2-[7-(diethylamino)-2-oxo-2H-1-benzopyran-3-yl]benzoxazole-5-sulphonamide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames Test: Under the conditions of the study, it is concluded that the test material is mutagenic in the Salmonella typhimurium reverse mutation assay and not mutagenic in the Escherichia coli reverse mutation assay. The mutagenicity was confined only to incubations with metabolic activation in one strain.

Chromosome Aberation Assay: Under the conditions of this study, the test material is not considered to be a chromosome damaging (clastogenic) agent in vitro using human lymphocytes.

Mouse Lymphoma Assay:Under the conditions of this study, it is concluded that the test material is not mutagenic in the TK mutation test system.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 July 2016 to 03 August 2016

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

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

- Histidine requirement in the Salmonella typhimurium strains (Histidine operon).
- Tryptophan requirement in the Escherichia coli strain (Tryptophan operon).

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

- Type and identity of media: Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid LTD, Hampshire, England) and incubated in a shaking incubator (37 ± 1 °C, 150 rpm), until the cultures reached an optical density of 1.0 ± 0.1 at 700 nm (10^9 cells/mL). Freshly grown cultures of each strain were used for testing.
- Properly maintained: Yes. The Salmonella typhimurium strains are regularly checked to confirm their histidine requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV sensitivity and the number of spontaneous revertants. Stock cultures of the strains were stored in liquid nitrogen (-196 °C).

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

- Type and identity of media: Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid LTD, Hampshire, England) and incubated in a shaking incubator (37 ± 1 °C, 150 rpm), until the cultures reached an optical density of 1.0 ± 0.1 at 700 nm (10^9 cells/mL). Freshly grown cultures of each strain were used for testing.
- Properly maintained: Yes. The strain is regularly checked to confirm the tryptophan requirement, UV-sensitivity and the number of spontaneous revertants. Stock cultures were stored in liquid nitrogen (-196 °C).

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (rat liver S9-mix induced by Aroclor 1254)

Test concentrations with justification for top dose:

- Dose range finding study (TA100 and WP2uvrA only): 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate (absence and presence of S9-mix)
- Experiment 1 (TA1535, TA1537 and TA98): 5.4, 17, 52, 164, 512 and 1600 μg/plate (absence and presence of S9-mix)
- Experiment 2 (all strains): 86, 154, 275, 492, 878 and 1568 μg/plate (absence and presence of S9-mix)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: A solubility test was performed. The test material could not be dissolved in water. A homogeneous suspension of 50 mg/mL was obtained in dimethyl sulfoxide. At concentrations of 16 mg/mL and lower the test material was dissolved in dimethyl sulfoxide. The stock solution was treated with ultrasonic waves to obtain an homogeneous suspension (Dose range finding test) or until the test material had completely dissolved (mutation experiments). The lower test concentrations were prepared by subsequent dilutions in DMSO.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191; 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DOSE RANGE FINDING TEST/ MUTATION ASSAY
Selection of an adequate range of doses was based on a dose range finding test with the strains TA100 and WP2uvrA, both with and without 5 % (v/v) S9-mix and reported as part of the first mutation experiment.

MUTATION ASSAY
At least five different doses (increasing with approximately half-log steps) of the test material were tested in each strain both in the absence and presence of 5 % (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. In a follow-up experiment with additional parameters, the test material was tested both in the absence and presence of 10 % (v/v) S9-mix in all tester strains.
Top agar in top agar tubes was melted by heating to 45 ± 2 °C. The following solutions were successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture (10^9 cells/mL) of one of the tester strains, 0.1 mL of a dilution of the test material in DMSO and either 0.5 mL S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted.

NUMBER OF REPLICATIONS: Testing was performed in triplicate

COLONY COUNTING
The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test material precipitate to interfere with automated colony counting were counted manually. Evidence of test material precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.

DETERMINATION OF CYTOTOXICITY
- Method: To determine the toxicity of the test material, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined.

