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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a key Bacterial Reverse Mutation Assay according to OECD guideline 471, the results indicate that the test item, under the experimental conditions described, was not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and E.coli strain WP2 uvr A in the presence and absence of a metabolic activation.


 


In a key in vitro Mammalian Cell Micronucleus Test according to OECD guideline 487, the test item did not induce micronuclei in cultured human peripheral blood lymphocytes following treatment in the absence and presence of a rat liver metabolic activation system (S-9), when tested up to cytotoxic concentrations.


 


According to a valid QSAR prediction using DEREK Nexus v. 6.2, no mutagenic properties of the test item were estimated. 


 


Overall conclusion: The test substance is neither mutagenic nor clastogenic in two in vitro genotoxicity tests and a QSAR prediction.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Justification for type of information:
Please refer to the QMRF and QPRF files provided under the section attached justification.
Qualifier:
no guideline available
Principles of method if other than guideline:
Estimates the mutagenic properties of chemicals using structural alert relationships.
Specific details on test material used for the study:
SMILES: O=C(C=Cc2ccc1OCOc1c2)N(c3ccccc3)c4ccccc4
Key result
Species / strain:
other: no alerts
Remarks on result:
no mutagenic potential (based on QSAR/QSPR prediction)
Conclusions:
Using Derek Nexus 6.2.0, no mutagenic properties of the test item were estimated.
Executive summary:

Mutagenicity was estimated using Derek Nexus 6.2.0. No mutagenic properties were estimated based on the described QSAR method (Derek, 2017).


 


The adequacy of a prediction depends on the following conditions:


a) the (Q)SAR model is scientifically valid: the scientific validity is established according to the OECD principles for (Q)SAR validation;


b) the (Q)SAR model is applicable to the query chemical


c) the (Q)SAR result is reliable


d) the (Q)SAR model is relevant for the regulatory purpose.


 


For assessment and justification of these 4 requirements the QMRF and QPRF files were developed and attached to this study record.


 


Description of the prediction Model


The prediction model was described using the harmonised template for summarising and reporting key information on (Q)SAR models. For more details please refer to the attached QSAR Model Reporting Format (QMRF) file. 


 


Assessment of estimation domain


The assessment of the estimation domain was documented in the QSAR Prediction Reporting Format file (QPRF). Please refer to the attached document for the details of the prediction and the assessment of the estimation domain.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-08-22 to 2014-02-14
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)
Version / remarks:
2010
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
lymphocytes: human peripheral blood lymphocytes
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: Human peripheral blood lymphocytes from two healthy, non-smoking female volunteers
- Suitability of cells: Yes, blood was received from donors who were not suspected of any virus infection or exposed to high levels of radiation or hazardous chemicals. All donors were non smokers and not heavy drinkers of alcohol. Donors were not taking any form of medication (contraceptive pill excluded).
- Normal cell cycle time: The measured cell cycle time of the donors falls within the range of 13±2 hours.

For lymphocytes:
- Sex, age and number of blood donors: Blood from 2 female donors was used in the range-finding and main study. The age of the donors was 28 and 31 in the range-finding study and 30 and 31 in the main experiment.
- Whether whole blood or separated lymphocytes were used: Whole blood cultures were used
- Whether blood from different donors were pooled or not: Blood was pooled using equal volumes from each donor prior to use.
- Mitogen used for lymphocytes: Phytohaemagglutinin (PHA, reagent grade)

MEDIA USED
- Type and composition of media: HEPES-buffered RPMI medium containing 10% (v/v) heat inactivated foetal calf serum and 0.52% penicillin / streptomycin
- Temperature: 37±1°C

Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- Source of S9: Obtained from Molecular Toxicology Incorporated, USA where it is prepared from male Sprague Dawley rats induced with Aroclor 1254.
- Method of preparation of S9 mix: Glucose-6-phosphate (180 mg/mL), β-Nicotinamide adenine dinucleotide phosphate (25 mg/mL), Potassium chloride (150 mM) and rat liver S-9 were mixed in the ratio 1:1:1:2.
- Concentration or volume of S9 mix and S9 in the final culture medium: The final concentration of the S9 mix in the test system was 5%.The final concentration of S9 in the test system was 2%.
- Quality controls of S9: Each batch was checked by the manufacturer for sterility, protein content, ability to convert known promutagens to bacterial mutagens and cytochrome P-450-catalyzed enzyme activities (alkoxyresorufin-O-dealkylase activities) as specified in the Certificate of Analysis.
Test concentrations with justification for top dose:
Range-finding study: 1.814 to 500 µg/mL (3-hour treatment +/- S9 and 20-hour treatment -S9);

Main study: 5 to 100 µg/mL (3-hour treatment -S9), 10 to 100 µg/mL (3-hour treatment +S9) and 2 to 40 µg/mL (20-hour treatment -S9)

Concentrations that were evaluated for micronuclei were: 10, 30 and 60 µg/mL (3-hour treatment -S9), 10, 30, 55 and 60 µg/mL (3-hour treatment +S9) and 8, 14 and 20 µg/mL (20-hour treatment -S9)
Vehicle / solvent:
- Vehicles used: DMSO (test substance, CPA), purified water (VIN, MMC)

- Justification for choice of solvent/vehicle: Solubility properties. DMSO is a commonly used solvent for this type of assay with a broad historical control database.

- Justification for percentage of solvent in the final culture medium: The maximum solvent concentration in the final culture medium was 1% which is known from historical control data to be not toxic and not mutagenic in this type of assay.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO (test substance, CPA), purified water (VIN, MMC)
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
vinblastine
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: For the range-finding study: Single (test substance) and duplicate (solvent control); for the main experiment: Duplicate (test substance and positive control) and quadruplicate (solvent control)
- Number of independent experiments: 1 range-finding assay and 1 main assay (each consisting of 3 tested exposure conditions)

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in: Medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 3 hours (+/- S9) and 20 hours (- S9)
- Harvest time after the end of treatment (sampling/recovery times): 24 hours for all test conditions. For the 3-hour treatments, cytochalasin B was added after the 3-hour treatment period and incubated for further 21 hours. For the 20-hour treatment, cytochalasin B was added at the beginning od the treatment period and incubated, together with the test substance for 24 hours.

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Identity of cytokinesis blocking substance, concentration, and duration and period of cell exposure: Cytochalasin B was added (0.1 mL per culture) to the 24-hour treatment culture at the time of treatment. Cytochalasin B was added (0.1 mL per culture) to the 3-hour treatment cultures directly after the treatment period. The final concentration was 6 μg/mL per culture.
- Methods of slide preparation and staining technique used including the stain used: Fixed lymphocytes were centrifuged and resuspended in a minimal amount of fresh fixative. Several drops of cell suspension were gently spread onto multiple clean, dry microscope slides. Slides were air-dried then stored protected from light at room temperature prior to staining. Slides were stained by immersion in 125 μg/mL Acridine Orange in phosphate buffered saline (PBS), pH 6.8 for approximately 10 seconds, washed with PBS (with agitation) for a few seconds before transfer and immersion in a second container of PBS for approx. 10 minutes. Slides were air-dried and stored protected from light at room temperature prior to analysis.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): Slides from the cytotoxicity range-finder experiment were examined for
proportions of mono-, bi- and multinucleate cells, to a minimum of 200 cells per concentration. In the main experiment, slides were examined for RI to a minimum of 500 cells per culture.1000 binucleate cells from each culture (2000 per concentration) were analysed for micronuclei.
- Criteria for scoring micronucleated cells: See "Any other information on materials and methods incl. tables"

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Replication index

METHODS FOR MEASUREMENTS OF GENOTOXICIY
The proportion of binucleate cells with micronuclei (MNBN) as compared to the proportion in vehicle controls
Rationale for test conditions:
Test concentrations were chosen based on a dose-range finding test.

