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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD 471): negative with and without metabolic activation in S. typhimurium TA 1535, 1537, 98 and 100, and E. coli WP2 uvr A


Micronucleus test (OECD 487): negative in primary human peripheral lymphocytes with and without metabolic activation


Gene mutation in mammalian cells (OECD 490): negative in mouse lymphoma L5178Y cells with and without metabolic activation

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 Apr - 02 Oct 2021
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)
Version / remarks:
adopted in 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Target gene:
TK locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: L5178Y TK+/− (Clone 3.7.2C) mouse lymphoma cells were obtained from American Type Culture Collection, Rockville, Maryland (ATCC code: CRL 9518).
- Suitability of cells: The use of the TK mutation system in L5178Y mouse lymphoma cells has been well characterised and validated and is accepted by many regulatory authorities.
- Normal cell cycle time (negative control): The generation time and mutation rates (spontaneous and induced) have been checked at ERBC.

For cell lines:
- Absence of Mycoplasma contamination: The cells are checked at regular intervals for the absence of mycoplasmal contamination.
- Number of passages if applicable: Not indicated
- Methods for maintenance in cell culture: Permanent stocks of the L5178Y TK+/− cells are stored in liquid nitrogen, and subcultures are prepared from the frozen stocks for experimental use.
- Cell cycle length, doubling time or proliferation index: Not indicated
- Modal number of chromosomes: Not indicated
- Periodically checked for karyotype stability: Not indicated
- Periodically ‘cleansed’ of spontaneous mutants: Prior to use, cells were cleansed of pre-existing mutants.


MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
Cytotoxicity test
Minimal medium A: 521 mL RPMI 1640 with L-glutamine (1X), 6.0 mL sodium pyruvate (100 mM), 5.4 mL non-essential amino acids (100X), 1.1 mL streptomycin sulphate 50000 IU/mL + Penicillin G 50000 IU/mL, 6.0 mL F 68 Pluronic, 1.1 mL penicillin G 50000 IU/mL.
Complete medium A (5%): 475 mL minimal medium A, 25 mL horse serum (heat-activated)
Complete medium A (10%): 450 mL minimal medium A, 50 mL horse serum (heat-activated)
Complete medium A (20%): 400 mL minimal medium A, 100 mL horse serum (heat-activated)
Complete medium B (20%): 391 mL RPMI 1640 with L-glutamine (1X), 4.45 mL sodium pyruvate (100 mM), 4.00 mL non-essential amino acids (100X), 0.818 mL streptomycin sulphate 50000 IU/mL + Penicillin G 50000 IU/mL, 100 mL horse serum (heat-inactived)

Main assays
Minimal medium A: 516.1 mL RPMI 1640 (1X), 5.4 mL L-glutamine (200 mM), 6.0 mL sodium pyruvate (100 mM), 5.4 mL non-essential amino acids (100X), 1.1 mL streptomycin sulphate 50000 IU/mL + Penicillin G 50000 IU/mL, 6.0 mL F 68 Pluronic
Minimal medium B: 522.1 mL RPMI 1640 (1X), 5.4 mL L-glutamine (200 mM), 6.0 mL sodium pyruvate (100 mM), 5.4 mL non-essential amino acids (100X), 1.1 mL streptomycin sulphate 50000 IU/mL + Penicillin G 50000 IU/mL
Complete medium (5%): 950 mL minimal medium A, 50 mL horse serum (heat-activated)
Complete medium (10%): 900 mL minimal medium A, 100 mL horse serum (heat-activated)
Complete medium A (20%): 800 mL minimal medium A, 200 mL horse serum (heat-activated)
Complete medium B (20%): 800 mL minimal medium B, 200 mL horse serum (heat-activated)

The plates were incubated at 37 °C in a 5% CO2 atmosphere (100% nominal relative humidity).
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9: Trinova Biochem GmbH; prepared from Sprague Dawley rats that had been induced with Phenobarbital – 5,6-Benzoflavone.
- method of preparation of S9 mix: The mixture of S9 tissue fraction and cofactors (S9 mix) was prepared as follows (for each 10 mL): 0.408 mL S9 tissue fraction; 0.204 mL NADP (100 mM); 0.204 mL G-6-P (100 mM); 0.204 mL KCL (330 mM); 8.98 mL Complete medium (5%).
- concentration or volume of S9 mix and S9 in the final culture medium: 9.8 mL S9 mix containing 0.4 mL S9 fraction.
- quality controls of S9: The S9 fraction was confirmed for protein content (35 mg/mL), positive enzymatic activity, absence of contaminating microorganisms, and negative promutagen activity.
Test concentrations with justification for top dose:
Migrated Data from field(s)
Field "Justification for deviation from the high dose level" (Path: ENDPOINT_STUDY_RECORD.GeneticToxicityVitro.MaterialsAndMethods.Method.JustificationForDeviationFromTheHighDoseLevel): Preliminary cytotoxicity study: 19.5, 39.1, 78.1, 156, 313, 625, 1250, 2500, 5000 μg/mL
Experiment 1 and 2: 16.0, 40.0, 100, 250, 625 μg/mL

Doses were selected based on the results of the preliminary cytotoxicity study
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Acetone (batch nos.: 17A304006 and 21A154014 obtained from VWR)

- Justification for choice of solvent/vehicle: The test item was found to be soluble up to 500 mg/mL in acetone.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
methylmethanesulfonate
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: 2 (Experiment 1 with and without metabolic activation and Experiment 2 without metabolic activation)

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 1E6 cells/mL
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 3 h (Experiment 1; with and without metabolic activation); 24 h (Experiment 2; without metabolic activation)