Evaluation criteria:

ACCEPTABILITY OF THE ASSAY
The assay is considered acceptable if it meets the following criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at the testing facility.
b) The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5 % of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.

DATA EVALUATION
In addition to the criteria stated below, any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range.
A test material is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.
A test material is considered positive (mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three (3) times the concurrent vehicle control.
b) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Statistics:

No formal hypothesis testing was done.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium, other: TA1535, TA1537 and TA100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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:

not applicable

Positive controls validity:

valid

Additional information on results:

DOSE RANGE FINDING TEST/FIRST MUTATION EXPERIMENT
- Precipitate: In the dose range finding test, precipitation of the test material on the plates was observed at the start of the incubation period at concentrations of 512 μg/plate and upwards. At the end of the incubation period, precipitation was observed at 164 μg/plate and above (absence of S9-mix) and at 512 and 1600 μg/plate and upwards (TA100 and WP2uvrA, respectively). In the first experiment, precipitation of the test material on the plates was observed at the start and at the end of the incubation period at concentrations of 512 μg/plate and upwards.
- Toxicity: No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed. In strain TA98 (absence of S9-mix) a fluctuation in the number of revertant colonies below the laboratory historical control data range was observed at the lowest dose level tested. However, since no dose-relationship was observed, this reduction is not considered to be caused by toxicity of the test material. It is more likely this reduction is caused by an incidental fluctuation in the number of revertant colonies.
- Mutagenicity: In the presence of S9-mix, the test material induced an up to 3.8-dose related increase in tester strain TA98. In all other tester strains, no increase in the number of revertants was observed upon treatment with the test material under all conditions tested.

MUTATION EXPERIMENT 2
- Precipitate: Precipitation of the test material on the plates was observed at the start of the incubation period at the concentration of 492 μg/plate. At the end of the incubation period, precipitation was observed at 1568 μg/plate in the absence of S9-mix, except in tester strain TA100. In the presence of S9-mix, precipitation was only observed in tester strain TA1535 at 1568 μg/plate.
- Toxicity: The bacterial background lawn was reduced in all tester strains in the presence of S9-mix at the highest tested concentration. In addition, a reduction in the bacterial background lawn was observed in tester strain TA100 in the absence of S9-mix and a biologically relevant decrease in the number of revertants was observed in tester strain TA1537 in the absence of S9-mix.
- Mutagenicity: In the presence of S9-mix, the test material induced an up to 4.4-dose related increase in tester strain TA98. In all other tester strains, no increase in the number of revertants was observed upon treatment with the test material under all conditions tested.

DISCUSSION
In the presence of S9-mix, the test material induced dose related increases in tester strain TA98. The increases observed were above the laboratory historical control data range, in two independently repeated experiments and were up to 4.4-fold the concurrent vehicle controls and therefore considered to be biologically relevant.
In the other tester strains (TA1535, TA1537, TA100 and WP2uvrA), the test material did not induce a significant dose-related increase in the number of revertant colonies in any experiment.
The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Table 1: Dose Range-finder and Experiment 1 (Plate incorporation assay 5 % S9)

+/- S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		Base-pair Substitution Type		Frameshift Type
		TA100	TA1535	WP2uvrA	TA98	TA1537
-	PC DMSO 1.7 5.4 17 52 164 512 1600 5000	711 76 83 68 77 92 NP 90 SP 88 SP 71 MP 68 n MP	815 9 - 6 7 12 15 NP 10 SP 10 n MP -	1367 24 25 24 28 25 NP 26 SP 19 SP 21 MP 13 n MP	1227 10 - 6 14 10 13 NP 11 SP 16 n MP -	1187 4 - 4 4 6 7 NP 6 SP 4 n MP -
+	PC DMSO 1.7 5.4 17 52 164 512 1600 5000	1003 75 60 73 71 80 79 NP 82 SP 79 MP 72 n MP	201 12 - 8 7 5 11 NP 14 SP 9 n MP -	390 32 34 32 38 33 33 34 NP 43 SP 19 n MP	743 22 - 14 18 23 48 NP 58 SP 84 n MP -	362 6 - 5 11 7 12 NP 7 SP 14 n MP -

