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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro gene mutation in bacteria (OECD 471): positive


In vitro chromosomal aberration (OECD 473): positive


In vitro gene mutation in mammalian cells (OECD 476): negative

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In vivo micronucleus test (OECD 474): negative


COMET assay: negative

Additional information

In vitro tests


Ames test (OECD 471)


The test item was tested for mutagenic effects in vitro in histidine-requiring strains of Salmonella typhimurium
and in a tryptophan-requiring strain of Escherichia coli, according to the OECD guideline 471. The following strains were used: S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2 uvrA.
The test was performed with and without the addition of rat-liver post mitochondrial supernatant (S9 fraction) as an extrinsic metabolic activation system. Each test item concentration, the negative and positive and controls were tested in triplicates. The test item was dissolved in bidistilled water and tested at five concentrations: 312.5, 625, 1250, 2500 and 5000 µg/plate.
ln order to confirm the results, the experiment was repeated with and without metabolic activation at the same concentrations used in the first experiment. The test with metabolic activation was carried out as pre-incubation assay.
Since a weak increase in the number of revertant colonies was observed in strain WP2 uvrA in the experiment with metabolic activation (pre-incubation assay), the experiment with this strain was performed in addition with the same concentrations tested before.
Each strain was additionally tested in the presence and in the absence of a metabolic activation system with a suitable, known mutagen as positive control.
Previously, a pre-experiment for toxicity (range finding test) was carried out with strains S. typhimurium TA 100 and E. coli WP2 uvrA to determine the highest concentration to be used in the mutagenicity assay. The experiment was performed with and without metabolic activation with the concentrations of 20.6, 61 .7, 185.2, 555.6, 1666.7 and 5000 µg/plate.
Normal background growth was observed with both strains. The number of revertant colonies was not reduced at any concentrations tested. The test item did not precipitate on the surface of the agar plates.
From the results obtained, the highest concentration suitable for the first mutagenicity test was selected to be 5000.0 µg/plate with and without metabolic activation.
ln the first mutagenicity test performed with and without metabolic activation, no substantial increase in the number of revertant colonies was observed after treatment with Navy MGi 1571 at any concentrations.
ln the second mutagenicity test carried out with metabolic activation, treatment of strain E. coli WP2 uvrA with the test item led to an increase in the number of revertant colonies at the concentrations of 1250 and 5000 µg/plate. No similar effects were observed with strains of S. typhimurium.
ln the supplement experiment carried out as pre-incubation assay with metabolic activation with E. coli WP2 uvrA again an increase in the number of revertant colonies occurred at the concentrations of 625 to 5000 µg/plate.
ln case of the increased number of revertant colonies reached the threshold for a positive response colonies from one plate of each respective concentration group were tested on selective agar plates for their mutant properties, in order to avoid false positive or false negative results.


Toxic effects, evident as a reduction in the number of revertants, were visible in the first experiment with activation in strain TA 1535 at the concentrations of 2500 and 5000 µg/plate and in strain TA 1537 at the concentration of 5000 µg/plate. ln the experiment without activation in strain TA 1537 a reduction in the number of reveftants occurred at the concentrations of 2500 and 5000 µg/plate.
ln both mutagenicity tests normal background growth was observed with all strains at all concentrations. The test item did not precipitate on the surface of the agar plates.
ln the experiments negative (solvent) and positive control treatments were included for all strains. The mean numbers of revertant colonies on negative control plates were found to be within acceptable ranges. The positive controls induced appropriate increases in the number of revertant colonies in all experiments, thus demonstrating the correct strain functioning and the activity of the S9-mix.


ln conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item induced gene mutations by base-pair changes and others in the genome of strain E. coliWP2 uvrA.
Therefore, Navy MGi 1571 is considered to be mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.


