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Genetic toxicity in vitro

Description of key information

An OECD487 and OECD490 in vitro assays and several Ames tests and a data from a SOS Chromotest and a QSAR ccaluculation are available

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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
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
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
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 / 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
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.
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.
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.

Endpoint:
in vitro gene mutation study in mammalian cells
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
Qualifier:
according to guideline
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
GLP compliance:
yes
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
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%
CAS No. 479-27-6
Purity 99.4 %
Homogeneity homogeneous
Expiry date 29. Oct. 2021
CAS No. 479-27-6
EINECS-No. 207-529-8
Target gene:
This in vitro assay was performed to assess the potential of the test item 1,8-Diaminonaphthalene chip to induce forward gene mutations at the autosomal thymidine kinase locus (Tk1) and/or structural chromosomal aberrations in heterozygous mouse lymphoma L5178Y Tk+/- cells.
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
S9 (liver enzyme mixture used for the test with metabolic activation) was produced from the livers of male Sprague-Dawley rats which were treated with 500 mg Aroclor 1254/kg body weight intraperitoneally.
Test concentrations with justification for top dose:
Experimental Setup Pre-test nominal concentrations of the test solutions (mM)
2000 1000 500 250 125 63 31
resulting concentrations in the experiment (mM)
10 5 2.5 1.25 0.63 0.31 0.16
All concentrations are given as nominal and partially rounded concentrations. Concentrations may differ ± 10%.
Vehicle / solvent:
acetone
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Details on test system and experimental conditions:
Cleansed and for mycoplasma contamination screened stocks of cells were stored in liquid nitrogen in the cell bank of LAUS GmbH to allow a continuous stock of cells, which guarantees similar parameters of the experiment and reproducible characteristics of the cells. Cells were thawed 7 ± 1 d prior treatment and cultivated in RPMI 1640 complete culture medium with 5 % HS in cell culture flasks at 37.0 ± 1.0 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2.
with and without S9 - two replicates per culture 7 concentrations for Pre-Test (Cytotoxicity):
 Treatment for 4 h in the presence and in the absence of S9
 Remove treatment medium (with/without S9) and test item
 Add fresh medium after washing
 Transfer of 2 cells per well into 96 well microtiter plates
 Incubation for 10-12 days
 Evaluation of relative cloning efficiency

Experiment I with S9 - two replicates per culture min. 4 concentrations (Main experiment I)
 Treatment for 4 h in the presence of S9
 Remove treatment medium, S9 and test item
 Add fresh medium after washing
 Measure cell density directly after treatment as well as 24 h and 48 h after treatment
 Evaluation of RSG in %
 Transfer of cells into 96 well microtiter plates, 2 cells per well for viability, 4000 cells per well for mutagenicity (with TFT)
 Incubation for 10-12 days
 Evaluation of cytotoxicity and mutagenicity (min. 4 concentrations)

Experiment I without S9 - two replicates per culture min. 4 concentrations (Main experiment I)
 Treatment for 4 h in the absence of S9
 Remove treatment medium, S9 and test item
 Add fresh medium after washing
 Measure cell density directly after treatment as well as 24 h and 48 h after treatment
 Evaluation of RSG in %
 Transfer of cells into 96 well microtiter plates, 2 cells per well for viability, 4000 cells per well for mutagenicity (with TFT)
 Incubation for 10-12 days
 Evaluation of cytotoxicity and mutagenicity (min. 4 concentrations)

Experiment II without S9, extended exposure, two replicates per culture, min 4 concentrations (as Experiment I was negative)
 Treatment for 24 h in the absence of S9
 Remove treatment medium and test item
 Add fresh medium after washing
 Measure cell density directly after treatment as well as 24 h and 48 h after treatment
 Evaluation of toxicity and RSG in %
 Transfer of cells into 96 well microtiter plates, 2 cells per well for viability, 4000 cells per well for mutagenicity (with TFT)
 Incubation for 10-12 days
 Evaluation of cytotoxicity and mutagenicity (min. 4 concentrations)