Mean number of revertant colonies/3 replicate plates

PC = Positive control

MP = Moderate Precipitate

NP = No precipitate

SP = Slight Precipitate

n = Normal bacterial background lawn

 

Table 2: Experiment 2 (Plate incorporation assay 10 % S9)

+/- S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		Base-pair Substitution Type		Frameshift Type
		TA100	TA1535	WP2uvrA	TA98	TA1537
-	PC DMSO 86 154 275 492 878 1568	1049 98 128 115 133 105 103 n 122 s NP	947 18 17 11 14 18 13 NP 12 n SP	1494 23 20 22 28 21 23 NP 21 n SP	1466 16 15 17 21 14 14 NP 16 n SP	1029 5 7 5 3 4 4 NP 1 n SP
+	PC DMSO 86 154 275 492 878 1568	1062 97 111 114 117 112 108 n 111 s NP	138 14 12 16 16 18 16 n NP 12 s SP	435 32 30 29 26 22 n 37 s 33 s NP	475 20 24 38 34 66 n 70 s 88 s NP	480 10 7 9 6 6 12 n 13 s NP

Mean number of revertant colonies/3 replicate plates

PC = Positive control

NP = No precipitate

SP = Slight Precipitate

n = Normal bacterial background lawn

s = Bacterial background lawn slightly reduced

Conclusions:

Under the conditions of this study, it is concluded that the test material is mutagenic in the Salmonella typhimurium reverse mutation assay and not mutagenic in the Escherichia coli reverse mutation assay. The mutagenicity was confined only to incubations with metabolic activation in one strain.

Executive summary:

The potential of the test material to cause mutagenic effects in bacteria was assessed in accordance with the standardised guidelines OECD 471 and EU Method B.13/14 under GLP conditions.

The test material was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by Aroclor 1254).

In the dose range finding test, the test material was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. The test material was tested up to or beyond a precipitating dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. Results of this dose range finding test were reported as part of the first mutation assay.

Based on the results of the dose range finding test, the test material was tested in the first mutation assay at a concentration range of 5.4 to 1600 μg/plate in the absence and presence of 5 % (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. The test material was precipitated on the plates at concentrations of 512 and 1600 μg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

In a follow-up experiment of the assay with additional parameters, the test material was tested at a concentration range of 86 to 1568 μg/plate in the absence and presence of 10 % (v/v) S9-mix in all strains. Precipitation of the test material on the plates was observed at 1568 μg/plate in the absence of S9-mix, except in tester strain TA100 and in the presence of S9-mix in tester strain TA1535 only. In the presence of S9-mix, the bacterial background lawn was reduced in all tester strains at the highest tested concentration. In the absence of S9-mix, a reduction in the bacterial background lawn was observed in tester strain TA100 and a biologically relevant decrease in the number of revertants was observed in tester strain TA1537.

In the presence of S9-mix, the test material induced dose related increases in tester strain TA98. The increases observed were above the laboratory historical control data range, in two independently repeated experiments and were up to 4.4-fold the concurrent vehicle controls and therefore considered to be biologically relevant. In the other tester strains (TA1535, TA1537, TA100 and WP2uvrA), the test material did not induce a significant dose-related increase in the number of revertant colonies in any experiment.