Chromosomal aberration test (OECD 473)


The test item Navy MGi 1571, dissolved in deionised water, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro according to the OECD guideline 473.


ln each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations.
Dose selection of the cytogenetic experiments was performed considering the toxicity data. 
Toxic effects indícated by reduced cell numbers of below 50 % of control were observed in the experimental part with S9 mix. However, in the absence of S9 mix the highest applied concentration showing strong toxic effects was not evaluable for cytogenetic damage.
ln the presence of S9 mix, statistically significant and biologically relevant increases in the number of cells carrying structural chromosomal aberrations were observed after treatment with the test item with 937.5 and 1250 µg/ml. At higher concentrations the aberration rate was withín the historical control data range (0.0 - 4.0 %). lt have to be considered that the cytogenetic damage at this stage led to cell lethality.
No increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls.
Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.
ln conclusion, it can be stated that under the experimental conditions reported, the test item induced structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro.
Therefore, Navy MGi 1571 is considered to be clastogenic in this chromosome aberration test in the presence of S9 mix.


In vitro gene mutation in mammalian cells (OECD 476)


The study was performed to investigate the potential of Navy MGi 1571 to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster.
The treatment time was 4 h in the fìrst experiment with and without metabolic activation. ln the second experiment the cells were exposed to the test item for 24 h without metabolic activation. The first experiment with metabolic activation was repeated (experiment lb) since the positive control showed no response in the second culture.
The cell cultures were evaluated at the following concentrations:


Experiment l: 


without 39 mix: 75; 150;300;600;900; and 1200 µg/mL
with 59 mix: 75; 150; 300;450; and 600 µg/mL

Experiment lb
with 59 mix: 75; 150;300;600; and 1200 µg/mL


Experiment ll:
without 59 mix: 150; 300;600; 900; and 1200 µg/mL


No precipitation visible to the unaided eye occurred in the pre-experiment or in the main experiments up to the maximum concentration.
The concentration range of the main experiments was selected according to data on toxicity generated in the range fìnding pre-experiment. The data on toxicity generated in the first experiment with metabolic activation were considered in the dose selection of the repeat experiment.
Up to the highest investigated concentration no relevant increase of the mutation frequency was observed in both independent experiments.
Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies.
ln conclusion it can be stated that under the experimental conditions reported the test substance did not induce gene mutations at the HPRT locus in V79 cells. Therefore, Navy MGi 1571 is considered to be non-mutagenic in this HPRT assay.


In vivo genetic toxicity


In vivo micronucleus test (OECD 474)


This study was performed to investigate the potential of Navy MG¡ 1571 to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse.
The test item was formulated in deionised water, which was also used as vehicle control. The volume administered orally was 10 ml/kg b.w.. 24 h and 48 h after a single administration of the test item the bone marrow cells were collected for micronuclei analysis.
Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. At least 2000 polychromatic erythrocytes (PCEs) per animal were scored for micronuclei.
To describe a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and total erythrocytes was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes.
The following dose levels of the test item were investigated: 
24 h preparation interval: 500, 1000, and 2000 mg/kg b.w
48 h preparation interval: 2000 mg/kg b.w.
The highest dose (2000 mg/kg; maximum guideline-recommended dose) was estimated by a pre-experiment to be suitable.
After treatment with the test item the number of PCEs was not substantially decreased as compared to the mean value of PCEs of the vehicle control thus indicating that Navy MGi 1571 did not exert any cytotoxic effects in the bone marrow. However, the urine of the treated animals was blue, indicating the systemic distribution of the test item and thus its bioavailability.
ln comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in tne frequency of the detected micronuclei at any preparation inierval after administration of the test item and with any dose level used.
40 mg/kg b.w. cyclophosphamide administered orally was used as positive control which showed a substantial increase of induced micronucleus frequency.
ln conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse
Therefore, Navy MGi 1571 is considered to be non-mutagenic in this micronucleus assay.


COMET assay


The test item was assessed in the In vivo alkaline single cell gel electrophoresis analysis for its potential to induce primary DNA damage in cells prepared from the liver, urinary bladder and small intestine of treated Wistar HsdCpb: WU (SPF)rats.


