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Genetic toxicity in vitro

Description of key information

Not mutagenic, bacterial reverse mutation assay (Ames test), S. typhimurium and E. coli, OECD TG 471, Thompson 2021


Not mutagenic (not clastogenic, not aneugenic), in vitro micronucleus assay, human lymphocytes, OECD TG 487, Morris 2021

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experiment start: 21 August 2019; experiment end: 12 September 2019
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:
1997
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine-dependent auxotrophic mutants of S. typhimurium and a tryptophan-dependent mutant of E. coli were used in the test.
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
The S9 Microsomal fraction (Sprague-Dawley) was purchased from Moltox and stored at approximately -196 °C in a liquid nitrogen freezer; Lot No. 4123 was used in this study and the protein level was adjusted to 20 mg/mL. For a 10% S9-mix, an appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution, to result in a final concentration of approximately 10% (v/v) in the S9-mix. The S9 mix contained MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium orthophosphate buffer (100 mM, pH 7.4) and was prepared using sterilized co-factors immediately prior to use and maintained on ice for the duration of the test.
Test concentrations with justification for top dose:
Plate incorporation test (experiment 1): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate
Pre-incubation test (experiment 2): 15, 50, 150, 500, 1500 and 5000 μg/plate
Vehicle / solvent:
Sterile distilled water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-aminoanthracene
Remarks:
Without S9 mix: 4-nitroquinoline-N-oxide, 9-aminoacridine, N-ethyl-N-nitro-N-nitrosoguanidine
With S9 mix: 2-aminoanthracene, benzo(a)pyrene
Details on test system and experimental conditions:
Bacterial cultures were prepared from frozen stocks by incubating for 10 hours at 37 °C in a shaking incubator. The bacteria were obtained from Trinova Biochem GmbH on 27 June 2017 and British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987.
Media: Top agar was prepared using 0.6% w/v Bacto agar (lot number 8255817 07/2023) and 0.5% w/v sodium chloride with 5 mL of 1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan solution added to each 100 mL of top agar. Vogel-Bonner Minimal agar plates were purchased from SGL Ltd (lot number 51673 09/2019).
Pre-cultures: A culture of each of the bacterial strains was prepared by inoculating nutrient broth with the appropriate coded stock culture and incubated, with shaking, for approximately 10 hours at 37 ± 3 °C. The bacterial cell count for each culture was determined by viable count analysis on nutrient agar plates on the day of test and was as follows
TA100 - 3.1 and 4.0E09/mL (experiment 1 and 2)
TA1535 - 1.3 and 3.5E09/mL (experiment 1 and 2)
WP2 uvrA pKM101 - 2.4 and 4.4E09/mL (experiment 1 and 2)
TA98 - 2.2 and 2.2E09/mL (experiment 1 and 2)
TA1537 - 1.7 and 2.409/mL (experiment 1 and 2)
Experiment 1: Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
Conditions experiment 1, without S9 mix: 0.1 mL of the appropriate concentration of test item, solvent vehicle or appropriate positive control was added to 2 mL of molten, trace amino-acid supplemented media containing 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer. These were then mixed and overlaid onto a Vogel-Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.
Conditions experiment 1, with S9 mix: The procedure was the same as described previously (see 3.5.2.2) except that following the addition of the test item formulation and bacterial culture, 0.5 mL of S9-mix was added to the molten, trace amino-acid supplemented media instead of phosphate buffer.
Incubation and scoring, experiment 1: All of the plates were incubated at 37 ± 3 °C for between 48 and 72 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Sporadic manual counts were performed due to spreading colonies which prevented an accurate automated count.
Experiment 2: The concentration range used for Experiment 2 was determined by the results of Experiment 1 and was 15, 50, 150, 500, 1500 and 5000 μg/plate.
Conditions experiment 2, without S9 mix: 0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.1 mL of the test item formulation, solvent vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3 °C for 30 minutes (with shaking) prior to addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating onto Vogel-Bonner plates. Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method. All testing for this experiment was performed in triplicate.
Conditions experiment 2, with S9 mix: The procedure was the same as described previously (see 3.5.3.2) except that following the addition of the test item formulation and bacterial strain culture, 0.5 mL of S9-mix was added to the tube instead of phosphate buffer, prior to incubation at 37 ± 3 °C for 30 minutes (with shaking) and addition of molten, trace amino-acid supplemented media. All testing for this experiment was performed in triplicate.
Incubation and scoring experiment 2: All of the plates were incubated at 37 ± 3 °C for between 48 and 72 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).
Rationale for test conditions:
The maximum concentration in experiment 1 was 5000 μg/plate (the maximum recommended concentration). In experiment 2, six test item concentrations per bacterial strain were selected in the second mutation test in order to achieve both a minimum of four non-toxic concentration levels and the maximum recommended concentration following the change in test methodology from plate incorporation to pre-incubation. No toxicity was observed up to the maximum concentration, with and without S9 mix. No test substance precipitate was observed on the plates at any of the concentrations tested with our without S9 mix in experiments 1 and 2.
Evaluation criteria:
If exposure to a test item produces a reproducible increase, in one or more concentration, in mean revertant colony numbers of at least twice that of the concurrent vehicle controls, with some evidence of a positive concentration-response relationship in at least one strain with or without metabolic activation system, it will be considered to exhibit mutagenic activity in this test system (Mortelmans and Zeiger 2000). No statistical analysis was performed.
If exposure to a test item does not produce an increase in mean revertant colony numbers, it will be considered to show no evidence of mutagenic activity in this test system. No statistical analysis was performed.
If the results obtained fail to satisfy the criteria for a clear “positive” or “negative” response, even after additional testing, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers. The statistical procedures used will usually be Dunnett’s test followed, if appropriate, by trend analysis (Mahon et al, 1989). Biological significance will be considered along with statistical significance. In general, treatment-associated increases in mean revertant colony numbers below twice those of the concurrent vehicle controls (as described above) will not be considered biologically important. It should be noted that it is acceptable to conclude an equivocal response if no clear results can be obtained.
Occasionally, these criteria may not be appropriate to the test data and, in such cases, the Study Director will use his/her scientific judgment.
Statistics:
No statistical analysis was performed.
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium 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
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid