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Under the conditions of the study, it is concluded that the test material is mutagenic in the Salmonella typhimurium reverse mutation assay and not mutagenic in the Escherichia coli reverse mutation assay. The mutagenicity was confined only to incubations with metabolic activation in one strain.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 July 2016 to 28 September 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

2014 and 2016

Deviations:

no

GLP compliance:

yes

Type of assay:

other: chromosome aberration in peripheral human lymphocytes

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Blood was collected from healthy adult, non-smoking volunteers (approximately 18 to 35 years of age). Blood samples were collected by venepuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin (Vacuette, Greiner Bio-One, Alphen aan den Rijn, The Netherlands). Immediately after blood collection lymphocyte cultures were started.
- Suitability of cells: Cultured peripheral human lymphocytes were used as test system as recommended in the guideline.
- Average Generation Time (AGT): The AGT of the cells and the age of the donor at the time the AGT was determined are: Dose range finding study/ First cytogenetic assay: age 26, AGT = 12.8 h; Second cytogenetic assay: age 27, AGT = 13.5 h; and Cytogenetic assay 2A: age 33, AGT = 12.7 h

MEDIA USED
- Type and identity of media: Culture medium consisted of RPMI 1640 medium, supplemented with 20 % (v/v) heat-inactivated (56 °C; 30 min) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 μg/mL respectively) and 30 U/mL heparin.
- Lymphocyte cultures: Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 mL (9 mg/mL) phytohaemagglutinin was added.
- Environmental conditions: All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100 % containing 5.0 ± 0.5 % CO2 in air in the dark at 37.0 ± 1.0 °C.

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

The highest concentration analysed was selected based on the solubility of the test material in the culture medium or on inhibition of the mitotic index of about 50 % or greater.

- Dose range finding test / First cytogenetic assay: At a concentration of 52 μg/mL, the test material precipitated in the culture medium. At the 3 h exposure time, blood cultures were treated in duplicate with 5.4, 17 and 52 μg test material/mL culture medium with and without S9-mix (first cytogenetic assay). At the 24 hour and 48 hour exposure time single blood cultures were treated with 0.55, 1.7, 5.4, 17, 52 and 164 μg/mL culture medium without S9-mix (dose range finding test).

- Second cytogenetic assay (without S9-mix): 1, 10 and 52 μg/mL culture medium (24 h exposure time, 24 h fixation time) and 1, 52, 100, 150, 200 and 250 μg/mL culture medium (48 h exposure time, 48 h fixation time).

- Cytogenetic assay 2A (without S9-mix): 1, 50, 150, 200, 250 and 300 μg/mL culture medium (48 h exposure time, 48 h fixation time). Further investigation showed that a concentration of 300 μg/mL precipitated in the culture medium after 48 hours.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

DOSE RANGE FINDING TEST / FIRST CYTOGENETIC ASSAY
In order to select the appropriate dose levels, cytotoxicity data were obtained in a dose range finding test in the absence and in the presence of 1.8 % (v/v) S9-fraction in duplicate.
Lymphocytes (0.4 mL blood of a healthy donor was added to 5 mL or 4.8 ml culture medium, without and with metabolic activation, respectively, and 0.1 mL (9 mg/mL) Phytohaemagglutinin) were cultured for 48 ± 2 h and thereafter exposed to selected doses for 3, 24 and 48 h in the absence of S9-mix or for 3 h in the presence of S9-mix. A negative control was included at each exposure time.
The highest tested concentration was determined by the solubility of the test material in the culture medium. The test material precipitated at concentrations of 52 μg/mL and upwards. The lymphocytes were cultured in duplicate at the 3 h exposure time and appropriate positive controls were included.
The cytogenetic assay was carried out as described by Evans, 1984 with minor modifications.
After 3 h exposure to the test material in the absence or presence of S9-mix, the cells were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and cells were rinsed with 5 mL Hanks’ Balanced Salt Solution (HBSS). After a second centrifugation step, HBSS was removed and cells were re-suspended in 5 mL culture medium and incubated for another 20 - 22 h (24 h fixation time). The cells that were exposed for 24 and 48 h in the absence of S9-mix were not rinsed after exposure but were fixed immediately (24 and 48 h fixation time).
Cytotoxicity of the test material in the lymphocyte cultures was determined using the mitotic index. No cytotoxicity was observed in the duplicate cultures of the 3 h exposure time and the slides were scored for chromosome aberrations. The pilot study (short term exposure period) was used as the first cytogenetic assay.
Based on the results of the dose range finding test an appropriate range of dose levels was chosen for the second cytogenetic assay considering the highest dose level was determined by the solubility. As clear negative results were obtained in the presence of metabolic activation, the repetition of the experiment was not considered necessary.

