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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Constituent 1
Chemical structure
Reference substance name:
1,8-naphthylenediamine
EC Number:
207-529-8
EC Name:
1,8-naphthylenediamine
Cas Number:
479-27-6
Molecular formula:
C10H10N2
IUPAC Name:
naphthalene-1,8-diamine
Details on test material:
purity: 99.5%
Specific details on test material used for the study:
Name 1,8-Diaminonaphthalene chip
Batch no. F71006
Appearance reddish purple to dark reddish brown(chips/flakes)
Composition 1,8-Diaminonaphthalene ≧99.0%
Purity 99.4 %
Homogeneity homogeneous
Expiry date 29. Oct. 2021
AS No. 479-27-6
EINECS-No. 207-529-8

Method

Target gene:
The in vitro micronucleus assay is a genotoxicity test for the detection of micronuclei in the
cytoplasm of interphase cells. Micronuclei may originate from acentric chromosome
fragments (i.e. lacking a centromere), or whole chromosomes that are unable to migrate to
the poles during the anaphase stage of cell division. The assay provides a comprehensive
basis for investigating chromosome damaging potential in vitro because both aneugens
and clastogens can be detected; however, differentiation of chemicals inducing polyploidy
from those inducing clastogenity is not possible. The addition of the actin polymerisation
inhibitor cytochalasin B prior to the targeted mitosis allows the identification and selective
analysis of micronucleus frequency in cells that have completed one mitosis because such
cells are binucleated.
Species / strain
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
Primary cultures of human peripheral lymphocytes
Metabolic activation:
with and without
Metabolic activation system:
S9 (liver enzyme mixture used for the test with metabolic activation) was obtained from Trinova Biochem GmbH, Gießen, and stored at – 80 ± 5°C.
Batch nos.: 3801, 3852
Specification: produced from the livers of male Sprague-Dawley rats which were treated with 500 mg Aroclor 1254/kg body weight intra- peritoneally.
Test concentrations with justification for top dose:
Experimental Setup nominal concentrations of the test solutions (mM)
2000 1000 500 250 125
resulting concentrations in the experiment (mM)
10 5 2.5 1.25 0.63
All concentrations are given as nominal and partially rounded concentrations. Concentrations may differ ± 10%.
Vehicle / solvent:
Acetone
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: Colchicine
Details on test system and experimental conditions:
Cell Cultivation
The blood cultures were set up in defined time intervals within 24 h after collection in sterile culture vessels, each containing 1 part of heparinised blood and 9 parts of complete
culture medium RPMI 1640 for cell proliferation. The cultures were incubated for 48 h (pre-exp.) or 72 h (exp. I and II) at 37 ± 1 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2.
Cell Treatment
Pre-Experiment, Experiment I and Experiment II with Metabolic Activation
After the initial cell cultivation, duplicate cultures were prepared for each test group. After centrifugation (10 min, 500 * g), the cells were resuspended in minimal culture medium
and solvent control, positive control or the single test item concentrations were added.
In the case of metabolic activation, 50 µL S9 mix per mL medium were used. The cell cultures were incubated at 37 ± 1 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2 for
an exposure period of 4 h (pre-exp. and exp. I) or 6 h (exp. II with metabolic activation).
After the exposure time of 4 h or 6 h, the cells were spun down by gentle centrifugation for 5 min (500 * g). The supernatant was discarded, the cells were re-suspended in 5 mL
Saline G and centrifuged again. The washing procedure was repeated once as described.
After washing, the cells were re-suspended in complete culture medium RPMI 1640, cytochalasin B (final concentration 5 µg/mL) was added and the cells were incubated at
37 ± 1 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2 for 19 h until preparation.

In experiment II without metabolic activation, 72 h after seeding, duplicate cultures were prepared for each test group. The blood cultures were centrifuged (10 min, 500 * g). The
cell pellet was re-suspended in complete culture medium RPMI 1640, cytochalasin B (final concentration 5 µg/mL) and medium control, solvent control, positive control or the test
item concentrations were added. The exposure duration was 21.5 h.
Harvesting Procedure
Each cell culture was harvested and processed separately. The cells were spun down by gentle centrifugation (10 min, 500 * g). The supernatant was discarded and the cells were
re-suspended in 12 ml hypotonic KCl solution. The cell suspension was allowed to stand for 10 min at room temperature (20 ± 5°C). After removal of the hypotonic solution by
centrifugation (10 min, 500 * g), the cell pellet was fixed with a mixture of methanol and glacial acetic acid (3:1). After fixation at 2 – 8 °C for minimum 30 min, the cell suspension
was spun down by gentle centrifugation (10 min, 500 * g), the supernatant was discarded and the cell pellet was re-suspended in fixative again. The washing procedures were
repeated until the cell pellet was white.
Preparation of Slides
The slides were prepared by dropping the cell suspension onto a clean microscope slide.
The cells were then stained with a 10% solution of Giemsa. All slides were independently coded before microscopic analysis.
Determination of the Cytokinesis-Block Proliferation Index
In all replicates, the cytokinesis-block proliferation index (using at least 500 cells per culture) was determined in order to assess the cytotoxicity of the test item. From these
determinations, the test item concentrations which were evaluated for scoring of micronuclei were defined.
Determination of Binucleated Cells with Micronuclei
At least 1000 binucleated cells per culture were scored for micronuclei. Only cells with sufficiently distinguishable cytoplasmic boundaries and clearly visible cytoplasm were
included in the analysis.
Rationale for test conditions:
Solvent Controls
Acetone was used as solvent control for the test item.
MCM was also carried along to show the suitability of Acetone as solvent, because there are no historical data for Acetone available.
0.9% NaCl was used as solvent control for the positive controls Cyclophosphamide monohydrate (CPA), Mitomycin C (MMC) and Colchicine.
Evaluation criteria:
Evaluation of the slides was performed using Zeiss microscopes with 40 x- and 100 x - oil immersion objectives. Cytotoxicity was calculated as reduction in CBPI compared to the CBPI of the concurrent
solvent control.
Statistics:
The number of binucleated cells with micronuclei in each treatment group was compared with the solvent control. Statistical significance was tested using Fisher’s exact test at the five per cent level (p <0.05).
For positive controls with high values of binucleated cells with micronuclei, the chi-square-test was used.

