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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames Test (OECD 471, GLP, K, rel. 1): non mutagenic up to cytotoxic concentrations in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 & E.coli WP2uvrA.

- Ames Test (similar to OECD 471, rel.2): non mutagenic up to cytotoxic concentration in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 & E.coli WP2uvrA.

- Chromosome aberration test (OECD 473, K, rel. 1): non clastogenic up to cytotoxic concentrations.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 April to 26 May, 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Well conducted and well described study in accordance with GLP and OECD Guideline 471 without any deviation. The substance is considered to be adequately characterised. Therefore full validation applies.
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
Directive 2000/32/EC
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
Inspected 2004-12-16 / Signed 2005-O3-11.
Type of assay:
bacterial reverse mutation assay
Target gene:
Salmonella typhimurium histidine (his) reversion system and the Escherichia coli tryptophan (trp) reversion system
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
10 % v/v S9; S9 fraction was prepared from liver homogenates of Sprague Dawley rat induced with Aroclor 1254
Test concentrations with justification for top dose:
Assay No. 1 (plate incorporation):
1 500, 450, 150, 45 and 15 μg/plate, with or without metabolic activation in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 (uvr A-)
Assay No. 2 (pre-incubation):
1 500, 450, 150, 45 and 15 μg/plate, with or without metabolic activation in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 (uvr A-)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Test substance preparation:
For the first bacteriostatic activity control, the highest dose studied is 5 000 μg/plate. A solution at 500 mg/mL is prepared with acetone.
For the two other bacteriostatic activity controls and mutagenic assays, the highest dose studied is 1 500 μg/plate. A solution at 150 μg/mL is prepared with acetone.
The test substance is stable in solvent used, under similar assay conditions, according to the sponsor.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
other: See Remarks
Remarks:
without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
yes
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
other: 2-Anthramine
Remarks:
with metabolic activation
Details on test system and experimental conditions:
SOURCE OF TEST SYSTEM: Strains of Salmonella typhimurium and Escherichia coli are obtained from "Unité de Programmation Moléculaire et Toxicologie Génétique" (CNRS UA 144) (Institut Pasteur).

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation

DURATION
- Preincubation period: Pre-incubation assay where the test substance was preincubated with the test strain, and 500 μL of S9-mix fraction for 1 hour at 37° C prior to mixing with the overlay agar and pouring onto the surface of the minimal agar plate.
- Exposure duration: Plates were incubated at 37 °C over a 48 hour period

NUMBER OF REPLICATIONS: 3 plates/dose for all groups

DETERMINATION OF CYTOTOXICITY
- Method: reduced number of spontaneous reversions indicating the bacteriostatic activity.

- OTHER:
- Checking strains: The genotype of bacterial strains was checked for Histidine and tryptophan requirements; Loss of cell wall LPS (rfa mutation) measuring crystal violet inhibition for Salmonella typhimurium strains; Ampicillin resistance for the strains which have the pKM 101 plasmide; Δuvr B mutation i.e. U.V.B sensitivity for Salmonella typhimurium and Δuvr A mutation i.e. U.V.A sensitivity for Escherichia coli; Spontaneous revertant rate
- After a 48 hour incubation period at 37° C, revertant colonies per plate are counted (n = 3).
- Sterility tests: The highest concentration and dilutions of the test substance (100 μL) were tested for sterility.
- Data were presented as the number of revertant colonies (mean ± standard deviation) per plate. The following ratio was calculated: R = Number of revertant colonies in the presence of the product / Number of revertant colonies in the absence of the product.
Rationale for test conditions:
Tested up to cytotoxicity limit
Evaluation criteria:
The result of the test is considered as negative if the revertant number is below three fold the number of spontaneous reversions, for TA 1535 and TA 1537 strains, and below two fold the number of spontaneous reversions for TA 98, TA 100 and Escherichia coli WP2(uvrA-) (pKM 101) strains with and/or without metabolic activation.
The validity criteria are as follows :
- bacteriostatic activity of the highest concentration shall be equal to or less than 75 %, - the spontaneous reversion rate of the absolute negative control shall comply with the laboratory’s historical control data.
- the mean number of revertant colonies obtained for each strain and the corresponding positive control, with and/or without metabolic activation shall comply with laboratory’s historical control data.
The result of the test is considered positive if a concentration – related increase is obtained in one, or several of the 5 strains, with and/or without metabolic activation; a mutagenic effect is taken into account for a given concentration of the test substance if the number of revertant colonies is at least two fold that of spontaneous revertant colonies number for TA 98, TA 100 and Escherichia coli WP2(uvrA-) (pKM 101), and three fold for TA 1535 and TA 1537.
All results must be confirmed in an independent experiment.
Statistics:
None
Key result
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
BACTERIOSTATIC ACTIVITY CONTROL (STRAIN TA 100)
In the first control, an important bacteriostatic activity of 99 % is observed in the presence of the test substance at 5 000 μg/plate.
The test substance is used at the following doses 1 500, 450, 150, 45 and 15 μg/plate for bacteriostatic activity control 2 and 3.
Then a bacteriostatic activity of 73 % compatible with the maximum level acceptable 75 % is observed, in the presence of the test substance at 1 500 μg/plate.
The test substance is tested at the following doses : 1 500, 450, 150, 45 and 15 μg/plate.