SECOND CYTOGENETIC ASSAY
To confirm the results of the first cytogenetic assay a second cytogenetic assay was performed with an extended exposure time of the cells in the absence of S9-mix. To be able to select appropriate dose levels for scoring of chromosome aberrations a repeat assay had to be performed.
Lymphocytes were cultured for 48 ± 2 h and thereafter exposed in duplicate to selected doses of the test material for 24 and 48 h in the absence of S9-mix. The cells were not rinsed after exposure but were fixed immediately after 24 and 48 h (24 and 48 h fixation time). Appropriate negative and positive controls were included in the second cytogenetic assay.

CHROMOSOME PREPARATION
During the last 2.5 - 3 h of the culture period, cell division was arrested by the addition of the spindle inhibitor colchicine (0.5 μg/mL medium). Thereafter the cell cultures were centrifuged for 5 min at 365 g and the supernatant was removed. Cells in the remaining cell pellet were swollen by a 5 min treatment with hypotonic 0.56 % (w/v) potassium chloride solution at 37 °C. After hypotonic treatment, cells were fixed with 3 changes of methanol:acetic acid fixative (3:1 v/v).

PREPARATION OF SLIDES
Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96 % (v/v) ethanol/ether and cleaned with a tissue. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10 - 30 min with 5 % (v/v) Giemsa (Merck) solution in Sörensen buffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry. The dry slides were automatically embedded in a 1:10 mixture of xylene/pertex and mounted with a coverslip in an automated cover slipper.

MITOTIC INDEX/DOSE SELECTION FOR SCORING OF THE CYTOGENETIC ASSAY
The mitotic index of each culture was determined by counting the number of metaphases from at least 1000 cells (with a maximum deviation of 5 %). At least three analysable concentrations were used for scoring of the cytogenetic assay. Chromosomes of metaphase spreads were analysed from those cultures with an inhibition of the mitotic index of 55 ± 5 %, whereas the mitotic index of the lowest dose level was approximately the same as the mitotic index of the solvent control. Also cultures treated with an intermediate dose were examined for chromosome aberrations. After 3 and 24 hours exposure time, the test material was not cytotoxic and difficult to dissolve in aqueous solutions, the highest concentration analysed was determined by the solubility in the culture medium.

ANALYSIS OF SLIDES FOR CHROMOSOME ABERRATIONS
One hundred and fifty metaphase chromosome spreads per culture were examined by light microscopy for chromosome aberrations. In case the number of aberrant cells, gaps excluded, was ≥ 38 in 75 metaphases, no more metaphases were examined. Only metaphases containing 46 ± 2 centromeres (chromosomes) were analysed. The number of cells with aberrations and the number of aberrations were calculated. Since the lowest concentration of MMC-C resulted in a positive response the highest concentration was not examined for chromosome aberrations.

Evaluation criteria:

ACCEPTABILITY OF THE ASSAY
A chromosome aberration test is considered acceptable if it meets the following criteria:
a) The concurrent negative control data are considered acceptable when they are within the 95 % control limits of the distribution of the historical negative control database.
b) The concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database.
c) The positive control material induces a statistically significant increase in the number of cells with chromosome aberrations.

DATA EVALUATION
A test material is considered positive (clastogenic) in the chromosome aberration test if:
a) At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the 95 % control limits of the historical control data range.
A test material is considered negative (not clastogenic) in the chromosome aberration test if:
a) None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are inside the 95 % control limits of the negative historical control data range.

