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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames Test: non mutagenic up to cytotoxic concentrations in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 & E.coli WP2uvrA in a key study (OECD 471, GLP, rel. 1) and a supporting study (similar to OECD 471, rel.2).

- Chromosome aberration test (OECD 473, K, rel. 2): non clastogenic up to precipitating 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:
4 April to 5 May, 2006
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.
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 on 2004-12-16 / Signed on 2005-03-11.
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 method):
30, 90, 300, 900 and 3000 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):
30, 90, 300, 900 and 3000 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: Ethanol
- Test substance preparation:
For the first bacteriostatic activity control, the highest dose studied is 5000 μg/plate. A solution at 200 mg/mL is prepared with ethanol.
For the two other bacteriostatic activity controls and mutagenic assays, the highest dose studied is 3000 μg/plate. A solution at 120 mg/mL is prepared with ethanol.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
other: cis-Platinum (II) Diammine Dichloride
Remarks:
without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other:
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:
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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
BACTERIOSTATIC ACTIVITY CONTROL (STRAIN TA 100)
Bacteriostatic activity control (strain TA 100): In the first control, an important bacteriostatic activity of 94 % is observed in the presence of the test substance at 5000 μg/plate. The test substance is used at the following doses 3000, 900, 300, 90 and 30 μg/plate for bacteriostatic activity control 2 and 3. Then a bacteriostatic activity of 72 % compatible with the maximum level acceptable 75 % is observed, in the presence of the test substance at 3000 μg/plate.
The test substance is tested at the following doses: 3000, 900, 300, 90 and 30 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 (3000, 900, 300, 90 and 30 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:
- In the presence of the test substance at 3000 μg/plate we observe for some bacterial strains like Salmonella typhimurium TA 1535, TA 98 and Escherichia coli WP2(uvrA-) (pKM 101) a significant decrease of the number of spontaneous reversions without or with metabolic activation which confirmed the bacteriostatic activity observed at this dose.

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): 30, 90, 300, 900 and 3000 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): 30, 90, 300, 900 and 3000 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 (3 000, 900, 300, 90 and 30 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).

In the presence of the test substance at 3 000 μg/plate we observe for some bacterial strains like Salmonella typhimurium TA 1535, TA 98 and Escherichia coli WP2(uvrA-) (pKM 101) a significant decrease of the number of spontaneous reversions without or with metabolic activation which confirmed the bacteriostatic activity observed at this dose. 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 cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 04 March to 10 April, 2019.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The substance is adequately characterised but purity is out of specifications. Therefore validation applies with restrictions.
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: Room temperature in the dark until 19 February 2019 and approximately 4°C, in the dark thereafter.
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 21 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 approximately -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, 5.55, 11.11, 22.22, 44.44, 88.88, 177.75, 355.5, 711 and 1422 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, 22.5, 45, 90, 180, 360, and 720 μg/mL, 4(20)-hour with (2%) and without S9-mix and 24-hour without S9-mix.
Justification: The selection of the maximum dose level used in the Main Experiment was based on the lowest precipitating dose level (preliminary toxicity test) and was 720 μg/mL for all three exposure groups.
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 14.22 mg/mL but was soluble in DMSO at 142.2 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 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): 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:
no cytotoxicity, but tested up to precipitating concentrations
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 = 6.48 Pa at 25°C)
- Water solubility: Insoluble at 14.22 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 711 μg/mL, in the 4(20)-hour exposure groups and at and above 355.5 μg/mL in the continuous exposure group. Precipitate was also observed in the blood cultures at the end of the exposure period at and above 355.5 μg/mL in all three exposure groups.
- Microscopic assessment of the slides prepared from the exposed cultures showed that there was no obvious toxicity observed and metaphase cells were present up to 1422 μg/mL in all three exposure groups. The maximum dose level selected for Mitotic Index evaluation in each exposure group was based on the lowest precipitating dose level. The test item induced no evidence of toxicity in any of the exposure groups.
The selection of the maximum dose level used in the Main Experiment was based on the lowest precipitating dose level and was 720 μg/mL for all three exposure groups.
MAIN STUDY RESULTS
-The qualitative assessment of the slides determined that there was no marked toxicity as in the Cell Growth Inhibition Test and that there were metaphases suitable for scoring present up to 720 μg/mL in all three exposure groups.
- Precipitate observations were made at the end of exposure in the parallel blood-free cultures and was noted at and above 360 μg/mL in the 4(20)-hour exposure groups and at 720 μg/mL in the 24-hour exposure group. Precipitate was also observed in the blood cultures at the end of the exposure period at and above 360 μg/mL in both exposure groups in the absence of S9 and at 720 μg/mL in the presence of S9. Based on these observations the lowest precipitating dose level was considered to be 360 μg/mL for all three exposure conditions.
- The results of the mitotic indices (MI) confirm the qualitative observations in that no marked inhibition of mitotic index was observed. In the 4(20)-hour exposure group in the absence of S9, modest toxicity was observed at 360 μg/mL with 27% mitotic inhibition. In the presence of S9, and in the 24-hour exposure group no inhibition of mitotic index was observed.
The maximum dose level selected for metaphase analysis was the lowest precipitating dose level (360 μg/mL).
- Genotoxicity results:
- 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. There was no indication of endoreduplication noted.