Rationale for test conditions:
The L5178Y is a murine T-cell lymphoma cell line, which grows as single or aggregated round cells in suspension. This cell line is characterized by a high sensitivity to chemical mutagens, by a high proliferation rate (doubling time 10-12 h in stock cultures), a high cloning efficiency (CE) and a stable spontaneous mutant frequency. The L5178Y consists of a stable karyotype and shows a diploid chromosome number (40 ± 2). The cells were purchased by ATCC (Wesel, Germany) and were sold under the name L5178Y TK+/- clone (3.7.2C) [TK+/- (clone 3.7.2C)] (ATCC® CRL-9518™).
In the first pre-test relevant cytotoxic effects were determined at all concentrations following 4 h treatment with and without metabolic activation. No precipitation was observed. To determine a concentration for the main experiments, a second pre-test had to be performed. In the second pre-test no precipitation was visible at the tested concentrations. Relevant cytotoxic effects were determined at 39.1, 19.5, 9.8 and 4.9 µM following 4 h treatment with metabolic activation and at 39.1 µM following 4 h treatment without metabolic activation.
Because the results of experiment I did not give an indication for a mutagenic effect of the test item at the tested concentrations, a subsequent experiment (without metabolic activation and with longer exposition) was performed in order to verify these results.
Evaluation criteria:
Cell numbers in suspension were determined with a cell counter. Afterwards, the total suspension growth (TSG) and the relative suspension growth (RSG) were calculated. Viability - Number of empty wells per microwell plate was counted manually. All generated data of each experiment (test item, solvent controls and positive control) were recorded in the raw data. Afterwards the relative cloning efficiency (RCE) and the relative total growth (RTG) were calculated.
Mutant Colonies
Colonies were counted manually under a binocular magnifying glass. In accordance with their size, the colonies were classified into two groups:
Less than 25 % of the well’s diameter = small colony
More than 25 % of the well’s diameter = large colony
All generated data of each experiment (test item, solvent controls, positive control and medium control) were recorded in the raw data. Afterwards the mutant frequency (MF) was calculated
Key result
Species / strain:
other: mouse lymphoma L5178
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
1,8-Diaminonaphthalene is considered to be “not mutagenic under the conditions of the mouse lymphoma assay”.
Executive summary:

The study was performed to investigate the potential of the test item 1,8-Diaminonaphthalene chip to induce mutations at the thymidine kinase locus (Tk1) on chromosome 11 and/or structural chromosomal aberrations in mouse lymphoma L5178Y Tk +/-cells.  

The assay was performed in three pre-tests and five independent experiments (experimentI (two times), II, I_2 and I_3). The first pre-test was invalid. The second and the third pre-

test were performed to detect a potential cytotoxic effect of the test item. Based on the results of these pre-tests the concentrations for the experiments were determined. The first

Experiment I was invalid, furthermore three experiments I (experiment I, experiment I_2 andexperiment I_3) and one experiment II were performed. The invalid pre-test and the invalid first experiment I are not reported; all documentation is kept with the raw data and will be archived at the test facility. Experiment I (exposure date: 31. Jul. 2018) was performed with

and without metabolic activation (liver enzyme S9 fraction / “liver S9 mix from male rats, treated with Aroclor 1254”) and a treatment period of 4 h. Experiment II (exposure date: 14.

Aug. 2018) was performed with a treatment period of 24 h in the absence of metabolic activation. Experiment I_2 (exposure date: 04. Sep. 2018) and I_3 (exposure date: 09. Oct.

2018) were performed with metabolic activation and a treatment period of 4 h.

The highest nominal concentration applied was chosen with regard to the solubility of the test item in organic solvents and aqueous media, the cytotoxicity, respectively the results of

the previous experiment(s). None of the real treatment concentrations in both experiments deviated more than 10 % from the nominal concentration. Precipitation of the test item was

not visible up to the highest concentration.

In experiment I 6 concentrations of the test item were tested (see table 9-a) with and without metabolic activation and a treatment period of 4 h. Afterwards, 4 concentrations were evaluated for mutant frequency. A cytotoxic effect only was observed with metabolic activation at the concentration 4.9 µM. There is an increase of the mutation frequency in + and – S9 in the highest test item concentration, but did not reach or exceed the threshold of 126 above the corresponding solvent control. To establish the biological relevance of the negative result without metabolic activation, experiment II was performed.  

In experiment II (for deviations see chapter 10, page 32) 6 concentrations of the test item were tested without metabolic activation and a treatment period of 24 h. Afterwards, 4 concentrations were evaluated for mutant frequency. The mutation frequency did not reach or exceed the threshold of 126 above the corresponding solvent control.