Statistics:

Graphpad Prism version 4.03 (Graphpad Software, San Diego, USA) was used for statistical analysis of the data.
Statistical significance was evaluated by the Fisher’s exact test (one-sided, p<0.05).

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

Positive controls validity:

valid

Additional information on results:

DOSE RANGE FINDING TEST / FIRST CYTOGENETIC ASSAY
At a concentration of 52 μg/mL the test material precipitated in the culture medium. Both in the absence and presence of S9-mix, the test material did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations (Tables 1 and 2).
Both in the absence and presence of S9-mix, the test material did not increase the number of polyploid cells and cells with endoreduplicated chromosomes.

SECOND CYTOGENETIC ASSAY
After 48 hours exposure time, no appropriate dose levels could be selected for scoring of chromosome aberrations since at the highest concentration of 250 μg/mL not enough cytotoxicity was observed (23 %) nor precipitate was observed. The experiment was repeated in cytogenetic assay 2A.
Based on observations of mitotic index the following doses were selected for scoring of chromosome aberrations: 1, 10 and 52 μg/mL culture medium (24 h exposure time, 24 h fixation time) and 1, 50 and 150 μg/mL culture medium (48 h exposure time, 48 h fixation time).
The test material did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations (Tables 3 and 4). The test material did not increase the number of polyploid cells and cells with endoreduplicated chromosomes.

EVALUATION OF THE RESULTS
The scores for the number of aberrant cells (gaps included and excluded) and the number of the various types of chromosome aberrations at the various concentrations of the test material are presented in the tables.
The number of cells with chromosome aberrations found in the solvent control cultures was within the 95 % control limits of the distribution of the historical negative control database. The number of polyploid cells and cells with endoreduplicated chromosomes in the solvent control cultures was within the 95 % control limits of the distribution of the historical negative control database. The positive control chemicals (MMC-C and CP) both produced statistically significant increases in the frequency of aberrant cells. In addition, the number of cells with chromosome aberrations found in the positive control cultures was within the 95 % control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.
Both in the absence and presence of S9-mix the test material did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments.
No effects of the test material on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix.
Therefore it can be concluded that the test material does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under these experimental conditions.

Duplicate cultures are indicated by A and B in the following tables.

Key:

g’ = Chromatid gap

g’’ = Chromosome gap

b’ = Chromatid break

b’’ = Chromosome break

m’ = Minute

m’’ = Double minutes

exch. = Exchange figure

dic = Dicentric chromosome

d’ = Chromatid deletion

misc. = (miscellaneous) aberrations not belonging to the ones mentioned above

p = Pulverised chromosomes

 

Table 1: Chromosome aberrations in human lymphocyte cultures in the absence of S9-mix in the first cytogenetic assay (3 h exposure time, 24 h fixation time)

Conc.	DMSO (1.0 % v/v)			5.4 μg/mL			17 μg/mL			52 μg/mL			MMC-C 0.5 μg/mL
Culture	A	B	A+B	A	B	A+B	A	B	A+B	A	B	A+B	A	B	A+B
Mitotic Index (%)	100			99			74			60			84
No. of cells scored	150	150	300	150	150	300	150	150	300	150	150	300	150	135	285
No. of cells with aberrations (+ gaps)	0	0	0	0	0	0	2	0	2	0	0	0	13	20	33***
No. of cells with aberrations (- gaps)	0	0	0	0	0	0	2	0	2	0	0	0	13	20	33***
g’
g’’
b’							1						5	6
b’’													2	9
m’
m’’
exch.													8	13
dic							1
d’
misc.
total aberr (+ gaps)	0	0		0	0		2	0		0	0		15	28
total aberr (- gaps)	0	0		0	0		2	0		0	0		15	28

*Significantly different from control group (Fisher’s exact test), * p<0.05, **p< 0.01 or *** p <0.001