HISTORICAL CONTROL DATA
- See historical control table in "Attached background material" section

MUTAGENICITY RESULTS
There is no significant difference between the number of spontaneous reversions, the number of reversions obtained in the positive controls (without and with metabolic activation), and the mean of corresponding experimental historic values obtained in the laboratory.
There is no evidence of any increase in the number of revertant colonies in the presence of the test substance (1500 μg - 450 μg - 150 μg - 45 μg - 15 μg) without and with metabolic activation for bacterial strains in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and Escherichia coli WP2(uvrA-) (pKM 101).
Results were confirmed in a second independent experiment.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
None

OTHERS:
Sterility test did not show any bacterial growth in presence of all substance doses / S9 mix.
Genotoxicity testing: see tables 3.1 to 3.4 for TA 1535; 4.1 to 4.4 for TA 1537; 5.1 to 5.4 for TA 98; 6.1 to 6.4 for TA 100; 7.1 to 7.4 for Escherichia coli in "Attached background material" section.

None

Conclusions:
Under the test conditions, test substance is not considered as mutagenic with and without metabolic activation in S. typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and E. coli (WP2uvrA) strains.
Executive summary:

In a reverse gene mutation assay in bacteria, performed according to the OECD Guideline 471 and in compliance with GLP, strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and Escherichia coli (WP2uvrA) were exposed to test substance at the following concentrations. 

Assay No. 1 (plate incorporation method): 15, 45, 150, 450, 1500 μg/plate, with or without metabolic activation in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 (uvr A-)

Assay No. 2 (pre-incubation method): 15, 45, 150, 450, 1500 μg/plate, with or without metabolic activation in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 (uvr A-) 

 

Metabolic activation system used in this test was10 % (v/v) S9 mix; S9 fraction prepared from liver homogenates of rats induced with Aroclor 1254. Negative, vehicle and positive control groups were also included in mutagenicity tests.

 

There is no significant difference between the number of spontaneous reversions, the number of reversions obtained in the positive controls (without and with metabolic activation), and the mean of corresponding experimental historic values obtained in the laboratory.

There is no evidence of any increase in the number of revertant colonies in the presence of the test substance (1500 - 450 - 150 - 45 - 15 µg) without and with metabolic activation for bacterial strains in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and Escherichia coli WP2(uvrA-) (pKM 101).

Results were confirmed in a second independent experiment.

 

Under the test conditions, test substance is not considered as mutagenic with and without metabolic activation in these bacterial systems.

This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
24 April to 26 May, 2008
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Study conducted similarly to OECD 471 Guideline with deviations: details of toxicity, evaluation criteria not reported
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
details of toxicity, evaluation criteria not reported
Principles of method if other than guideline:
Not applicable
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction prepared from liver homogenates of male Sprague Dawley rats induced with Phenobarbital and 5,6-Benzoflavone
Test concentrations with justification for top dose:
Test 1: 1.2, 4.9, 20, 78, 313, 1250 and 5000 µg/plate in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2, with and without S9 mix
Test 2: 9.8, 20, 39, 78, 156, 313 µg/plate in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2, with and without S9 mix
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Solubility of test material in DMSO is more than 5% and stable. Also it is not soluble in water at 5%.
- Shelf time and temperature after preparation of solution: Max. 2 hours 10 minutes at 25 °C
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
furylfuramide
other: ICR-191
Remarks:
without S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: Preincubation

DURATION
- Preincubation period: 20 minutes at 37 °C
- Exposure duration: Plates were inverted and incubated at 37 °C for 48 hours.

