for scoring. In the second cytogenetic assay, the highest concentration used for scoring of micronuclei (30 μg /ml) showed 43% cytostasis whereas the protocol stated about 55 ± 5%. Since the percentage cytostasis of this concentration, determined by a second scorer was 54% (quality control) a concentration of 30 μg/ml was used for scoring. The next higher concentration of 50 μg/ml was too toxic for scoring (73% cytostasis).

Applicant's summary and conclusion

Conclusions:

An in vitro micronucleus assay with the substance in cultured peripheral human lymphocytes was performed according to OECD 487 guideline and in accordance with GLP principles. The substance induced a dose dependent increase in the number of binucleated cells with micronuclei in both the first and second cytogenetic assays which reached statistical significance at the highest concentrations tested. The increases in the formation of micronuclei in human lymphocytes induced by the substance were just above the historical control data range, or at high cytotoxic concentrations and therefore considered equivocal.

Executive summary:

An in vitro micronucleus assay with the substance in cultured peripheral human lymphocytes was performed according to OECD 487 guideline and in accordance with GLP principles. In the first cytogenetic assay, the substance was tested up to 50 and 125 μg/ml for a 3 hours exposure time with a 27 hours harvest time in the absence and presence of S9-fraction, respectively. In the second cytogenetic assay, the substance was tested up to 30 μg/ml for a 24 hours exposure time with a 24 hours harvest time in the absence of S9-mix. Appropriate toxicity were reached at these dose levels.

The number of mono- and binucleated cells with micronuclei found in the solvent control cultures was within the laboratory historical control data range with the exception of the first cytogenetic assay in which the number of micronucleated binucleated cells was 10 to 11 per 1000 cells (laboratory historical control data: 0-7 per 1000 cells). However since a clear positive response was observed in the highest test substance concentration, the test conditions were considered adequate. The positive control chemicals, mitomycin C, cyclophosphamide and colchicine induced appropriate respnses. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

The substance induced a dose dependent increase in the number of binucleated cells with micronuclei in both the first and second cytogenetic assays. The increase was statistically significant at the highest concentrations tested, 50 and 125 μg/ml in the absence and presence of S9-mix, respectively in the first assay and 30 μg/ml in the second assay. In addition, a statistically significant dose dependent increase in the number of mononucleated cells with micronuclei was observed in the presence of S9-mix in the first assay.

Although the increases were just above the historical control data range, or at high cytotoxic concentrations, a dose relationship was observed in both cytogenetic assays. The increase in the formation of micronuclei in human lymphocytes induced by the substance in DMSO at a concentration range from 3 to 125 μg/ml both in the absence and presence of S9 metabolic activation is therefore considered equivocal under the experimental conditions described in this report.