 

Table 2: Chromosome aberrations in human lymphocyte cultures in the presence of S9-mix in the first cytogenetic assay (3 h exposure time, 24 h fixation time)

Conc.	DMSO (1.0 % v/v)			5.4 μg/mL			17 μg/mL			52 μg/mL			CP 0.5 μg/mL
Culture	A	B	A+B	A	B	A+B	A	B	A+B	A	B	A+B	A	B	A+B
Mitotic Index (%)	100			95			73			75			32
No. of cells scored	150	150	300	150	135	285	150	150	300	150	150	300	150	150	300
No. of cells with aberrations (+ gaps)	0	0	0	1	1	2	1	1	2	0	0	0	19	17	36***
No. of cells with aberrations (- gaps)	0	0	0	1	1	2	1	1	2	0	0	0	19	16	35***
g’														4
g’’
b’				1									11	5
b’’							1	1					4	10
m’					4
m’’
exch.													5	4
dic													1
d’
misc.
total aberr (+ gaps)	0	0		1	4		1	1		0	0		21	23
total aberr (- gaps)	0	0		1	4		1	1		0	0		21	19

*Significantly different from control group (Fisher’s exact test), * p<0.05, **p< 0.01 or *** p <0.001

 

Table 3: Chromosome aberrations in human lymphocyte cultures in the absence of S9-mix in the second cytogenetic assay (24 h exposure time, 24 h fixation time)

Conc.	DMSO (1.0 % v/v)			1.0 μg/mL			10 μg/mL			52 μg/mL			MMC-C 0.2 μg/mL
Culture	A	B	A+B	A	B	A+B	A	B	A+B	A	B	A+B	A	B	A+B
Mitotic Index (%)	100			93			93			92			52
No. of cells scored	150	150	300	150	150	300	150	150	300	150	150	300	150	150	300
No. of cells with aberrations (+ gaps)	1	0	1	0	2	2	0	0	0	1	0	1	57	49	106***
No. of cells with aberrations (- gaps)	0	0	0	0	1	1	0	0	0	0	0	0	56	49	105***
g’										1
g’’	1				1								2
b’					1								28	28
b’’													23	22
m’													1	1
m’’													3	3
exch.													14	4
dic
d’													1	1
misc.
total aberr (+ gaps)	1	0		0	2		0	0		1	0		72	59
total aberr (- gaps)	0	0		0	1		0	0		0	0		70	59

*Significantly different from control group (Fisher’s exact test), * p<0.05, **p< 0.01 or *** p <0.001

 

Table 4: Chromosome aberrations in human lymphocyte cultures in the absence of S9-mix in the second cytogenetic assay (48 h exposure time, 48 h fixation time)

Conc.	DMSO (1.0 % v/v)			1 μg/mL			50 μg/mL			150 μg/mL			MMC-C 0.1 μg/mL
Culture	A	B	A+B	A	B	A+B	A	B	A+B	A	B	A+B	A	B	A+B
Mitotic Index (%)	100			87			82			51			54
No. of cells scored	150	150	300	150	150	300	150	150	300	150	150	300	150	150	300
No. of cells with aberrations (+ gaps)	1	0	1	0	1	1	0	1	1	0	0	0	43	53	96***
No. of cells with aberrations (- gaps)	0	0	0	0	0	0	0	1	1	0	0	0	43	52	95***
g’					1
g’’	1												5	6
b’													25	19
b’’								1					18	33
m’
m’’													1
exch.													8	8
dic
d’													1
misc.													p	2p
total aberr (+ gaps)	1	0		0	1		0	1		0	0		59	68
total aberr (- gaps)	0	0		0	0		0	1		0	0		54	62

*Significantly different from control group (Fisher’s exact test), * p<0.05, **p< 0.01 or *** p <0.001

Conclusions:

Under the conditions of this study, the test material is not clastogenic in human lymphocytes in both the presence and absence of metabolic activation.