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 2 d
- Selection time: 14 days after treatment with 3 µg/mL of 5-trifluorothymidine (TFT)
- Method used: 96-well plates were incubated at 37 °C in a 5% CO2 atmosphere (100% nominal relative humidity) for 14 days and wells containing clones were identified by eye using background illumination and counted. In addition, the number of wells containing large colonies as well as the number of those containing small colonies were scored..
- Number of cells seeded and method to enumerate numbers of viable and mutants cells:
Treatment with test concentrations: 1E6 cells / mL
Expression period: 2E5 cells / mL
Plating with TFT: 2E3 cells / mL
Plating for viability: 1.6 cells / well
- Criteria for small (slow growing) and large (fast growing) colonies:
Small colonies: Colonies having a diameter less than 25% of the diameter of the well. A small colony should also have a compact morphology.
Large colonies: Colonies having a diameter greater than 25% of the diameter of the well. Morphology is diffused, totally or in periphery.
For the unteated and positive controls, the small and large colony mutant frequencies were estimated and the proportion of small mutant colonies was calculated.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
A preliminary cytotoxicity test was performed in order to select appropriate dose levels for the mutation assays. In this test a wide range of dose levels of the test item was used and the survival of the cells was subsequently determined.
Treatments were performed in the absence and presence of S9 metabolic activation for
3 hrs and for 24 hrs only in the absence of S9 metabolic activation. A single culture was used at each test point. After washing in Phosphate Buffered Saline (PBS), cells wereresuspended in 20 mL of complete medium (10%). Cell concentrations were adjusted to 8 cells/mL using complete medium (20%) and, for each dose level, 0.2 mL was plated into 96 microtitre wells. The plates were incubated at 37°C in a 5% CO2 atmosphere (100% nominalrelative humidity) for 8 days. Wells containing viable clones were identified by eye using background illumination and then counted.
In order to aid toxicity data interpretation, the relative total growth (RTG), expressed as a percentage of the concurrent negative control, was also calculated. This is a product of the relative suspension growth (RSG) and the Day 2 relative cloning efficiency (RCE), expressed as a percentage of the concurrent negative control, for each culture, as follows:
RTG = RSG x RCE (Day 2)
RSG = (Total suspension growth (TSG)test/TSGcontrol) x 100
TSG = (post-treatment cell count/pre-treatment cell count) x (Day 1 cell count/2E5) x (Day 2 cell count/2E5)
The main experiment and the preliminary toxicity test used the same methodology to determine cytotoxitiy.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
Mutant frequency (MF) = ((-ln (ym/nm))/fm) / (-ln (ys/ns))/fs)) x 10^6
ym = number of empty wells (mutant plates); nm = total number of wells (mutant plates); fm = number of cells plated per well (2E3 cells/well); ys = number of empty wells (viability plates); ns = total number of wells (viability plates); fs = number of cells plated per well (1.6 cells/well)
Induced mutant frequency (IMF) = MFtest - MFcontrol
Rationale for test conditions:
Based on OECD test guideline 490 (2016).
Evaluation criteria:
For a test item to be considered mutagenic in this assay, it is required that:
1. The induced mutant frequency (IMF) is higher than the global evaluation factor (GEF) suggested for the microwell method (126E−6) at one or more doses.
2. There is a significant dose-relationship as indicated by the linear trend analysis.

Results which only partially satisfy the above criteria will be dealt with on a case-by-case basis. Similarly, positive responses seen only at high levels of cytotoxicity will require careful interpretation when assessing their biological significance. Any increase in mutant frequency should lie outside the historical control range to have biological relevance.
Statistics:
Statistical analysis was performed according to UKEMS guidelines.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
Upon addition of the test item to the cultures, precipitation was observed at the highest dose level, while dose related opacity of treatment mixtures was noticed in all treatment series from 156 μg/mL onwards.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH and osmolality: The addition of the test item solution did not have any obvious effect on the osmolality or pH of the treatment medium.
- Precipitation and time of the determination: Upon addition of the test item to the cultures, precipitation was observed at the highest dose level, while dose related opacity of treatment mixtures was noticed in all treatment series from 156 μg/mL onwards. By the end of the treatment, dose related opacity was seen in all treatment series starting from 156 μg/mL.

RANGE-FINDING/SCREENING STUDIES:
A preliminary cytotoxicity test was performed in order to select appropriate dose levels for the mutation assays. In this test a wide range of dose levels of the test item was used and the survival of the cells was subsequently determined.
Treatments were performed in the absence and presence of S9 metabolic activation for 3 h and for 24 h only in the absence of S9 metabolic activation. A single culture was used at each test point. After washing in Phosphate Buffered Saline (PBS), cells were resuspended in 20 mL of complete medium (10%). Cell concentrations were adjusted to 8 cells/mL using complete medium (20%) and, for each dose level, 0.2mL was plated into 96 microtiter wells. The plates were incubated at 37 °C in a 5% CO2 atmosphere (100% nominal relative humidity) for 8 days. Wells containing viable clones were identified by eye using background illumination and then counted.
Both in the absence and presence of S9 metabolic activation, the test item was assayed at dose levels of 19.5, 39.1, 78.1, 156, 313, 625, 1250, and 5000 μg/mL. Slight or no relevant toxicity was noted at all concentrations tested, in each treatment series.

STUDY RESULTS.
- Genotoxicity results: No statistically significant or biologically relevant increase in mutant frequency values was observed in any experiment, at any concentration tested, in the absence or presence of S9 metabolism, using the short or long treatment time. The IMF was not higher than the GEF and there was not a significant dose-relationship. See 'Any other information on results incl. tables', Table 1.
Untreated, solvent and positive control cultures were included in each mutation experiment. The mutant frequencies in the untreated control cultures fell within the normal range (50 − 170E−6 viable cells).
Both in Experiment 1 and 2, the positive control item induced an increase in the small colony mutation frequency higher than 150E-6 above that seen in the concurrent solvent control, demonstrating that one criterion of acceptance for the small colony mutant frequency was achieved. The study was accepted as valid.