3-hour treatment (-S9): Precipitation occured at concentration of 38.88 µg/mL and above. At this concentration, 51% cytotoxicity was reached.

3-hour treatment (+S9): Precipitation occured at concentration of 38.88 µg/mL and above. At this concentration, 41% cytotoxicity was reached. At 64.80 µg/mL, 57% cytotoxicity was reached.

24-hour treatment (-S9): Marked cytotoxicity occured at concentrations of 8.398 µg/mL (36%), 14 µg/mL (79%) and 23.33 µg/mL (100%). Precipitation occured at concentrations of 38.88 µg/mL and above.

On this basis, test concentrations for the main assay were selected. The highest concentration selected for micronucleus analysis following all treatment conditions induced 55±5% cytotoxicity. Slides from the highest selected concentration and two or three lower concentrations were taken for microscopic analysis, such that a range of cytotoxicity from maximum to little was covered.
Evaluation criteria:
For valid data, the test article was considered to induce clastogenic and/or aneugenic events if:
1. A statistically significant increase in the frequency of MNBN cells at one or more concentrations was observed.
2. An incidence of MNBN cells at such a concentration that exceeded the normal range in both replicates was observed.
3. A concentration-related increase in the proportion of MNBN cells was observed. The test article was considered positive in this assay if all of the above criteria were met.

The test article was considered negative in this assay if none of the above criteria were met.

Results which only partially satisfied the above criteria were dealt with on a case-by-case basis. Evidence of a concentration-related effect was considered useful but not essential in the evaluation of a positive result
Statistics:
The proportions of MNBN cells in each replicate were used to establish acceptable heterogeneity between replicates by means of a binomial dispersion test.

The proportion of MNBN cells for each treatment condition was compared with the proportion in vehicle controls by using Fisher's exact test.

Probability values of p≤0.05 were accepted as significant. Additionally, the number of micronuclei per binucleate cell were obtained and recorded.
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
The limit cytotoxicity of 55±5% was reached at 60 µg/mL (3-hour treatment with and without S9) and at 20 µg/mL (24-hour treatment without S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: No marked changes in pH were observed at the highest concentration tested (500.0 μg/mL) in the Range Finder Experiment as compared to the concurrent vehicle controls.
- Data on osmolality: No marked changes in osmolality were observed at the highest concentration tested (500.0 μg/mL) in the Range Finder Experiment as compared to the concurrent vehicle controls (individual data not reported).
- Water solubility: The test item was soluble in DMSO up to a concentration of 175.0 mg/mL.
- Precipitation and time of the determination: Precipitation occured at the beginning of treatment from 38.88 µg/L onwards under all test conditions. Precipitation occured at the end of tretment from 108 and 180 µg/mL onwards for the 3-hour treatment times (-/+S9). Precipitation occured at harvest from 64.80 µg/mL (3-hours, -S9), from 108 µg/mL (3-hours, +S9) and from 38.88 µg/mL (24-hours, -S9) onwards under all test conditions.
- Definition of acceptable cells for analysis: See "Any other information on materials and methods incl. tables"

RANGE-FINDING/SCREENING STUDIES: Yes, cytotoxicity was tested in a range-finding study at concentrations between 1.814 to 500 µg/mL in duplicate (vehicle control) or single cultures (test item) under all three incubation conditions (3-hour treatment with and without S9 and 24-hour treatment without S9). Dose-dependent cytotoxic effects were observed in all cultures (see "Rationale for test conditions"). On this basis, test concentrations for the main experiment were selected.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: See "Attached background material"

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship: No concentration-response relationship was observed.
- Statistical analysis: No statistical significance was observed for any of the the test substance concentrations and negative controls. Positive control yielded the expected statistically significant responses.