NUMBER OF REPLICATIONS: 2 plates/dose

OTHER:
Counting method: Manual and mechanical counting
Correction:
Mechanical measuring: Surface and miscounting correction
Manual measuring: Surface correction for >300
Colony counts >1500: measured manually
Rationale for test conditions:
Tested up to cytotoxicity limit
Evaluation criteria:
No data
Statistics:
No data
Key result
Species / strain:
bacteria, other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 1250 and 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Tables of results : see "Attached background material" section.

None

Conclusions:
Under the test conditions, test item is not considered as mutagenic in S. typhimurium (TA1535, TA1537, TA98, TA100) and E. coli (WP2uvrA) strains.
Executive summary:

In a reverse gene mutation assay in bacteria, performed similarly to the OECD Guideline 471, strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and Escherichia coli (WP2uvrA) were exposed to test substance at the following concentrations: 

Test 1: 1.2, 4.9, 20, 78, 313, 1250 and 5000 µg/plate in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2, with and without S9 mix

Test 2: 9.8, 20, 39, 78, 156, 313 µg/plate inS. typhimurium TA 1535, TA 1537, TA 98, TA 100 andE. coliWP2, with and without S9 mix

 

S9 fraction prepared from liver homogenates of male Sprague Dawley rats induced with Phenobarbital and 5,6-Benzoflavone. Vehicle and positive control groups were also included in mutagenicity tests.

 

The mean numbers of revertant colonies are fell within acceptable ranges for vehicle control treatments, and were elevated by positive control treatments. 

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation. 

 

Under the test conditions, test item is not considered as mutagenic in these bacterial systems.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 04 March to 25 April, 2019.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD TG 473 without any deviation.
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
Adopted 29 July 2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: The Japanese Ministry of Health, Labour and Welfare (MHLW), Ministry of Economy Trade and Industry (METI), and Ministry of the Environmental (MOE).
Version / remarks:
Guidelines of 31 March 2011.
Deviations:
no
Principles of method if other than guideline:
Not applicable.
GLP compliance:
yes (incl. QA statement)
Remarks:
UK GLP Compliance Programme (Inspected on 2018-08-21 / Signed on 2018-11-19).
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
- Physical state: extremely pale yellow liquid
- Storage condition of test material: approximately 4°C, in the dark.
Target gene:
Not applicable.
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
For lymphocytes:
- Sex, age and number of blood donors: male, aged 21 years (preliminary toxicity test). male, aged 29 years (Main experiment).
- Whether whole blood or separated lymphocytes were used: for each experiment, sufficient whole blood was drawn from the peripheral circulation of a non-smoking volunteer who had been previously screened for suitability.
- Whether blood from different donors were pooled or not: no, one donor for each experiment.
- Mitogen used for lymphocytes: phytohaemagglutinin (PHA).

MEDIA USED :
Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10 % fetal bovine serum (FBS), at approximately 37 ºC with 5 % CO2 in humidified air.
Additional strain / cell type characteristics:
other: Not applicable.
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : Covance laboratory (Lot No. PB/βNF S9 31/08/18), stored at -196°C.
- method of preparation of S9 mix: S9 fraction was obtained from the liver homogenates of male rats treated with Phenobarbitone/Beta-naphthoflavone.
- concentration or volume of S9 mix and S9 in the final culture medium : the final concentration of S9, when dosed at a 10% volume of S9-mix into culture media, was 2%.
Test concentrations with justification for top dose:
- Preliminary Toxicity Test (Cell Growth Inhibition): 0, 6.89, 13.77, 27.54, 55.08, 110.16, 220.33, 440.65, 881.3 and 1762.6 μg/mL, 4h exposure time with and without metabolic activation followed by a 20h recovery period (4(20)-hour with (2%) and without S9-mix), and a continuous exposure of 24h without metabolic activation (24-hour without S9-mix).
Justification: The maximum dose was the maximum recommended dose level, the 10 mM concentration.

- Main Experiment:
0, 27.5, 55, 110, 165, 220, 275, 330 and 440 μg/mL, 4(20)-hour without S9-mix;
0, 27.5, 55, 110, 165, 220, 275, 330, 385 and 440 μg/mL, 4(20)-hour with (2%) S9-mix;
0, 13.75, 27.5, 55, 82.5, 110, 137.5, 165 and 220 μg/mL, 24-hour without S9-mix.
Justification: Mitotic index data was used to estimate test item toxicity and for selection of the dose levels for the main experiment.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)