For the determination of the biological relevance of the results in the first experiment I with metabolic activation, an additional experiment I (only +S9) was performed using more

closely spaced test item concentrations to get more detailed information about the potential mutagenic effects of the test item in the concentration area between 1.4 – 4.9 µM.  

In experiment I_2 8 concentrations of the test item were tested with metabolic activation and a treatment period of 4 h. No concentration could be evaluated because of cytotoxicity.

Therefor another experiment I was performed.

In experiment I_3 9 concentrations of the test item were tested with metabolic activation and a treatment period of 4 h, 5 concentrations were evaluated for mutant frequency. In all evaluated concentrations of the test item no substantial and reproducible dose dependent increase in mutant colony numbers was observed. No relevant shift of the ratio of small versus large colonies was observed up to the maximal concentration of the test item. The mutation frequency did not reach or exceed the threshold of 126 above the corresponding solvent control.

MMS (19.5 µg/mL for all experiments I and 12.5 µg/mL for experiment II) and CPA monohydrate (5.5 µg/mL) were used as positive controls and showed a distinct increase in induced total mutant colonies and exceeded the number of mutant colonies of more than 300 in comparison to the corresponding solvent control.  

Since no historical control data are available for acetone, an additional untreated control (medium control) was tested in parallel to demonstrate the lack of genotoxicity of Acetone

at the concentration used.

In all experiments of this study (experiment I*, II, I_2* and I_3) the solvent controls were in the range of the historical data as well as in the range of the spontaneous mutant frequency of the L5178Y cells or the historical control data. The Mutant Frequency of Acetone in relation to the untreated control (medium control) was calculated.

Acetone didn´t show any genotoxicity at the concentration used, Since all further acceptability criteria of the assay were also met the study is valid

In conclusion, it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the thymidine kinase locus (Tk1) in heterozygous mouse lymphoma L5178Y Tk+/- cells.

Therefore, the test item 1,8-Diaminonaphthalene chip is considered to be “not mutagenic under the conditions of the mouse lymphoma assay”.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: scientifically acceptable and well documented - but only 4 strains tested
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
1,8-naphthylenediamine was investigated using the Salmonella/microsome test (Ames Test) for point mutagenic effects in doses up to 12500 µg per plate on four Salmonella typhimurium LT2 mutants. These comprised the histidine-auxotrophic strains TA 1535, TA 100, TA 1537 and TA 98.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: all strains are partly deficient in lipopolysaccharide side chains
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix
Test concentrations with justification for top dose:
first test: 0, 20, 100, 500, 2500, 12500 µg/plate
repeat test: 0, 0,8, 4, 20, 100, 500 µg/plate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: endoxan, trypaflavin, 2-aminoanthracen
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 500 µg with and without S-9 mix
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Remarks:
Migrated from field 'Test system'.

The test substance revealed a mutagenic effect on S. typhimurium strains TA 98, TA100 and TA1537. The lowest reproducible and effective doses for S. typhimurium TA 98, TA 100. and TA 1537 were 20 µg/plate.

1,8 -Naphthylenediamine is positive under the conditions of this Ames test with and without metabolic activation.

Conclusions:
Interpretation of results:
positive
Executive summary:

1,8-Naphthylenediamine was investigated using the Salmonella/microsome test (Ames Test) for point mutagenic effects in doses up to 12500 µg per plate on four Salmonella typhimurium LT2 mutants. These comprised the histidine-auxotrophic strains TA 1535, TA 100, TA 1537 and TA 98.

The test substance revealed a mutagenic effect on S. typhimurium strains TA 98, TA100 and TA1537. The lowest reproducible and effective doses for S. typhimurium TA 98, TA 100. and TA 1537 were 20 µg/plate.

1,8 -Naphthylenediamine is positive under the conditions of this Ames test with and without metabolic activation.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Additional information

Additional information from genetic toxicity in vitro:
The results from the OECD 487 (human lymphocytes) test were equivocable.

The results from the OECD 490 (gene mutation) were negative

In the Ames tests, the SOS Chromotest and the QSAR calculation 1,8-naphthylenediamine are positive


Justification for selection of genetic toxicity endpoint
key study used

Justification for classification or non-classification

The compound is positive in bacterial reverse mutation assays, negative in in vitro gene mutation and equivocable in human lymphocytes