HISTORICAL CONTROL DATA
See 'Any other information on results incl. tables', Table 2.

Table 1: L5178Y TK+/- mouse lymphoma cell mutation assay










































































































































































































































Dose


(µg/mL)



RTG



MF §



P-value



IMF §



Proportion small colony mutants



Precipitation



Main assay 1 – 3 h without S9



Untreated



100%



55.1



-



-



0.32



-



0.00



100%



57.2



-



-



0.26



-



16.0



83%



51.0



NS



-



-



-



40.0



81%



52.4



NS



-



-



-



100



105%



46.4



NS



-



-



-



250



69%



48.5



NS



-



-



+



625



89%



45.0



NS



-



-



+



Main assay 1 – 3 h with S9



Untreated



100%



53.5



-



-



0.31



-



0.00



100%



67.0



-



-



0.27



-



16.0



103%



47.8



NS



-



-



-



40.0



103%



43.7



NS



-



-



-



100



88%



66.1



NS



-



-



-



250



106%



53.7



NS



-



-



+



625



94%



42.0



NS



-



-



+



B(a)P 2.00



26%



1136.6



-



1083.1@



0.37



-



Main assay 2 – 24 h without S9



Untreated



100%



55.8



-



-



0.29



-



0.00



100%



75.5



-



-



0.28



-



16.0



94%



56.6



NS



-



-



-



40.0



93%



79.9



NS



4.39



-



-



100



82%



69.8



NS



-



-



-



250



50%



101.6



NS



26.12



-



+



625



74%



66.7



NS



-



-



+



MMS 5.00



49%



573.3



-



517.5@



0.40



-



§ = per 106 viable cells


B(a)P = benzo(a)pyrene


IMF = induced mutation frequency


MF = mutation frequency


MMS = methylmethansulfonate


NS = not statistically significant


RTG = relative total growth


+ = Opacity of treatment medium


@ = IMF > global evaluation factor (GEF = 126 x 10-6)



Table 2: Historical data of negative / solvent and positive controls (Mutation frequencies per million surviving cells)













































































 



Without S9; 3 h



With S9; 3h



Without S9; 24 h



Negative control



Positive control



Negative control



Positive control



Negative control



Positive control



Mean Value



81.0



447



83.4



759



81.1



635



Standard Deviation



24.2



140



26.0



266



25.1



285



Upper Confidence Limit (p < 5%)



128



NC



135



NC



130



NC



Min



41.9



209



44.9



187



50.4



221



Max



234



1103



234



1507



217



2325



n



172



136



262



262



118



118



NC = Not calculated


n = number of experiments



 

Conclusions:
Under the experimental conditions reported the test item did not induce gene mutations at the TK locus in L5178Y mouse lymphoma cells in the absence or presence of S9 metabolic activation. Therefore, the test item is considered to be non-mutagenic in the TK assay.
Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 Apr - 24 Jun 2021
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:
adopted in 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
Not applicable
Species / strain / cell type:
lymphocytes: Human primary culture
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells:
human peripheral blood lymphocytes
- Suitability of cells:
selected according to OECD guideline 487

For lymphocytes:
- Sex, age and number of blood donors:
donor 1: female, 31 yrs; donor 2: female, 23 yrs
- Whether whole blood or separated lymphocytes were used:
Human lymphocyte cultures were prepared from human whole blood batches obtained from Biopredic International (France).
- Whether blood from different donors were pooled or not:
Blood from different donors were pooled.
- Mitogen used for lymphocytes: phytohaemagglutinin (PHA)

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
The culture medium for the lymphocytes had the following composition: 500 mL RPMI 1640 1x (Dutch modification), 100 mL Foetal Calf Serum, 6.25 mL L-Glutamine (200 mM), 1.25 mL Antibiotic solution. The foetal calf serum was heat-inactivated at 56 °C for 20 min before use. For the initiation of the cultures, medium with the addition of phytohaemagglutin (PHA) was used in the following proportion: 10 mL of PHA was added to 500 mL of medium. For the treatment series, culture medium without PHA was used. The cultures were incubated at 37 °C.
Cytokinesis block (if used):
6 µg/mL Cytochalasin B
Metabolic activation:
with and without
Metabolic activation system:
- source of S9: Phenobarbital - 5,6-Benzoflavone induced rat liver S9 was used as the metabolic activation system. One batch of S9 tissue fraction, provided by Trinova Biochem GmbH, was used in this study.
- method of preparation of S9 mix: The mixture of S9 tissue fraction and cofactors (S9 mix) was prepared as follows (for each 10 mL): 1.0 mL S9 tissue fraction; 0.4 mL NADP (100 mM); 0.5 mL G-6-P (100 mM); 0.2 mL MgCl2 (100 mM); 5.0 mL Phosphate buffer (pH 7.4, 200 mM); 2.9 mL Distilled water.
- concentration or volume of S9 mix and S9 in the final culture medium: 1 mL S9 mix (0.1 mL S9) in 5 mL treatment medium;
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): The S9 fraction was confirmed for protein content (35 mg/mL), positive enzymatic activity, absence of contaminating microorganisms, and negative promutagen activity.
Test concentrations with justification for top dose:
Migrated Data from field(s)
Field "Justification for deviation from the high dose level" (Path: ENDPOINT_STUDY_RECORD.GeneticToxicityVitro.MaterialsAndMethods.Method.JustificationForDeviationFromTheHighDoseLevel): Experiment I: 78.0, 117, 176, 263, 395, 593, 889, 1330, and 2000 µg/mL (with and without metabolic activation, 3 h exposure)
Experiment II: 52.0, 78.0, 117, 176, 263, 395, 593, 889, 1330, and 2000 µg/mL (without metabolic activation, 31 h exposure)

Based on solubility features, the maximum dose level was 2000 µg/mL. The maximum dose level of 5000 µg/mL for UVCB substances, as indicated in the OECD 487, was therefore not reached.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Acetone

- Justification for choice of solvent/vehicle:
The test item was found soluble in acetone at the concentration of 500 mg/mL.