Micronucleus test in mammalian cells:
- Results from cytotoxicity measurements: See "Attached background material"
o In the case of the cytokinesis-block method: CBPI or RI; distribution of mono-, bi- and multi-nucleated cells: See "Attached background material"

- Genotoxicity results
o Number of cells with micronuclei separately for each treated and control culture and defining whether from binucleated or mononucleated cells, where appropriate: See "Attached background material"

HISTORICAL CONTROL DATA
- Positive historical control data: See "Attached background material"
- Negative (solvent/vehicle) historical control data: See "Attached background material"
Conclusions:
In an in vitro Mammalian Cell Micronucleus Test according to OECD guideline 487, the test item did not induce micronuclei in cultured human peripheral blood lymphocytes following treatment in the absence and presence of a rat liver metabolic activation system (S-9), when tested up to cytotoxic concentrations.
Executive summary:

The test item was tested in an in vitro micronucleus assay according to OECD guideline 487 and GLP using duplicate human lymphocyte cultures prepared from the pooled blood of two female donors in a single experiment. Treatments covering a broad range of concentrations, separated by narrow intervals, were performed both in the absence and presence of metabolic activation (S-9) from Aroclor 1254-induced rats. The test article was formulated in anhydrous analytical grade dimethyl sulphoxide (DMSO) and the highest concentrations used in the Micronucleus Experiment, were determined following a preliminary cytotoxicity Range-Finder Experiment. Treatments were conducted (as detailed in the following summary table) 48 hours following mitogen stimulation by phytohaemagglutinin (PHA). The test article concentrations for micronucleus analysis were selected by evaluating the effect of the test item on the replication index (RI). Micronuclei were analysed at three or four concentrations and a summary of the data is presented in the following table:


 

































































































































Treatment



Concentration


(µg/mL)



Cytotoxicity


(%)



Mean


MNBN Cell


Frequency


(%)



Historical


Control


Range (%)



Statistical


Significance



3+21 hour -S9



Vehicle



-



0.80



0.10 – 1.10



-



10.00



11



1.05



 



NS



30.00



27



0.80



 



NS



60.00



55



0.65



 



NS



MMC, 0.80



ND



7.35



 



p ≤0.001



3+21 hour +S9



Vehicle



-



0.80



0.20 – 1.20



-



10.00



7



0.90



 



NS



30.00



27



0.85



 



NS



55.00



57



0.40



 



NS



60.00



50



0.45



 



NS



CPA, 6.25



ND



2.90



 



p ≤0.001



24+0 hour -S9



Vehicle



-



1.10



0.10 – 1.50



-



8.00



10



1.25



 



NS



14.00



24



1.00



 



NS



20.00



55



1.05



 



NS



VIN, 0.02



ND



16.92



 



p ≤0.001



 


Appropriate negative (vehicle) control cultures were included in the test system under each treatment condition. The proportion of micronucleated binucleate (MNBN) cells in the vehicle cultures fell within, or very close to, the 95th percentile of the current observed historical vehicle control (normal) ranges. Mitomycin C (MMC) and Vinblastine (VIN) were employed as clastogenic and aneugenic positive control chemicals, respectively, in the absence of rat liver S-9. Cyclophosphamide (CPA) was employed as a clastogenic positive control chemical in the presence of rat liver S-9.


 


Cells receiving these were sampled in the Micronucleus Experiment at 24 hours after the start of treatment; all compounds induced statistically significant increases in the proportion of cells with micronuclei. All acceptance criteria were considered met and the study was therefore accepted as valid. Treatment of cells with the test item in the absence and presence of S-9 resulted in frequencies of MNBN cells that were generally similar to and not significantly higher than those observed in concurrent vehicle controls for all concentrations analysed under all three treatment conditions. The MNBN cell frequency of all treated cultures fell within normal ranges with the exception of a single culture at 10 μg/mL, the lowest concentration analysed following 3+21 hours treatment in the presence of S-9. This isolated marginal increase of 1.3% compared to the normal range of 0.2-1.2% was not reproduced in the replicate culture at 10  g/mL or at any other concentration tested. In addition, as the mean MNBN cell frequency fell within the normal range and was not statistically significantly higher than the concurrent vehicle controls, it was considered of no biological relevance.