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 the test conditions, 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, 5.55, 11.11, 22.22, 44.44, 88.88, 177.75, 355.5, 711 and 1422 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, 22.5, 45, 90, 180, 360, and 720 μg/mL;

4(20)-hour with S9 (2%): 0, 22.5, 45, 90, 180, 360, and 720 μg/mL;

24-hour without S9-mix: 0, 22.5, 45, 90, 180, 360, and 720 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 did not demonstrate any marked toxicity and did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that was the lowest precipitating dose level.

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:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
24 April 22 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
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability under test conditions: Stable at room temperature
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: 39, 78, 156, 313, 625, 1250 and 2500 µg/plate in S. typhimurium TA 100 and TA 1535, without S9 mix
Test 2: 156, 313, 625, 1250, 2500 and 5000 µg/plate in S. typhimurium TA 1537, TA 98 and E. coli WP2, without S9 mix
Test 2: 156, 313, 625, 1250, 2500 and 5000 µg/plate in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2, with S9 mix
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Solubility of Figolide 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. 1 hour 45 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 limit concentration
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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
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: 39, 78, 156, 313, 625, 1250 and 2500 µg/plate in S. typhimurium TA 100 and TA 1535, without S9 mix

Test 2: 156, 313, 625, 1250, 2500 and 5000 µg/plate in S. typhimurium TA 1537, TA 98 and E. coli WP2, without S9 mix

Test 2: 156, 313, 625, 1250, 2500 and 5000 µg/plate in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2, with 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 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, 2006

Ames Test

(OECD 471)

K, rel. 1)

Gene mutation

TA 1535, TA 1537, TA 98,

TA 100,

E. coli WP2

-S9

+S9

Up to cytotoxic concentration - i.e. 3000 µg/plate

(in ethanol)

-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 limit concentration

(in DMSO)

 -S9 : non mutagenic

+S9 : non mutagenic

 3

COVANCE, 2019

CAT

 (OECD 473)

K, rel.2

  chromosomal aberration

 Human lymphocytes

  

 -S9

+S9

Up to precipitating concentrations

 

-S9 : non clastogenic

+S9 : non clastogenic

 

Gene mutation Assays (Tests n° 1 and 2) :

A Key study was identifed (Lemi, 2006). 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 anin vitro chromosome aberration test in human lymphocytes (OECD 473), which measures the potential of a substance to increase the incidence of structural chromosome aberrations in cultured human lymphocytes.

None of the dose levels up to the precipitating concentrations 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

Harmonised classification:

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

Self classification:

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