- Justification for percentage of solvent in the final culture medium: According to OECD 487 organic solvents should not exceed 1%.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
colchicine
cyclophosphamide
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: two

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

TREATMENT AND HARVEST SCHEDULE:
- Pretreatment period: Lymphocyte cultures were prepared by adding aliquots of whole blood (0.5 mL) to 4.5 mL of culture medium with PHA, in appropriately labelled conical screw-cap tissue culture tubes. The cultures were incubated at 37 °C and were treated 48 h after they were initiated.
Before treatment, cultures were centrifuged at 1000 rpm for 10 minutes and the culture medium was decanted and replaced with treatment medium.
- Exposure duration/duration of treatment: Short treatment: 3 h with and without metabolic activation (Experiment 1). Continuous treatment: 31 h without metabolic activation (Experiment 2).
- Harvest time after the end of treatment (sampling/recovery times): At the end of treatment time of the short term treatment series (Experiment 1), the cell cultures were centrifuged and washed twice with Phosphate Buffered Saline (PBS) Solution. Fresh medium was added and the cultures were incubated for a further 29.5 h (Recovery Period) before harvesting. At the same time, Cytochalasin-B was added to achieve a final concentration of 6 μg/mL.
For the continuous treatment series, 3 h after beginning of treatment, Cytochalasin-B was also added and the cultures were incubated for a further 28 h before harvesting.

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- If cytokinesis blocked method was used for micronucleus assay: Cytochalasin-B was used as a cytokinesis blocking substance (6 μg/mL) for 29.5 h (Experiment 1) and 28 h (Experiment 2).
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): The lymphocyte cultures were centrifuged for 10 min at 1000 rpm and the supernatant was removed. The cells were resuspended in hypotonic solution. Fresh methanol/acetic acid fixative was then added. After centrifugation and removal of this solution, the fixative was changed several times by centrifugation and resuspension. A few drops of the cell suspension obtained in this way were dropped onto clean, wet, grease free glass slides. Three slides were prepared for each test point and each was labelled with the identity of the culture. The slides were allowed to air dry and kept at room temperature prior to staining with a solution of Acridine Orange (0.10 mg/mL in PBS).
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): For the selected doses, for the untreated and solvent control and the positive control Cyclophosphamide, 1000 binucleated cells per cell culture (i.e. 2000 in total) were scored to assess the frequency of micronucleated cells. Concerning cultures treated with Colchicine, since it is a known mitotic spindle poison which induces mitotic slippage and cytokinesis block, a greater magnitude of response was observed in mononucleated cells. For this reason, 1000 mononucleated cells per cell culture (i.e. 2000 in total) were scored.
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification): The criteria for identifying micronuclei were as follows: 1. The micronucleus diameter was less than 1/3 of the nucleus diameter; 2. The micronucleus diameter was greater than 1/16 of the nucleus diameter; 3. No overlapping with the nucleus was observed; 4. Micronuclei were non-refractile and had the same staining intensity as the main nuclei.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Cytokinesis-block proliferation index (CBPI); 5 dose levels (500 cells per culture) were analysed.
Rationale for test conditions:
The test was performed according to the specifications of the OECD guideline 487 (2016).
Evaluation criteria:
In this assay, the test item is considered as clearly positive if the following criteria are met:
– Significant increases in the proportion of micronucleated cells over the concurrent controls occur at one or more concentrations.
– The proportion of micronucleated cells at such data points exceeds the normal range based on historical control values (95% control limits).
– There is a significant dose effect relationship.

The test item is considered clearly negative if the following criteria are met:
– None of the dose levels shows a statistically significant increase in the incidence of micronucleated cells.
– There is no concentration related increase when evaluated with the Cochran-Armitage trend test.
– All the results are inside the distribution of the historical control data (95% control limits).
Statistics:
For the statistical analysis, a modified χ2 test was used to compare the number of cells with micronuclei in control and treated cultures.
Cochran-Armitage Trend Test (one-sided) was performed to aid determination of concentration response relationship.
Species / strain:
lymphocytes: human primary culture
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
Precipitation (visible by eye) seen at the highest dose (2000 µg/mL)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH and osmolality: Following treatment with the test item, no remarkable variations of the pH or osmolality values over the concurrent negative controls were observed in any treatment series.
- Precipitation and time of the determination: Precipitation (visible by eyes) seen at the highest dose (2000 µg/mL).

STUDY RESULTS
Micronucleus test in mammalian cells:
- Results from cytotoxicity measurements: The CBPI was calculated for each of the treatment series. Following treatment with the test item no cytotoxicity was observed at any dose level in the absence or presence of S9 metabolism. The dose levels of 0.00, 395, 889, and 2000 μg/mL were selected for scoring of micronuclei.
- Genotoxicity results: Following treatment with the test item, no statistically significant increase over the concurrent solvent control value was observed at any dose level, in any treatment series. All the results were inside the distribution of the historical control data (95% confidence limits) and no dose effect relationship was seen. Please see Table 1 and 2 under 'Any other information on results incl. tables'.
Statistically significant increases in the incidence of micronucleated cells were observed following treatments with the positive controls Cyclophosphamide and Colchicine and the responses were compatible with those generated in the historical control database of the lab, indicating the correct functioning of the test system.