 


It is concluded that the test item did not induce micronuclei in cultured human peripheral blood lymphocytes following treatment in the absence and presence of a rat liver metabolic activation system (S-9), when tested up to cytotoxic concentrations.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-08-18 to 2009-09-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP guideline study - expiration date missing
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted 1997-07-21
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
signed 2005-10-21
Type of assay:
bacterial reverse mutation assay
Target gene:
TA1537: his C 3076
TA98: his D 3052
TA1535 & TA100: his G 46
E. coli WP2 uvrA: trp-
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction prepared from livers of male Wistar rats orally receiving phenobarbitone/β-naphthoflavone on three consecutive days
Test concentrations with justification for top dose:
Standard plate test (experiment 1): 0, 22, 110, 550, 2750 and 5500 μg/plate (with and without metabolic activation)
Preincubation test (experiment 2): 0, 22, 110, 550, 2750, and 5500 μg/plate (with and without metabolic activation)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: due to the limited solubility of the test item in water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
Positive control with metabolic activation: concentrations: 2.5 μg/plate (strains TA 1535, TA 100, TA 1537, & TA 98) & 60 μg/plate (strain E. coli WP2 uvrA)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-methyl-N'-nitro-N-nitrosoguanidine
Remarks:
Positive control without metabolic activation: concentration: 5 μg/plate (strains TA 1535 & TA 100)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylenediamine
Remarks:
Positive control without metabolic activation: concentration: 10 μg/plate (strain TA 98)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Positive control without metabolic activation: concentration: 100 μg/plate (strain TA 1537)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Positive control without metabolic activation: concentration: 5 μg/plate (strain E. coli WP2 uvrA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: Experiment 1: in agar (plate incorporation); Experiment 2: preincubation

DURATION
- Preincubation period (experiment 2): about 20 minutes
- Exposure duration (experiments 1 & 2): 48 -72 hours at 37 °C

NUMBER OF REPLICATIONS (experiments 1 & 2): 3

DETERMINATION OF CYTOTOXICITY
Toxicity detected by a
- decrease in the number of revertants
- clearing or diminution of the background lawn (= reduced his- or trp- background growth)
- reduction in the titer
is recorded for all test groups both with and without S9 mix in all experiments.

Precipitation of the test material is recorded.
Evaluation criteria:
The test chemical is considered positive in this assay if the following criteria are met:
- a dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.

A test item is generally considered non-mutagenic in this test if:
- the number of revertants for all tester strains were within the historical negative control range under all experimental conditions in at least two experiments carried out independently of each other.
Statistics:
no data
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
please refer to the field "Additional information on results" below
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
please refer to the field "Additional information on results" below
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
please refer to the field "Additional information on results" below
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
please refer to the field "Additional information on results" below
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
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:
cytotoxicity
Remarks:
please refer to the field "Additional information on results" below
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
STANDARD PLATE TEST (experiment 1):
No increase in the number of his+ or trp+ revertants was observed with and without metabolic activation.

PREINCUBATION TEST (experiment 2):
No increase in the number of his+ or trp+ revertants was observed with and without metabolic activation.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A weak bacteriotoxic effect (slight decrease in the number of his+ revertants) was occasionally observed in the standard plate test (experiment 1) depending on the strain and test conditions at the highest applied concentration (5500 μg/plate).
In the preincubation assay (experiment 2) bacteriotoxicity (slight decrease in the number of his+ revertants, slight reduction in the titer) was occasionally observed depending on the strain and test conditions from about 2750 μg/plate onward.