- Justification for choice of solvent/vehicle: The test item was insoluble in Minimal Essential Medium at 17.63 mg/mL but was soluble in DMSO at 176.3 mg/mL in solubility checks performed in-house.
- Formulation preparation: The test item was accurately weighed, dissolved in DMSO and serial dilutions prepared. The test item was formulated within two hours of it being applied to the test system; the test item formulations were assumed to be stable for this duration. No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation because it is not a requirement of the guidelines.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Without metabolic activation.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With metabolic activation (2% S9-mix).
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: quadruplicate cultures for the control; duplicate culture per dose levels
- Number of independent experiments: 2

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: Exp. 1: 4 hours (± S9) / Exp. 2: 24 hours (-S9)
- Harvest time after the end of treatment (sampling/recovery times): 24 hours

FOR CHROMOSOME ABERRATION:
- Spindle inhibitor (cytogenetic assays): Mitotic activity was arrested by addition of demecolcine (Colcemid 0.1 μg/mL), two hours before the harvest time.
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): After incubation with demecolcine, the cells were centrifuged, the culture medium was drawn off and discarded, and the cells re-suspended in 0.075M hypotonic KCl. After approximately fourteen minutes (including centrifugation), most of the hypotonic solution was drawn off and discarded. The cells were re-suspended and then fixed by dropping the KCl cell suspension into fresh methanol/glacial acetic acid (3:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 ºC to ensure complete fixation prior to slide preparation.
The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re-suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Each slide was permanently labeled with the appropriate identification data.
When the slides were dry, they were stained in 5 % Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): Where possible 1000 cells per culture were evaluated for the incidence of metaphase cells and expressed as the mitotic index and as a percentage of the vehicle control value.
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): Where possible, 300 consecutive well-spread metaphases from each concentration were counted, 600 from the vehicle control (150 per replicate), where there were at least 15 cells with aberrations (excluding gaps), slide evaluation was terminated. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing and the ISCN (1985). Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides
- Determination of polyploidy / endoreplication: cells with 69 chromosomes or more were scored as polyploid cells (including endoreduplicated cells) and the incidence of polyploid cells (%) reported. Many experiments with human lymphocytes have established a range of aberration frequencies acceptable for control cultures in normal volunteer donors.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: mitotic index (MI)


Rationale for test conditions:
Human peripheral blood lymphocytes are recognized in the OECD 473 guidelines as being a suitable cell line for the Mammalian Chromosome Aberration Test.
Evaluation criteria:
The following criteria were used to determine a valid assay:
• The frequency of cells with structural chromosome aberrations (excluding gaps) in the vehicle control cultures was within the laboratory historical control data range.
• Concurrent positive control chemicals should induce responses that are compatible with those generated in historical positive control data base and produce a statistically significant increase compared with the concurrent negative control.
• The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline.
• The required number of cells and concentrations were analyzed.

Statistics:
The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test. (Richardson et al. 1989).
A toxicologically significant response is recorded when the p value calculated from the statistical analysis of the frequency of cells with aberrations excluding gaps is less than 0.05 when compared to its concurrent control and there is a dose-related increase in the frequency of cells with aberrations which is reproducible. Incidences where marked statistically significant increases are observed only with gap-type aberrations will be assessed on a case by case basis.
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data of pH: No significant change in pH when the test item was added into media.
- Data of osmolality: Osmolality did not increase by more than 50 mOsm.
- Possibility of evaporation from medium: not expected (vapour pressure = 3.46 Pa at 25°C)
- Water solubility: insoluble at 17.63 mg/mL

PRELIMINARY TOXICITY TEST (CELL GROWTH INHIBITION TEST):
- A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure, at and above 440.65 μg/mL, in all three exposure groups.
- Hemolysis was observed following exposure to the test item at and above 55.08 μg/mL in the 4(20)-hour exposure group in the absence of S9 and at and above 110.16 μg/mL in the presence of S9 and in the 24-hour exposure group. Hemolysis is an indication of a toxic response to the erythrocytes and not indicative of any genotoxic response to the lymphocytes.
- A reduced cell pellet was also noted at the end of the exposure period at and above 440.65 μg/mL in the 4(20)-hour exposure group in the absence of S9 and at and above 220.33 μg/mL in the presence of S9 and in the 24-hour exposure group. This is considered to be indicative of toxicity to the cell population as a whole.
- Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 220.33 μg/mL in the 4(20)-hour exposures in the presence and absence of metabolic activation (S9). The maximum dose with metaphases present in the 24-hour continuous exposure was 110.16 μg/mL. The test item induced marked evidence of toxicity in all three exposure groups.