HISTORICAL CONTROL DATA
- Positive historical control data: Please see Table 4 under 'Any other information on results incl. tables'.
- Negative (solvent/vehicle) historical control data: Please see Table 3 under 'Any other information on results incl. tables'.

Table 1: Micronucleus test – 3 h (32.5 h sampling time; Experiment 1)























































































Dose


(µg/mL)



% Mn cells



Significance



% Cytotoxicity



Without S9



Untreated



0.45



-



-7



Solvent 1%



0.55



-



0



395



0.70



N.S.



2



889



0.55



N.S.



-4



2000



0.60



N.S.



-4



With S9



Untreated



0.50



-



-1



Solvent 1%



0.50



-



0



395



0.65



N.S.



2



889



0.45



N.S.



0



2000



0.70



N.S.



4



Cyclophosphamide


15.0 µg/mL



4.65



***



56



Mn = micronuclei


N.S. = not significant


*** = significant difference (p < 0.001)



 


Table 2: Micronucleus test – 31 h (31 h sampling time; Experiment 2)






















































Dose


(µg/mL)



% Mn cells



Significance



% Cytotoxicity



Without S9



Untreated



0.25



-



-9



Solvent 0.5%



0.15



-



0



395



0.45



N.S.



-10



889



0.35



N.S.



-4



2000



0.45



N.S.



-6



Colchicine


0.0800 µg/mL



2.65



***



100



Mn = micronuclei


N.S. = not significant


*** = significant difference (p < 0.001)



 


Table 3: Historical control data – Untreated and solvent controls (2011 - 2022)























































 



Without S9



With S9



Short treatment time (3 h)



Long treatment time (31 – 32 h)



Short treatment time (3 h)



Untreated



-



-



-



Mean



0.35



0.42



0.42



SD (σn-1)



0.21



0.30



0.27



n



84



81



84



UCL



0.77



1.00



0.95



LCL



0.00



0.00



0.00



SD = Standard deviation


UCL = Upper confidence limit (mean value + 2 SD)


LCL = Lower confidence limit (mean value – 2 SD)


n = number of experiments



 


Table 4: Historical control data – Positive controls (2011 - 2022)














































 

With S9



Without S9



Shot treatment time (3 h)



Long treatment time (31 - 32 h)



Cyclophosphamide



Colchicine



Mean



3.12



3.51



SD (σn-1)



1.44



1.7



n



72



66



Minimum



0.95



1.1



Maximum



7.65



9.60



SD = Standard deviation


n = number of experiments


Conclusions:
The study was performed according to OECD guideline 487 and under GLP conditions. The test substance was found not to induce micronuclei in cultured human peripheral blood lymphocytes neither in the presence nor absence of a metabolic activation system up to the concentration where precipitation occurred. Therefore, the test substance is considered to be neither clastogenic nor aneugenic under the test conditions used.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 - 23 April 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted in 1997
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon (for S. typhimurium strains)
trp operon (for the E.coli strain)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
other: rfa wall mutation; uvrB mutation; TA98 and TA100 contain the pKM101 plasmid
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
other: uvrA DNA repair deficiency
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9: Trinova Biochem GmbH; prepared from Sprague Dawley rats that had been induced with Phenobarbital – 5,6-Benzoflavone.
- method of preparation of S9 mix: The mixture of S9 tissue fraction and cofactors (S9 mix) was prepared as follows (for each 10 mL): 1.0 mL S9 tissue fraction; 0.4 mL NADP (100 mM); 0.5 mL G-6-P (100 mM); 1.0 mL KCL (330 mM); 0.8 mL MgCl2 (100 mM); 5.0 mL Phosphate buffer (pH 7.4, 200 mM); 1.3 mL Distilled water.
- concentration or volume of S9 mix and S9 in the final culture medium: 0.5 mL S9 mix containing 0.05 mL S9 fraction.
- quality controls of S9: The S9 fraction was confirmed for protein content (37 mg/mL), positive enzymatic activity, absence of contaminating microorganisms, and negative promutagen activity.
Test concentrations with justification for top dose:
Migrated Data from field(s)
Field "Justification for deviation from the high dose level" (Path: ENDPOINT_STUDY_RECORD.GeneticToxicityVitro.MaterialsAndMethods.Method.JustificationForDeviationFromTheHighDoseLevel): Preliminary toxicity test:
- all strains: 50, 158, 500, 1580, 5000 µg/plate with and without metabolic activation

Experiment 1 (plate incorporation) and Experiment 2 (preincubation):
- all strains: 313, 625, 1250, 2500, 5000 µg/plate with and without metabolic activation

5000 µg/plate is the recommended maximum test concentration for soluble non-cytotoxic substances according to OECD 471.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Acetone (VWR, batch 17A304006)
- Justification for choice of solvent/vehicle: The test item is not soluble in water. The test item was found to be soluble up to 500 mg/mL in acetone.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
other:
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: three replicate plates / dose
- Number of independent experiments: 2 (direct plate and pre-incubation assay)

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 10^9 cells/mL
- Test substance added in agar (plate incorporation; Experiment 1); pre-incubation (Experiment 2)

TREATMENT AND HARVEST SCHEDULE:
Direct plate assay
- Exposure duration/duration of treatment: 72 h at 37 °C
Pre-incubation assay
- Preincubation period: 30 min at 37.0 °C
- Exposure duration/duration of treatment: 72 h at 37 °C

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Thinning of the background lawn was evaluated using a low magnification optical microscope.