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: test item precipitation was found from 110 μg/plate onward with and without S9 mix.

Please also refer to the field "Attached background material" below.
Conclusions:
In a Bacterial Reverse Mutation Assay according to OECD guideline 471, the results indicate that the test item, under the experimental conditions described, was not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and E.coli strain WP2 uvr A in the presence and absence of a metabolic activation.
Executive summary:

A bacterial reverse mutation assay was performed with the substance according to the OECD guideline 471 (1997) using S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 as well as E. coli WP2 uvr A. The study was carried out using the standard plate test (experiment 1) and the preincubation test (experiment 2). Both experiments were conducted with and without metabolic activation and each concentration was plated in triplicate. The substance was tested at the following concentrations in the both experiments: 22, 110, 550, 2750, and 5500 µg/plate. Additionally, a vehicle control and several positive controls were run concurrently.


In both experiments no increase in the number of his+ or trp+ revertants was observed with and without metabolic activation.


A weak bacteriotoxic effect (slight decrease in the number of his+ revertants) was occasionally observed in the standard plate test depending on the strain and test conditions at the highest applied concentration (5500 μg/plate). In the preincubation assay bacteriotoxicity (slight decrease in the number of his+ revertants, slight reduction in the titer) was occasionally observed depending on the strain and test conditions from about 2750 μg/plate onward. Test item precipitation was found from 110 µg/plate onward with and without metabolic activation.


The results of the negative as well as the positive controls performed corroborated the validity of this study, since the values fulfilled the acceptance criteria of this study.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Key, Bacterial Reverse Mutation Assay, RL1


A bacterial reverse mutation assay was performed with the substance according to the OECD guideline 471 (1997) and GLP using S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 as well as E. coli WP2 uvr A. The study was carried out using the standard plate test (experiment 1) and the preincubation test (experiment 2). Both experiments were conducted with and without metabolic activation and each concentration was plated in triplicate. The substance was tested at the following concentrations in the both experiments: 22, 110, 550, 2750, and 5500 µg/plate. Additionally, a vehicle control and several positive controls were run concurrently. In both experiments no increase in the number of his+ or trp+ revertants was observed with and without metabolic activation. A weak bacteriotoxic effect (slight decrease in the number of his+ revertants) was occasionally observed in the standard plate test depending on the strain and test conditions at the highest applied concentration (5500 μg/plate). In the preincubation assay bacteriotoxicity (slight decrease in the number of his+ revertants, slight reduction in the titer) was occasionally observed depending on the strain and test conditions from about 2750 μg/plate onward. Test item precipitation was found from 110 µg/plate onward with and without metabolic activation. The results of the negative as well as the positive controls performed corroborated the validity of this study, since the values fulfilled the acceptance criteria of this study.


 


Key, In Vitro Mammalian Cell Micronucleus Test, RL1


The test item was tested in an in vitro micronucleus assay according to OECD guideline 487 and GLP using duplicate human lymphocyte cultures prepared from the pooled blood of two female donors in a single experiment. Treatments covering a broad range of concentrations, separated by narrow intervals, were performed both in the absence and presence of metabolic activation (S-9) from Aroclor 1254-induced rats. The test article was formulated in anhydrous analytical grade dimethyl sulphoxide (DMSO) and the highest concentrations used in the Micronucleus Experiment, were determined following a preliminary cytotoxicity Range-Finder Experiment. Treatments were conducted (as detailed in the following summary table) 48 hours following mitogen stimulation by phytohaemagglutinin (PHA). The test article concentrations for micronucleus analysis were selected by evaluating the effect of the test item on the replication index (RI). Micronuclei were analysed at three or four concentrations and a summary of the data is presented in the following table:


 

































































































































Treatment



Concentration


(µg/mL)



Cytotoxicity


(%)



Mean


MNBN Cell


Frequency


(%)



Historical


Control


Range (%)