The selection of the maximum dose level for the Main Experiment was based on toxicity and was 440 μg/mL for the 4(20)-hour exposure groups in the absence and presence of S9 and was 220 μg/mL for the 24-hour continuous exposure group.

MAIN STUDY RESULTS
- Concurrent vehicle negative and positive control data: all of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range.
All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated.
- Results from cytotoxicity measurements:
- The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Cell Growth Inhibition Test and that there were metaphases suitable for scoring present up to 275 μg/mL in the 4(20)-hour exposure groups in the absence and presence of S9 and up to 165 μg/mL in the 24-hour exposure group.
- A reduced cell pellet was also noted at the end of the exposure period at and above 220 μg/mL and 275 μg/mL in the 4(20)-hour exposure groups in the absence and presence of S9, respectively.
- Hemolysis was observed in the blood cultures at the end of exposure at and above 110 μg/mL in the 4(20)-hour exposure in the absence of S9 and at and above 55 μg/mL in the presence of S9 and in the 24-hour exposure group.
- In the 4(20)-hour exposure group in the absence of S9, close to optimum toxicity was achieved at 275 μg/mL with 61% mitotic inhibition. This was the highest dose level with sufficient metaphases for scoring in this exposure group.
- In the presence of S9, dose-related toxicity was observed and toxicity approaching optimum was achieved at 275 μg/mL with 33% mitotic inhibition. The higher dose level of 330 μg/mL had no metaphases available for scoring.
- The 24-hour exposure group demonstrated a plateau in toxicity at 110 μg/mL, 137.5 μg/mL and 165 μg/mL with 43%, 44% and 41% mitotic inhibition, respectively. The dose level of 220 μg/mL in the 24-hour exposure group had no metaphases available for scoring.
- The maximum dose level selected for metaphase analysis was based on toxicity and was 275 μg/mL for the 4(20)-hour exposures in the absence and presence of S9 and 165 μg/mL for the 24-hour exposure group.
- Genotoxicity results:
- The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline. The maximum dose level scored in the 4(20)-hour exposure in the absence of S9 was considered to achieve optimum toxicity with 61% mitotic inhibition. The maximum dose levels scored in the 4(20)-hour exposure group in the presence of S9 and in the 24-hour exposure group achieved toxicity approaching optimum and were the maximum dose levels with metaphases available for scoring. The steepness of the toxicity curve in these two exposure groups prevented optimum toxicity being achieved and since the test item was tested to its toxic limits with the maximum dose levels scored for chromosome aberrations achieving toxicity approaching optimum it is considered to be adequately tested. The result of the 4(20)-hour exposure group in the absence of S9 where there was no increase in the frequency of cells with chromosome aberrations at optimum toxicity is supporting evidence for the absence of a response in the 24-hour exposure group in the absence of S9.
- The test item did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation.
- The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

HISTORICAL CONTROL DATA (mean ± standard deviation)
- Positive historical control data:
cells with aberrations (-gaps):
4(20)-hour exposure without S9%: 25.13 ± 13.14
4(20)-hour exposure with S9 (2%): 16.22 ± 7.00
24-hour exposure without S9: 26.81 ± 12.28
% cells with polyploids:
4(20)-hour exposure without S9%: 0.01 ± 0.06
4(20)-hour exposure with S9 (2%): 0.03 ± 0.12
24-hour exposure without S9: 0.02 ± 0.11

- Negative (solvent/vehicle) historical control data:
cells with aberrations (-gaps):
4(20)-hour exposure without S9%: 0.48 ± 0.40
4(20)-hour exposure with S9 (2%): 0.54 ± 0.53
24-hour exposure without S9: 0.36 ± 0.43

% cells with polyploids:
4(20)-hour exposure without S9%: 0.04 ± 0.13
4(20)-hour exposure with S9 (2%): 0.03 ± 0.10
24-hour exposure without S9: 0.02 ± 0.07



None

Conclusions:
Under test condition, test item did not induce any statistically significant increase in the frequency of cells with chromosome aberrations, in either the absence or presence of a liver enzyme metabolizing system. The test item was therefore considered to be non-clastogenic to human lymphocytes in vitro.
Executive summary:

In an in vitro chromosome aberration test performed according to OECD Guideline 473 and in compliance with GLP, cultured human lymphocytes were exposed to test item at the following concentrations:

 

Preliminary Toxicity Test (Cell Growth Inhibition Test)