METHODS FOR MEASUREMENTS OF GENOTOXICITY
- Plates from the preliminary toxicity test and Experiment 2 were immediately scored by counting the number of revertant colonies on each plate manually. Plate from Experiment 1 were held for 24 h at 4 °C before scoring. Precipitation of the test item on the plates was assessed by unaided eye.
Rationale for test conditions:
Based on OECD test guideline 471 (1997).
Evaluation criteria:
For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels.
Statistics:
Individual plate counts for these tests and the mean and standard error of the mean for each test concentration were given. Regression analysis was also performed.
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
Precipitation was observed at the end of the incubation period at the the two highest concentrations both in the absence and presence of S9 metabolism.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
Precipitation was observed at the end of the incubation period at the the two highest concentrations both in the absence and presence of S9 metabolism.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
Precipitation was observed at the end of the incubation period at the the two highest concentrations both in the absence and presence of S9 metabolism.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
Precipitation was observed at the end of the incubation period at the the two highest concentrations both in the absence and presence of S9 metabolism.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
Precipitation was observed at the end of the incubation period at the the two highest concentrations both in the absence and presence of S9 metabolism.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Dose related precipitation of the test item, which did not interfere with the scoring of revertant colonies and the observation of the background lawn, was observed at the end of the incubation period at the two highest concentrations both in the absence and presence of S9 metabolism.

RANGE-FINDING/SCREENING STUDIES:
In the preliminary toxicity test, there was no increase in the number of revertants observed upon treatment with the test material (refer to 'Any other information on results incl. tables', table 1).

STUDY RESULTS:
In the Plate Incorporation Assay (Experiment 1) and Pre-Incubation Assay (Experiment 2), there was no increase in the number of revertants observed upon treatment with the test material under all conditions tested (refer to 'Any other information on results incl. tables', table 2 and 3).
The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. There was no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants observed upon treatment with the test material under all conditions tested.

HISTORICAL CONTROL DATA
See 'Any other information on results incl. tables', table 4 and 5.

Table 1: Preliminary toxicity test










































































































































Dose


(µg/plate)



Mean number of revertant colonies / plate



TA1535



TA1537



TA98



TA100



WP2 uvr A



Without metabolic activation



Untreated



14



21



29



155



31



0.00



17



16



29



153



29



50.0



17



17



29



116



29



158



15



15



28



130



32



500



18



17



26



126



29



1580



14P



18P



29P



128P



31P



5000



19PP



16PP



31PP



132PP



31PP



With metabolic activation



Untreated



19



18



39



173



39



0.00



18



18



32



163



30



50.0



18



19



32



163



39



158



17



21



39



171



34



500



15



22



32



183



38



1580



16P



18P



39P



158P



33P



5000



20PP



20PP



36PP



164PP



37PP



P = Slight precipitate


PP = Moderate precipitate



 


Table 2: Experiment 1 – Plate incorporation method
















































































































































































































































































Dose


(µg/plate)



Mean number of revertant colonies / plate (± SE)



TA1535



TA1537



WP2 uvr A



TA98



TA100



Mean



± SE



Mean



± SE



Mean



± SE



Mean



± SE



Mean



± SE



Without metabolic activation



Negative control



-



-



19



1.9



-



-



28



2.4



-



-



Untreated



19



1.0



18



1.0



27



1.5



28



2.6



119



3.5



0.00



16



1.5



17



1.2



30



0.7



33



2.6



139



10.3



313



15



0.9



18



1.3



32



1.0



30



1.3



116



1.3



625



18



1.8



16



1.0



32



2.2



33



1.5



146



7.8



1250



17



1.7



18



0.9



26



1.5



30



0.7



151



6.7



2500



18P



2.2



19P



1.5



30P



0.7



31P



1.0



132P



3.7



5000



19PP



0.9



20PP



0.6



31PP



0.9



33PP



0.9



147PP



3.9



Positive control



557



28.9



121



16.4



147



2.6



142



9.0



566



21.1



With metabolic activation



Negative control



16



1.2



23



1.5



30



1.3



38



1.5



119



2.5



Untreated



14



0.6



16



0.9



28



0.3



40



1.0



120



3.2



0.00



17



2.2



16



1.0



36



2.3



34



2.7



146



7.3



313



19



0.6



23



1.2



39



0.6



40



0.7



143



3.3



625



18



1.0



21



1.3



37



2.4



37



2.6



156



6.8



1250



18



0.9



19



1.0



33



2.0



39



0.0



152



3.6



2500



16P



1.8



19P



0.3



40P



0.3



33P



1.2



167P



5.0



5000



16PP



1.5



23PP



1.2



39PP



0.9



36PP



1.8



146PP



7.9



Positive control



126



4.4



106



4.9



206



6.2



668



12.2



1142



57.7



P = Slight precipitate


PP = Moderate precipitate


 


Positive controls (without metabolic activation):


TA1535 and TA100 – sodium azide


TA1537 – 9-aminoacridine


TA98 – 2-nitrofluorene


WP2 uvrA - methylmethanesulfonate


 


Positive controls (with metabolic activation)


All strains – 2-aminoanthracene



 


Table 3: Experiment 2 – Preincubation method
















































































































































































































































































Dose


(µg/plate)



Mean number of revertant colonies / plate (± SE)