Table: summary of results of plate incorporation test

















































































































































































































































































































































































































































































































































 Number of revertants (mean) +/- SD
 Base-pair substitution strainsFrameshift strains
Concentration TA100TA1535WP2uvrApKM101TA98TA1537
Per PlateWithout S9 mixWith S9 mixWithout S9 mixWith S9 mixWithout S9 mixWith S9 mixWithout S9 mixWith S9 mixWithout S9 mixWith S9 mix
Solvent Control1071441018146114293877
(Water)106142781121082021128
 146140101112012437251416
1.5 µg1221361412117136109115
 1451358101151063020157
 11914111141031431720104
5 µg1111431011941431721149
 11212310141021422732114
 13512515141171301830108
15 µg10315110141171351518125
 110147138112136223067
 12212211161201421920154
50 µg1151171219136130914173
 1211167812815517121510
 10313110111321282813137
150 µg13011313101281332128139
 12913417231691142022312
 105148129121142202879
500 µg1041471212125141201655
 114126712144124162078
 10712412131371131114610
1500 µg10312512151311531522519
 9710110141321352722912
 11816312111311182528102
5000 µg10611579117153232557
 116114813121121194946
 11313717131291383120711
Positive controlsENNG ENNG ENNG 4NQO 9AA 
Concentration 3 µg 5 µg 0.5 µg 0.2 µg 80 µg 
 Without S9 mixWith S9 mixWithout S9 mixWith S9 mixWithout S9 mixWith S9 mixWithout S9 mixWith S9 mixWithout S9 mixWith S9 mix
 486 367 910 126 281 
 439 546 719 168 380 
 408 664 792 228 305 
Positive controls 2AA 2AA 2AA BP 2AA
Concentration  1 µg 2 µg 10 µg 5 µg 2 µg
  1639 295 1143 147 155
  1876 252 1115 130 160
  1869 268 1096 190 179