0, 6.89, 13.77, 27.54, 55.08, 110.16, 220.33, 440.65, 881.3 and 1762.6 μg/mL; 4 h exposure time with and without metabolic activation followed by a 20 h recovery period (4(20)-hour with and without S9-mix), and a continuous exposure of 24 h without metabolic activation (24-hour without S9-mix)

 

Main experiment

4(20)-hour without S9-mix: 0, 27.5, 55, 110, 165, 220, 275, 330 and 440 μg/mL ;

4(20)-hour with S9 (2%): 0, 27.5, 55, 110, 165, 220, 275, 330, 385 and 440 µg/mL;

24-hour without S9-mix: 0, 13.75, 27.5, 55, 82.5, 110, 137.5, 165 and 220 μg/mL;

 

Mitotic activity was arrested by addition of colcemid at 0.1 μg/mL for each culture, two hours before the harvest. The cells were then treated with a hypotonic solution, fixed, stained and examined for mitotic indices and chromosomal aberrations. Vehicle and positive controls were also included in this test.

 

All vehicle ( Dimethyl sulphoxide) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9- mix were validated.

 

The test item was markedly toxic and did not induce any statistically significant increases in the frequency of cells with aberrations in any of the three exposure groups. The 4(20)-hour exposure in the absence of S9 achieved marginally greater than optimum toxicity with 61% mitotic inhibition at the maximum dose level scored for aberrations. The 4(20)-hour exposure group in the presence of S9 and the 24-hour exposure group were limited by toxicity but did not achieve optimum toxicity due to the steepness of the toxicity curve and the maximum dose level selected for scoring in these two exposure groups was the highest dose level with sufficient metaphases for scoring. This was 275 μg/mL in the presence of S9 with 33% mitotic inhibition and 165 μg/mL in the 24-hour exposure group with 41% mitotic inhibition.

Under the test conditions, the test item was considered to be non-clastogenic to human lymphocytes in vitro.

This study is considered as acceptable and satisfies the requirement for chromosome aberration endpoint.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Table 7.6/1: Summary of genotoxicity tests

Test n°

Test / Guideline

Reliability

Focus

Strains tested

Metabolic activation

Test concentration

Statement

1

 LEMI, 2005

Ames Test

(OECD 471)

K, rel. 1)

Gene mutation

TA 1535, TA 1537, TA 98,

TA 100,

E. coli WP2

-S9

+S9

Up to cytotoxicity limit

(in acetone)

-S9 : non mutagenic

+S9 : non mutagenic

 2

Koei Techno, 2008

  Similar to Ames Test

(OECD 471)

S, rel. 2)

  Gene mutation

  TA 1535, TA 1537, TA 98,

TA 100,

E. coli WP2

  -S9

+S9

  Up to cytotoxicity limit

(in DMSO)

 -S9 : non mutagenic

+S9 : non mutagenic

 3

COVANCE

2019

 CAT (OECD 473)

K, rel.1

 Chromosomal

Aberration

 Human Lymphocytes

 

-S9

+S9

 
 Up to cytotoxicity limit   

 -S9 : non clastogenic

+S9 : non clastogenic

Gene mutation Assays :

A Key study was identifed (Lemi, 2005). A Bacterial Reverse mutation Assay (Ames test) was performed according to OECD guideline No. 471 with the registered substance (See Table 7.6/1). No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains under the test conditions, with any dose of the substance, either in the presence or absence of metabolic activation. The substance does not induce gene mutations in bacteria whereas all positive control chemicals (with and without metabolic activation) induced significant increase of colonies. These results were confirmed in a supporting study performed similarly to the OECD guideline No. 471 (See Table 7.6/1). The substance is therefore considered as non-mutagenic according to the Ames test.

Chromosomal aberration (Test n°3)

The clastogenic potential of the substance was determined using an in vitro chromosome aberration test in human lymphocytes (OECD 473), which measures the potential of a substance to increase the incidence the of structural chromosome aberrations in cultured human lymphocytes.

None of the dose levels up to the cytotoxicity limit with the substance, either in the presence or absence of metabolic activation, induced significant increases in the frequency of cells with aberrations in either of three experiments. The substance does not induce structural aberrations in the chromosomes of human lymphocytes under activation and non-activation conditions, whereas both positive control chemicals (with and without metabolic activation) induced significant increases in the frequency of aberrant cells.The substance is therefore considered as negative for inducing chromosomal mutations in human lymphocyte cells under activation and non-activation conditions used in this assay.

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification for human health according to the Regulation (EC) No. 1272/2008 (CLP).

Self classification:

Based on the available data, no classification is proposed regarding genetic toxicity according to the CLP and to the GHS.