TA1535



TA1537



WP2 uvr A



TA98



TA100



Mean



± SE



Mean



± SE



Mean



± SE



Mean



± SE



Mean



± SE



Without metabolic activation



Negative control



-



-



17



1.8



-



-



28



0.9



-



-



Untreated



17



1.5



15



0.6



26



2.1



27



1.3



149



4.3



0.00



17



1.2



15



0.7



29



1.2



28



1.0



157



9.3



313



15



0.3



17



0.9



30



0.9



25



1.0



143



5.0



625



16



0.6



17



1.5



29



2.3



29



1.5



139



9.2



1250



16



1.2



16



0.7



30



0.7



28



1.0



135



5.0



2500



14P



0.6



17P



0.6



25P



0.3



29P



1.7



142P



10.7



5000



15PP



0.3



16PP



0.7



26PP



1.2



28PP



1.5



132PP



4.1



Positive control



482



12.9



168



8.8



182



6.4



192



7.0



596



34.4



With metabolic activation



Negative control



15



0.9



20



2.0



34



1.5



37



0.9



162



3.8



Untreated



18



1.2



18



1.3



31



1.5



36



1.0



168



3.2



0.00



17



1.0



18



0.9



29



1.2



36



1.5



172



4.4



313



17



0.9



18



1.9



37



2.7



40



2.6



169



0.6



625



15



0.6



20



0.9



38



1.2



31



2.3



167



7.4



1250



19



0.6



21



1.2



35



2.2



38



0.9



185



4.6



2500



18P



2.1



20P



0.9



33P



2.7



37P



3.0



164P



3.2



5000



18PP



1.7



21PP



2.0



39PP



0.9



39PP



1.3



185PP



4.6



Positive control



114



4.4



99



3.6



216



6.1



653



30.4



1142



71.2



P = Slight precipitate


PP = Moderate precipitate


 


Positive controls (without metabolic activation):


TA1535 and TA100 – sodium azide


TA1537 – 9-aminoacridine


TA98 – 2-nitrofluorene


WP2 uvrA - methylmethanesulfonate


 


Positive controls (with metabolic activation)


All strains – 2-aminoanthracene



 


Table 4: Historical control data of the negative controls (2000 – 2020)







































































































































































































































 



TA 1535



TA 1537



TA 98



TA 100



WP2 uvr A



Plate incorporation method



S9-mix



-



+



-



+



-



+



-



+



-



+



Mean



18



17



17



21



31



38



140



150



28



34



SD



2.6



2.1



2.2



2.8



3.3



3.9



17.8



18.6



2.8



3.4



UCL



23.1



21.2



21.6



26.5



37.6



45.8



175.4



187.4



33.9



40.4



LCL



12.7



12.7



12.7



15.2



24.3



30.3



104.2



113.0



22.5



26.7



n



540



541



544



543



543



543



544



543



490



490



min



13



12



11



15



23



29



109



86



21



26



max



26



22



27



33



43



53



182



196



38



47



Preincubation method



S9-mix



-



+



-



+



-



+



-



+



-



+



Mean



18



17



18



21



31



39



143



150



28



33



SD



2.5



2.2



2.3



2.4



3.0



3.2



16.8



15.6



3.0



3.2



UCL



22.9



21.4



22.1



25.6



37.2



45.7



176.6



181.2



33.7



39.4



LCL



12.7



12.7



13.0



16.2



25.2



32.9



109.4



118.9



21.7



26.5



n



418



401



411



393



401



375



411



388



340



324



min



12



12



12



15



24



32



112



119



20



24



max



25



23



27



27



42



50



192



197



38



44



SD = Standard deviation


UCL = Upper confidence limit (mean value + 2 SD)


LCL = Lower confidence limit (mean value – 2 SD)


n = number of experiments



 


Table 5: Historical control data of the positive controls (2000 – 2020)







































































































































































































































 



TA 1535



TA 1537



TA 98



TA 100



WP2 uvr A



Plate incorporation method



S9-mix



-



+



-



+



-



+



-



+



-



+



Mean



512



136



175



114



162



509



630



1188



177



194



SD



81.1



27.3



39.5



20.8



28.8



153.9



153.2



222.2



19.9



35.7



UCL



673.8



190.6



254.0



155.6



219.4



817.2



936.3



1632.6



216.8



265.1



LCL



349.4



81.2



95.8



72.4



104.2



201.6



323.4



743.8



137.1



122.4



n



540



541



544



543



543



543



544



543



490



490



min



299



81



92



75



109



203



144



607



127



117



max



750



241



407



181



259



994



1081



2257



240



333



Preincubation method



S9-mix



-



+



-



+



-



+



-



+



-



+



Mean



523



98



166



98



163



624



673



1119



187



197



SD



78.6



14.7



46.6



12.2



26.5



176.8



151.9



196.9



41.2



33.3



UCL



680.5



127.8



259.6



122.1



216.5



977.3



977.0



1513.1



269.3



263.9



LCL



366.3



69.2



73.3



73.2



110.5



270.2



369.5



725.7



104.5



130.8



n



418



400



411



392



401



375



411



387



340



323



min



374



54



72



69



115



188



325



745



113



129



max



774



179



412



142



256



1377



1081



1701



351



322



SD = Standard deviation


UCL = Upper confidence limit (mean value + 2 SD)


LCL = Lower confidence limit (mean value – 2 SD)


n = number of experiments


Conclusions:
Under the present test conditions, the test item showed negative mutagenic responses in all bacterial strains over the entire dose-range.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Ames test (OECD 471): negative with and without metabolic activation in S. typhimurium TA 1535, 1537, 98 and 100, and E. coli WP2 uvr A


Micronucleus test (OECD 487): negative in primary human peripheral lymphocytes with and without metabolic activation


Gene mutation in mammalian cells (OECD 490): negative in mouse lymphoma L5178Y cells with and without metabolic activation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Genetic toxicity (mutagenicity) in bacteria in vitro