Table: summary of results of pre-incubation test



































































































































































































































































































































































































































































 Number of revertants (mean) +/- SD
 Base-pair substitution strainsFrameshift strains
Concentration TA100TA1535WP2uvrApKM101TA98TA1537
Per PlateWithout S9 mixWith S9 mixWithout S9 mixWith S9 mixWithout S9 mixWith S9 mixWithout S9 mixWith S9 mixWithout S9 mixWith S9 mix
Solvent Control108114911957727311211
(Water)129136191312111818291216
 134119108841482926914
15 µg1171319121009421441716
 109111499810427291416
 1271451568711032341713
50 µg109102912911192825129
 114136112013612431311814
 14112913101401042130117
150 µg1111207590116173698
 10710151017014626381714
 10112812116816130291713
500 µg10912918710614423281910
 11213117910614515261012
 1141337810714627451015
1500 µg1321341291038722431414
 11712613101061121935910
 12011511810812418241516
5000 µg971301112105124253999
 11813381211212522301614
 113129151910712720361211
Positive controlsENNG ENNG ENNG 4NQO 9AA 
Concentration 3 µg 5 µg 0.5 µg 0.2 µg 80 µg 
 Without S9 mixWith S9 mixWithout S9 mixWith S9 mixWithout S9 mixWith S9 mixWithout S9 mixWith S9 mixWithout S9 mixWith S9 mix
 716 2235 632 205 148 
 637 2753 603 195 197 
 657 2187 687 209 144 
Positive controls 2AA 2AA 2AA BP 2AA
Concentration  1 µg 2 µg 10 µg 5 µg 2 µg
  1491 322 1035 102 175
  1311 350 1067 154 191
  1283 313 1124 121 222
Conclusions:
The substance did not induce gene mutations by base pair changes or frameshifts in the genome of S. typhimurium and E. coli strains, and was considered to be non-mutagenic in the bacterial reverse mutation assay.
Executive summary:

The mutagenic potential of the substance was studied under GLP to OECD TG 471 with histidine-dependent auxotrophic mutants of Salmonella typhimurium, strains TA1535, TA1537, TA98 and TA100, and a tryptophan-dependent mutant of Escherichia coli, strain WP2uvrApKM101. The bacteria were exposed to the substance diluted in sterile distilled water. Two independent mutation experiments were performed in the presence and absence of liver preparations (S9-mix) from rats treated with phenobarbital and β-naphthoflavone. Experiment 1 was a standard plate incorporation assay and experiment 2 a pre-incubation test. The maximum concentration of the test item in experiment 1 was 5000 µg/plate, the standard limit concentration recommended in the regulatory guideline that this assay follows. This maximum concentration was also selected for experiment 2. There was no toxicity, evident as a reduction in the number of revertants (below an induction factor of 0.5) or a reduction in the background lawn, in any of the five tester strains either with or without S9 mix following exposure to the substance in either the plate incubation or pre-incubation experiments. No test item precipitate was observed on the plates at any of the concentrations tested in either the presence or absence of metabolic activation (S9-mix) in experiments 1 and 2. The vehicle (sterile distilled water) control plates gave counts of revertant colonies generally within the normal range. There were no biologically relevant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any concentration of the test item, either with or without metabolic activation (S9-mix) in experiments 1 and 2. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start: 18 September 2019, experimental end: 28 October 2019
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:
2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
Not applicable: The occurrence of micronuclei in interphase cells provides an indirect, but easy and rapid measure of structural chromosomal damage and aneugenicity in cells that have undergone cell division during or after exposure to the test substance. Micronuclei arise from chromosomal fragments or whole chromosomes and rarely occur spontaneously, but are inducible by clastogens or agents affecting the spindle apparatus (Countryman and Heddle, 1976; Obe and Beek, 1982, Rosefort et al, 2004).
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
Sufficient whole blood was drawn on the day of experiment from the peripheral circulation of a non-smoking volunteer (female aged 30 years in the preliminary test and female aged 22 in the main experiment).
Cytokinesis block (if used):
Cytochalasin B was formulated in DMSO and added to all cultures, after washing, at the end of the exposure period at a final concentration of 4.5 μg/mL for a period of 24 hours.
Metabolic activation:
with and without
Metabolic activation system:
The Rat S9 Microsomal fraction was purchased from Moltox, Lot No. 4061, Expiry 11 July 2021. The S9 was adjusted to a final protein content of 20 mg/mL before use. The S9-mix was prepared prior to the dosing of the test cultures and contained the S9 fraction (20% (v/v)), MgCl2 (8mM), KCl (33mM), sodium orthophosphate buffer pH 7.4 (100mM), glucose-6-phosphate (5mM) and NADP (5mM). 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:
The maximum concentration was 1311 μg/mL. This was calculated to be equivalent to 10mM (based on the molecular weight of 131.09 g/mol), the maximum recommended concentration.
Preliminary toxicity test: The dose range of test item used was 0, 5.12, 10.24, 20.48, 40.97, 81.94, 163.88, 327.75, 655.5 and 1311 μg/mL.
Main experiment: The dose range of test item used for all three exposure groups was 0, 40.97, 81.94, 163.88, 327.75, 655.5 and 1311 μg/mL.
Vehicle / solvent:
Minimal Essential Medium (MEM)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: Demecolcine
Remarks:
With S9 mix: cyclophosphamide
Without S9 mix: mitomycin C, demecolcine
Details on test system and experimental conditions:
Cell culture: 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 °C with 5% CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).
Culture conditions: Duplicate lymphocyte cultures (A and B) and quadruplicate for the vehicle were established for each concentration level by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture:
- 8.05-9.05 mL MEM, 10% (FBS)
- 0.1 mL Li-heparin
- 0.1 mL phytohaemagglutinin
- 0.75 mL heparinized whole blood
4-hour exposure with S9 mix: After 44 - 48 hours incubation at approximately 37 ºC, 5% CO2 in humidified air, the
cultures were transferred to tubes and centrifuged. Approximately 9 mL of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and 1.0 mL of the appropriate solution of vehicle control or test item was added to each culture. For the positive control, 0.1 mL of the appropriate solution was added to the cultures. 1mL of 20% S9 mix (i.e. 2% final concentration of S9 in standard co-factors) was added to the cultures of the Preliminary Toxicity Test and Main Experiment. After 4 hours at approximately 37 °C, 5% CO2 in humidified air, the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the original culture medium. The cells were then re-incubated for a further 24 hours at approximately 37 °C in 5% CO2 in humidified air in the presence of 4.5 μg/mL Cytochalasin B.
4-hour exposure without S9 mix: After 44 - 48 hours incubation at approximately 37 °C, 5% CO2 in humidified air, the
cultures were transferred to tubes and centrifuged. Approximately 9 mL of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and 1.0 mL of the appropriate solution of vehicle control or test item was added to each culture. For the positive control, 0.1 mL of the appropriate solution was added to the cultures. 1.0 mL of 20% S9-mix (i.e. 2% final concentration of S9 in standard co factors) was added to the cultures of the Preliminary Toxicity Test and the Main Experiment. All cultures were then returned to the incubator. The nominal total volume of each culture was 10 mL. After 4 hours at approximately 37 °C, the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium, supplemented with Cytochalasin B at a final concentration of 4.5 μg/mL, and then incubated for a further 24 hours.
24-hour exposure without S9 mix: The exposure was continuous for 24 hours in the absence of metabolic activation. Therefore, when the cultures were established the culture volume was a nominal 9 mL. After 44 - 48 hours incubation the cultures were removed from the incubator and dosed with 1.0 mL of vehicle control, test item dose solution or 0.1 mL of positive control solution. The nominal total volume of each culture was 10 mL. The cultures were then incubated for 24 hours, the tubes and the cells washed in MEM before resuspension in fresh MEM with serum. At this point Cytochalasin B was added at a final concentration of 4.5 μg/mL, and then the cells were incubated for a further 24 hours. The extended exposure detailed above does not follow the suggested cell treatment schedule in the Guideline. The OECD guideline permits modified treatment times where justified. This design avoids any potential interaction between Cytochalasin B and the test item during exposure to the cells and any effect this may have on the activity or response (Whitwell et al. 2019). Additionally, as the stability or reactivity of the test item is unknown prior to the start of the study this modification of the schedule is considered more effective and reproducible due to the in-house observations on human lymphocytes and their particular growth characteristics in this study type and also the significant laboratory historical control data using the above format. The Preliminary Toxicity Test was performed using the exposure conditions as described for the Main Experiment but using single cultures for the test item concentration levels and duplicate cultures for the vehicle controls, whereas the Main Experiment used duplicate