Results and discussion

Test results
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
ambiguous
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Pre-experiment Cytotoxicity test:
Strong cytotoxicity was observed at the 4 highest test item concentrations (1.5, 2.5, 5 and 10 mM). Therefore, no micronuclei scoring was performed. Only the CBPI of the controls was determined to show the validity of the experiment, as well as the CBPI of the lowest concentration.
Experiment I
Cytotoxicity Test - As neither critical cytotoxicity nor precipitation interfered with scoring, the three highest test item concentrations (1.25, 0.63 and 0.31 mM) were chosen for scoring of micronuclei.
In experiment I without metabolic activation, the highest test item concentration (1.25 mM) showed a statistically significant increased (p < 0.05) proportion of micronuclei compared with the concurrent solvent control. All values lay within the historical data and literature data for solvent controls, and no dose-response relationship was detected.
In experiment I with metabolic activation, all 3 evaluated test item concentrations showed statistically significant increased values of micronuclei lying above the historical data (also above the 95.5% control limits) for minimal culture medium and 0.9% NaCl and lying also above the literature data for solvents. No dose-response relationship was detected.
Experiment II
Cytotoxicity Test - Since the highest test item concentration (1.25 mM) showed quite predominantly morphologically irregular shaped cells and nuclei in the approach without metabolic activation, the determination of micronuclei according to chapter 15 (scoring criteria) was not possible, evaluation was started at the concentration 0.63 mM
In experiment II without metabolic activation, the concentrations 0.63 mM and 0.16 mM showed statistically significant increased (p < 0.05) values of micronuclei. The value for the concentration 0.63 mM lay also above the historical data (also above the 95.5% control limits) for minimal culture medium and 0.9% NaCl and also above the literature data for solvents. No dose-response relationship was detected.
In experiment II with metabolic activation, the concentrations 1.25 mM and 0.31 mM showed statistically significant increased (p < 0.01) values of micronuclei. These values lay also above the historical data (also above the 95.5% control limits) for minimal culture medium and 0.9% NaCl. All values were outside the literature data for solvents. However, no dose-response relationship was detected.
Remarks on result:
not determinable
Remarks:
The result of this in vitro micronucleus assay is therefore assessed as “equivocal” under the conditions of the test.

Applicant's summary and conclusion

Conclusions:
1,8-Diaminonaphthalene chip did not lead to a clear positive or negative resulting regarding the formation of micronucleiin human lymphocytes in vitro and thererfore the result of this in vitro micronucleus assay is therefore assessed as “equivocal” under the conditions of the test.
Executive summary:

This study was performed to assess the genotoxic potential of 1,8-Diaminonaphthalene chip to induce formation of micronuclei in human lymphocytes cultured in vitro in absence

and presence of an exogenous metabolic activation system (liver S9 mix from male rats, treated with Aroclor 1254).

For the analysis of the genotoxic potential of the test item, three independent experiments were performed.  

In the pre-experiment, started at the concentration 10 mM with 5 test item concentrations in total, no micronuclei scoring was performed due to strong cytotoxicity caused by the test

item. Therefore, experiment I and II were performed with adapted test item concentrations.

In experiment I without metabolic activation, the highest evaluated test item concentration (1.25 mM) showed a statistically significant increased (p < 0.05) proportion of micronuclei

compared with the concurrent solvent control. All values lay within the historical data and literature data for solvent controls, no dose-response relationship was detected.

In experiment I with metabolic activation, all 3 evaluated concentrations (1.25, 0.63 and 0.31 mM) showed statistically significant increased values of micronuclei lying above the

historical data (also above the 95.5% control limits) for minimal culture medium and 0.9% NaCl and lying also above the literature data for solvents. No dose-response relationship

was detected.

In experiment II without metabolic activation, the highest test item concentration (1.25 mM) showed quite predominantly morphologically irregular shaped cells and nuclei, possibly

necrotic or apoptotic. Therefore, evaluation was started at the concentration 0.63 mM. The concentrations 0.63 mM (p < 0.01) and 0.16 mM (p < 0.05) showed statistically significant increased values of micronuclei. The value for the concentration 0.63 mM lay also above the historical data (also above the 95.5% control limits) for minimal culture medium and 0.9% NaCl and also above the literature data for solvents. No dose-response relationship was detected.

In experiment II with metabolic activation, the concentrations 1.25 mM and 0.31 mM showed statistically significant increased (p < 0.01) values of micronuclei. These values lay

also above the historical data (also above the 95.5% control limits) for minimal culture medium and 0.9% NaCl. All values were outside the literature data for solvents. No dose-

response relationship was detected.

None of the experimental conditions led to a clear positive or negative result. At least one criterion for a positive result was fulfilled in every experimental part, but a dose-response

relationship could not be detected in any of the tested approaches.

In conclusion, under the experimental conditions reported, 1,8-Diaminonaphthalene chip did not lead to a clear positive or negative resulting regarding the formation of micronucleiin human lymphocytes in vitro.

The result of this in vitro micronucleus assay is therefore assessed as “equivocal” under the conditions of the test.