The ability of the registered substance to induce reverse mutations at the histidine locus (His+) in several strains of Salmonella typhimurium (S. typhimurium TA98, TA100, TA1535, and TA1537), and at the tryptophan locus (Trp+) of Escherichia coli (E. coli) strain WP2 uvr A, either in the presence or absence of a metabolic activation system (S9-mix) was examined according to OECD 471 and GLP principles (Croda Europe Limited, 2022). The test was performed in two independent experiments: a plate incorporation assay (Experiment 1) and a pre-incubation assay (Experiment 2). The vehicle of the test item was DMSO. The study started with a preliminary toxicity test where the test item was initially tested at concentrations of 50 - 5000 µg/plate in all test strains in a plate incorporation assay with and without metabolic activation. The test item precipitated on the plates at dose levels of 1580 μg/plate and upwards. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. The test concentrations for the two main tests were determined based on the results of the preliminary toxicity test. In Experiment 1, the test item was tested at concentrations of 313 - 5000 µg/plate in all test strains with and without metabolic activation. The test item precipitated on the plates at dose levels of 2500 μg/plate and upwards. No toxicity was observed at any of the dose levels tested, the bacterial background lawn was not reduced at any of the concentrations tested, and no biologically relevant decrease in the number of revertants was observed. In Experiment 2, the test item was also tested at concentrations of 313 - 5000 µg/plate in all test strains in a pre-incubation assay with and without metabolic activation. The test item precipitated on the plates at dose levels of 2500 μg/plate and upwards. No toxicity was observed at any of the dose levels tested, the bacterial background lawn was not reduced at any of the concentrations tested, and no biologically relevant decrease in the number of revertants was observed. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. The test item did not induce a biologically relevant, dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (S. typhimurium TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain E. coli WP2uvrA both in the absence and presence of S9-metabolic activation in two independent experiments. Based on the results of this study it was concluded that the registered substance was not mutagenic in the bacterial reverse mutation assay.


 


Genetic toxicity (cytogenicity) in mammalian cells in vitro


The ability of the registered substance to induce micronuclei in human lymphocytes, either in the presence or absence of a metabolic activation system (S9-mix), was examined according to OECD 487 and GLP principles (Croda Europe Limited, 2021). Three treatment conditions were performed: A short-term treatment, where the cells were treated for 3 h, in the absence and presence of S9 metabolism with a harvest time of approximately 32.5 h, corresponding to approximately two cell cycle lengths and a long-term (continuous) treatment only in the absence of S9 metabolism, until harvest at 31 h. The vehicle of the test item was acetone. Based on solubility features, the maximum dose level of 2000 μg/mL was selected as top dose for the study. Two replicate cell cultures were prepared at each test concentration In the short-term treatment, the test item was tested at concentrations of 78 - 2000 µg/mL (395 - 2000 µg/mL selected for scoring) with and without metabolic activation, along with an untreated control, a solvent (1%) control, and a positive control (cyclophosphamide 15.0 µg/mL) for with metabolic activation. The test item precipitated at the 2000 μg/mL dose level. No toxicity was observed at any of the dose levels tested and no significant increase in micronucleated cells was observed. In the long-term treatment, the test item was tested at concentrations of 78 - 2000 µg/mL (395 - 2000 µg/mL selected for scoring) without metabolic activation, along with an untreated control, a solvent (0.5%) control, and a positive control (colchicine 0.0800 µg/mL). The test item precipitated at the 2000 μg/mL dose level. No toxicity was observed at any of the dose levels tested and no significant increase in micronucleated cells was observed. The negative and positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate, and that the metabolic activation system functioned properly. Based on the results of this study it was concluded that the registered substance was not clastogenic in the in vitro micronucleus test.


 


Genetic Toxicity in vitro: gene mutation in mammalian cells


The ability of the registered substance to induce 5-trifluorothymidine resistant mutants in mouse lymphoma TK+/− L5178Y cells, either in the presence or absence of a metabolic activation system (S9-mix), was examined according to OECD 490 (In Vitro Mammalian Cell Gene Mutation Test – Fluctuation Method) and GLP principles (Croda Europe Limited, 2023). A preliminary solubility trial indicated that the maximum practicable concentration of the test item was 5000 µg/mL. The vehicle of the test item was acetone. A preliminary toxicity test was performed at concentrations of 19.5 - 5000 µg/mL. Slight or no remarkable toxicity was noticed at all concentrations tested. Precipitation was observed at the highest dose level, while dose related opacity of treatment mixtures was noticed in all treatment concentrations from 156 µg/mL and upwards. Based on the results obtained in the preliminary toxicity test, two independent assays for mutation at the TK locus were performed: a short-term treatment, where the cells were treated for 3 h, in the absence and presence of S9 metabolism and a long-term (24 h) treatment only in the absence of S9 metabolism. In the short-term treatment, the test item was tested at concentrations of 16.0 - 625 µg/mL with and without metabolic activation. The test item precipitated at 250 μg/mL and upwards. Slight or no remarkable toxicity was observed at all of the dose levels tested and no significant increase in mutant frequency was observed. In the long-term treatment, the test item was tested at concentrations of 16.0 - 625 µg/mL without metabolic activation. The test item precipitated at 250 μg/mL and upwards. Mild reduction of the relative total growth (RTG of 50%) was seen at 250 μg/mL; however, no toxicity was seen at any of the other dose levels tested. No significant increase in mutant frequency was observed. The negative and positive control values were within the normal ranges indicating that the test conditions were adequate, and that the metabolic activation system functioned properly. Based on the results of this study it was concluded that the registered substance was not mutagenic in mouse lymphoma TK+/− L5178Y cells.


 


Conclusion for genetic toxicity


The results of the available in vitro studies on mutagenicity in bacterial cells, cytogenicity in mammalian cells, and gene mutation in mammalian cells were consistently negative. Based on the available data performed with the target substance, no mutagenic or clastogenic potential is expected for the registered substance.

Justification for classification or non-classification

According to Regulation (EC) No. 1272/2008 the data on genotoxicity are conclusive but not sufficient for classification.