cultures for the test item and quadruplicate cultures for the vehicle controls.
Preliminary toxicity test: Three exposure groups were used:
i) 4-hour exposure to the test item without S9-mix, followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
iii) 24-hour continuous exposure to the test item without S9-mix, followed by a 24-hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test item precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods. Using a qualitative microscopic evaluation of the microscope slide preparations from each treatment culture, appropriate dose levels were selected for the evaluation of the frequency of binucleate cells and to calculate the cytokinesis block proliferation index (CBPI). Coded slides were evaluated for the CBPI. The CBPI data were used to estimate test item toxicity (cytostasis) and for selection of the dose levels for the exposure groups of the Main Experiment.
Main experiment: Three exposure groups were used for the Main Experiment:
i) 4-hour exposure to the test item without S9-mix, followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
iii) 24-hour continuous exposure to the test item without S9-mix, followed by a 24-hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
Cell harvest: At the end of the Cytochalasin B treatment period the cells were centrifuged, the culture medium was drawn off and discarded, and the cells resuspended in MEM. The cells were then treated with a mild hypotonic solution (0.0375M KCl) before being fixed with fresh methanol/glacial acetic acid (19:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 ºC prior to slide making.
Preparation of microscope slides: 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 with gentle warming. Each slide was permanently labeled with the appropriate identification data.
Staining: When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.
Rationale for test conditions:
Preliminary toxicity test: There was no precipitate of the test item observed in the parallel blood-free cultures at the end of the exposure, in any of the three exposure groups. The test item did not demonstrate any marked toxicity in any of the three exposure groups with and without S9 mix.
The maximum dose level selected for the main experiment was the maximum recommended dose level (1311 μg/mL equivalent to 10 mM) since there was no marked toxicity or precipitate observed in any of the three exposure groups.
Evaluation criteria:
Provided that all acceptance criteria have been met, the test item is considered to be clearly positive if, in any of the experimental conditions examined:
- At least one of the test concentrations exhibits a statistically significant increase in the frequency of micronucleated cells compared with the concurrent vehicle control.
- The increase in the frequency of micronucleated cells is dose-related when evaluated with an appropriate trend test.
- Any of the results are outside the distribution of the historical vehicle control data (above the upper 95% control limit).
If all of these criteria are met, the test item will be considered able to induce chromosome breaks and/or gain or loss in the test system.
Provided that all acceptance criteria have been met, a clearly negative response will be claimed if, in all of the experimental conditions examined:
- None of the test concentrations exhibits a statistically significant increase in the frequency of micronucleated cells compared with the concurrent vehicle control.
- There is no concentration-related increase when evaluated with an appropriate trend test.
- All results are inside the distribution of the historical vehicle control data (within the 95% control limits).
If all of these criteria are met, the test item will be considered unable to induce chromosome breaks and/or gain or loss in the test system.
In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations. The Study Director may make a judgement based on experience and the biological relevance of the data and any justification for acceptance of the data will be included in the report. Scoring additional cells (where appropriate) or performing a repeat experiment possibly using modified experimental conditions (e.g. concentration spacing, other metabolic activity.
Statistics:
When there is no indication of any increase at all dose levels tested then statistical analysis may not be necessary. In all other circumstances comparisons were made between the appropriate vehicle control and each individual dose level, using Chi-squared Test on observed numbers of cells with micronuclei (Hoffman et al., 2003). A statistically significant response was recorded when the p value calculated from the statistical analysis of the frequency of binucleate cells with micronuclei was less than 0.05 when compared to its concurrent control. The dose-relationship was assessed using a linear regression model. An arcsine square-root transformation was applied to the percentage of binucleated cells containing micronuclei (excluding positive control(s)). A linear regression model was applied to these transformed values with dose values fitted as the explanatory variable. The F-value from the model was assessed at the 5% statistical significance level.
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
Minimal toxicity was achieved at the maximum concentration following 24 hours of exposure.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Preliminary toxicity test: There was no precipitate of the test item observed in the parallel blood-free cultures at the end of the exposure, in any of the three exposure groups. Microscopic assessment of the slides prepared from the exposed cultures showed that binucleate cells were present at up to 1311 μg/mL in the 4-hour exposure, both in the presence and absence of metabolic activation (S9) and in the 24-hour continuous exposure group. The test item did not demonstrate any marked toxicity in any of the three exposure groups.
Main experiment: The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were binucleate cells suitable for scoring at the maximum dose level of test item, 1311 μg/mL in all three exposure groups. There was no precipitate of the test item observed in the blood cultures at the end of the exposure period.
The CBPI data confirm the qualitative observations in that there was no marked toxicity in the 4-hour exposure groups in the absence and presence of S9. Minimal toxicity was observed in the 24-hour exposure group at the maximum dose level where 21% cytostasis was achieved. The maximum concentration selected for analysis of binucleate cells was the maximum recommended concentration, 10 mM (1311 μg/mL).
The vehicle control cultures for all three exposure groups had frequencies of binucleate cells with micronuclei which were considered acceptable for addition to the laboratory’s historical negative control data and were within the 95% control limits. The positive control items induced statistically significant increases in the frequency of cells with micronuclei which were compatible with the laboratory’s historical positive control data base. The cytotoxicity achieved by the positive control items did not exceed the limits of acceptability recommended in the OECD 487 guideline. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item was soluble in DMSO at 131.1 mg/mL. The pH and osmolality measurements when the test item formulations were dosed into media were measured. There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm.