The test item was formulated in sterile water. Seven male rats per group were treated twice with a single oral dose of 1000 or 2000 mg /kg body weight (b.w.) 24 or 4 hours prior to preparation of the cells. The administration volume was 10 mL/kg body weight for the test item and vehicle control. After the end of the treatment period the animals were anaesthetised and sacrificed. Primary hepatocytes were isolated by liver perfusion, cells of the urinary bladder via enzymatic digestion and cells from the small intestine were isolated by mincing the tissue.


Dose selection and number of evaluated doses was based on the publication of Tice et al. (2000). At the highest dose clinical signs of toxicity such as ruffled fur and reduced spontaneous activity were observed.


The viability of the prepared hepatocytes or isolated cells of the urinary bladder were not affected by the in vivo treatment with the substance as determined by trypan blue dye exclusion.


The number ofnuclei from apoptotic or necrotic cells per 500 total nuclei was determined for each sample to indicate the quality of the slide preparation. For cells from the liver and small intestine theamountof dead cells was negligible, whereas for cells from the urinary bladder thepercentageof dead cells per group ranged between 31.4 and 53.6 % indicating high cytotoxicity. This might be due to slide preparation.


In each experimental group including the controls, hepatocytes, cells of the urinary bladder and small intestine from 7 males were assessed for the occurrence of DNA damage.For each dose group and at each treatment time 100 cells were evaluated for primary DNA damage. None of the tested dose levels revealed a biologically relevant or statistically significant increase in DNA damage in the hepatocytes or the cells from the small intestine in the treated animals as compared to thecorresponding vehicle controls in the evaluated parameter (% Tail Intensity). In addition, all % Tail Intensities of the treated animals were within the historical control data range for the hepatocytes (except animal no. 12) and even below the historical control range for the small intestine.


For cells from the urinary bladder the mean % Tail Intensity of all vehicle treated animals was 2.5 times higher than the upper limit of the laboratory’s historical control data. That, together with the high amount of dead cells (mean of 31.4%) the data for the urinary bladder do not meet the acceptance criteria and are, therefore, inconclusive. Considering the clear negative results for the liver and the small intestine and a high statistically significant increase in DNA damage in the positive control what cannot be seen for any tested dose of the test item it is very unlikely that the test item is genotoxic in the bladder, but under the mentioned circumstances a clear statement cannot be made.


For liver and small intestine the vehicle control was in the range to ensure a valid performance of the study. An appropriate reference mutagen [MMS, 25 mg/kg b.w. oral] was used as a positive control. Treatment with the positive control substance led to a distinct and statistically significant increase of DNA damage as detected by % Tail Intensity analysis.

Justification for classification or non-classification

The genetic toxicity of Navy MGi 1571 was investogated by in vitro and in vivo studies.


Positive results were obtained in the Ames test (OECD 471) and in the chromosomal aberration test (OECD 473) while no effects were observed in the HPRT study (OECD 476).


According to the ECHA guidance r.7a, Table R.7.7–5, in case positive results are obtained in the Ames test and in the chromosomal aberration study, both genotoxic endpoints should be further investigated through in vivo tests. The in vivo alkaline single cell gel electrophoresis assay for DNA strand breaks (comet assay), recognises primary DNA damage that would lead to gene mutations and/or chromosome aberrations, therefore would be sufficient to investigate both genotoxic end-points. In the available COMET assay, carried out with Navy MGi 1571 no signs of DNA-damage was detected in liver cells and cells from the small intestine. Cells of the urinary bladder were also investigated. Due to a high number of dead cells, the results for the urinary bladder are inconclusive. Navy MGi 1571 is therefore considered to be non-genotoxic in this in vivo alkaline assay.
An in vivo micronucleus test (OECD 474) is also available and under test consitions the substance did not induce micronuclei in bone marrow cells of the mouse.


Considering the overall results of the tests performed, the test substance is not classified as mutagenic according to the CLP Regulation n.1272/2008.