Statistical


Significance



3+21 hour -S9



Vehicle



-



0.80



0.10 – 1.10



-



10.00



11



1.05



 



NS



30.00



27



0.80



 



NS



60.00



55



0.65



 



NS



MMC, 0.80



ND



7.35



 



p ≤0.001



3+21 hour +S9



Vehicle



-



0.80



0.20 – 1.20



-



10.00



7



0.90



 



NS



30.00



27



0.85



 



NS



55.00



57



0.40



 



NS



60.00



50



0.45



 



NS



CPA, 6.25



ND



2.90



 



p ≤0.001



24+0 hour -S9



Vehicle



-



1.10



0.10 – 1.50



-



8.00



10



1.25



 



NS



14.00



24



1.00



 



NS



20.00



55



1.05



 



NS



VIN, 0.02



ND



16.92



 



p ≤0.001



 


Appropriate negative (vehicle) control cultures were included in the test system under each treatment condition. The proportion of micronucleated binucleate (MNBN) cells in the vehicle cultures fell within, or very close to, the 95th percentile of the current observed historical vehicle control (normal) ranges. Mitomycin C (MMC) and Vinblastine (VIN) were employed as clastogenic and aneugenic positive control chemicals, respectively, in the absence of rat liver S-9. Cyclophosphamide (CPA) was employed as a clastogenic positive control chemical in the presence of rat liver S-9.


 


Cells receiving these were sampled in the Micronucleus Experiment at 24 hours after the start of treatment; all compounds induced statistically significant increases in the proportion of cells with micronuclei. All acceptance criteria were considered met and the study was therefore accepted as valid. Treatment of cells with the test item in the absence and presence of S-9 resulted in frequencies of MNBN cells that were generally similar to and not significantly higher than those observed in concurrent vehicle controls for all concentrations analysed under all three treatment conditions. The MNBN cell frequency of all treated cultures fell within normal ranges with the exception of a single culture at 10 μg/mL, the lowest concentration analysed following 3+21 hours treatment in the presence of S-9. This isolated marginal increase of 1.3% compared to the normal range of 0.2-1.2% was not reproduced in the replicate culture at 10  g/mL or at any other concentration tested. In addition, as the mean MNBN cell frequency fell within the normal range and was not statistically significantly higher than the concurrent vehicle controls, it was considered of no biological relevance.


 


It is concluded that the test item did not induce micronuclei in cultured human peripheral blood lymphocytes following treatment in the absence and presence of a rat liver metabolic activation system (S-9), when tested up to cytotoxic concentrations.


 


QSAR, In Vitro Gene Mutation Study in Mammalian Cells, RL2


Mutagenicity was estimated using Derek Nexus 6.2.0. No mutagenic properties were estimated based on the described QSAR method (Derek, 2017).


 


The adequacy of a prediction depends on the following conditions:



  1. the (Q)SAR model is scientifically valid: the scientific validity is established according to the OECD principles for (Q)SAR validation;

  2. the (Q)SAR model is applicable to the query chemical

  3. the (Q)SAR result is reliable

  4. the (Q)SAR model is relevant for the regulatory purpose.


For assessment and justification of these 4 requirements the QMRF and QPRF files were developed and attached to the endpoint study record.


 


Description of the prediction Model


The prediction model was described using the harmonised template for summarising and reporting key information on (Q)SAR models. For more details please refer to the attached QSAR Model Reporting Format (QMRF) file. 


 


Assessment of estimation domain


The assessment of the estimation domain was documented in the QSAR Prediction Reporting Format file (QPRF). Please refer to the attached document for the details of the prediction and the assessment of the estimation domain.


 


Overall conclusion: The test substance is neither mutagenic nor clastogenic in two in vitro genotoxicity tests and a QSAR prediction.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on genetic toxicity, the test item is not classified and labelled according to Regulation (EC) No 1272/2008 (CLP), as amended for the eighteenth time in Regulation (EU) 2022/692.