Table: results of pH and osmolality readings












































Conc. in μg/mL05.1210.2420.4840.9781.94163.88327.75655.51311
pH7.387.387.397.387.387.377.347.307.166.90
Osmolality in mOsm314316N/A314N/A316N/A316N/A324

Table: Summary of results






















































































































Exposure conditionConcentration (μg/mL)Mean cytostasis (%)% binucleated cells containing micronuclei
Meanp-valueTrend test p-value
4-hour, without S9 mixMinimum essessentiel medium (MEM)00.13-0.660
327.75120.250.2623
655.580.150.8025
131170.10-
0.2 mitomycin C341.854.19E-14*-
4-hour, with S9 mixMEM00.30-0.253
327.7510.350.7452
655.550.550.1396
131100.400.5264
8 cyclophosphamide582.455.04E-15*-
24-hour, without S9 mixMEM00.25-0.791
327.7580.400.3166
655.570.20-
1311210.300.7233
0.075 demecolcine543.652.17E-26*-
* p <0.001
Conclusions:
The substance did not induce any statistically significant increases in the frequency of binucleated cells with micronuclei in the absence or presence of S9 mix. The substance was considered to be clearly non-mutagenic (not clastogenic, not aneugenic) to human lymphocytes in vitro.
Executive summary:

An in vitro study for the detection of the clastogenic and aneugenic potential of the substance on the nuclei of normal human lymphocytes was performed under GLP to OECD TG 487. Duplicate cultures of human lymphocytes obtained from healthy, non-smoking female volunteers aged 22 and 30 years were treated with the test item and evaluated for micronuclei in binucleate cells for at least three concentrations, together with vehicle (minimum essentiel medium, quadruplicate cultures) and positive controls (duplicate cultures). Three exposure conditions in a single experiment were used for the study using a 4 hour exposure in the presence of S9 mix at a 2% final concentration, and a 4 hour or 24-hour exposure in the absence of metabolic activation. At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 hours in the presence of Cytochalasin B. The preliminary toxicity test indicated that the maximum concentration of 10 mM (1311 µg/mL) did not lead to marked toxicity or precipitation, and this maximum concentration was also used in the main experiment. All Minimal Essential Medium vehicle controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes and matching the laboratory historical vehicle control data. The positive control items induced statistically significant increases in the frequency of cells with micronuclei, demonstrating the sensitivity of the assay and the efficacy of the S9-mix. The test substance did not induce any statistically significant increases in the frequency of cells with micronuclei at any test concentration. All micronucleus frequencies remained within the 95% control limits of the historical vehicle control and there were no concentration related trends. The criteria for a clearly negative response were met in each treatment condition.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

The substance has been found to be non-mutagenic in two valid and reliable in vitro genotoxicity studies conducted under GLP to OECD TG 471 and OECD TG 487. In the Ames study, the substance was tested with two independent experiments in the plate incorporation test and the pre-incubation test with and without S9 mix. The maximum test concentration was 5000 µg/plate in both experiments. No significant toxicity or precipitation occurred at this highest concentration. All strains were tested with triplicate plates at all concentrations of test substance, solvent and positive controls. There was no statistically significant increase in the number of revertant colonies at any test concentration in the two experiments with and without S9 mix and the substance was found to be clearly non-mutagenic. In the in vitro micronucleus assay, duplicate cultures of human lymphocytes were exposed for 4 hours in the absence and presence of S9 mix and for 24 hours in the absence of S9 mix, before cell cultures were washed and incubated for 24 hours after addition of cytochalasin B. The maximum test concentration was 10 mM (1311 µg/mL). The test substance was dissolved in minimum essentiel medium before application. No marked toxicity or precipitation was observed at any test concentration. All vehicle controls had frequencies of cells with micronuclei within the expected range. The positive control substances induced statistically significant increases in the frequency of cells with micronuclei, showing the sensitivity of the test system and the adequacy of the S9 mix. The test substance did not induce any statistically significant increases in the frequency of cells with micronuclei, and all observed micronucleus frequencies were within the 95% control limits of the historical vehicle control. The substance was found to be clearly non-mutagenic (i.e., non-clastogenic, non-aneugenic) in the valid study.

Additional information

Justification for classification or non-classification

Based on the negative results of the fully reliable and valid bacterial reverse mutation assay and the in vitro micronucleus assay with human lymphocytes the substance does not meet the classification criteria for mutagenicity under Regulation (EC) No. 1272/2008.