Executive summary:

A study was conducted to evaluate the test material for its ability to induce structural chromosome aberrations in cultured human lymphocytes in accordance with the standardised guideline OECD 473 under GLP conditions.

The test material in DMSO was investigated in both the presence and absence of a metabolic activation system (phenobarbital and β-naphthoflavone induced rat liver S9-mix). The possible clastogenicity of was tested in two independent experiments.

In the first cytogenetic assay, the test material was tested up to 52 μg/mL for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8 % (v/v) S9-fraction. The test material precipitated in the culture medium at this dose level.

In the second cytogenetic assay, the test material was tested up to the precipitating dose level of 52 μg/mL for a 24 h continuous exposure time with a 24 h fixation time and up to 150 μg/mL for a 48 h continuous exposure time with a 48 h fixation time in the absence of S9-mix. Appropriate toxicity was reached at this dose level.

The test material did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently performed experiments. No effects on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix.

Therefore it can be concluded that the test material does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions of this study.

The number of cells with chromosome aberrations found in the solvent control cultures was within the 95 % control limits of the distribution of the historical negative control database. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations. In addition, the number of cells with chromosome aberrations found in the positive control cultures was within the 95 % control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. The test is therefore considered to be valid.

Under the conditions of this study, the test material is not clastogenic in human lymphocytes in both the presence and absence of metabolic activation.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 November 2016 to 02 January 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Deviations:

no

GLP compliance:

yes

Type of assay:

other: in vitro mammalian cell gene mutation test

Target gene:

Induction of forward mutations at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: American Type Culture Collection (ATCC, Manassas, USA) 2001

MEDIA USED
- Basic medium: RPMI 1640 Hepes buffered medium containing penicillin/streptomycin (50 U/mL and 50 μg/mL, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin.
- Growth medium: Basic medium, supplemented with 10 % (v/v) heat-inactivated horse serum (= R10 medium). Horse serum was inactivated by incubation at 56 °C for at least 30 minutes.
- Exposure medium: For 3 hour exposure cells were exposed in basic medium supplemented with 5 % (v/v) heat-inactivated horse serum (R5-medium). For 24 hour exposure cells were exposed in basic medium supplemented with 10 % (v/v) heat-inactivated horse serum (R10-medium).
- Selective medium: Basic medium supplemented with 20 % (v/v) heat-inactivated horse serum (total amount of serum = 20 %, R20) and 5 μg/mL trifluorothymidine (TFT).
- Non-selective medium: Basic medium supplemented with 20 % (v/v) heat-inactivated horse serum (total amount of serum = 20 %, R20).
- Properly maintained: Yes. Stock cultures of the cells were stored in liquid nitrogen (-196 °C). Cell density was kept below 1 x 10^6 cells/mL.
- Periodically checked for Mycoplasma contamination: Yes
- Periodically 'cleansed' against high spontaneous background: Yes. The cells were grown for 1 day in R10 medium containing 10^-4 M hypoxanthine, 2 x 10^-7 M aminopterine and 1.6 x 10^-5 M thymidine (HAT-medium) to reduce the amount of spontaneous mutants, followed by a recovery period of 2 days on R10 medium containing hypoxanthine and thymidine only. After this period cells were returned to R10 medium for at least 1 day before starting the experiment.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Since the test material was not severely toxic and difficult to dissolve in aqueous solutions the highest concentration was determined by the solubility in the culture medium. Upon mixing with exposure medium the test material precipitated at concentrations of 5.2 mg/mL (= 52 μg/mL in the exposure medium) and above. The concentration used as the highest test material concentration for the dose range finding test was 164 μg/mL.

- Dose range finding test: 0.54, 1.7, 5.4, 17, 52 and 164 µg/mL
- Mutagenicity test: 0.78, 1.57, 3.13, 6.25, 12.5, 25, 50 and 100 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: No correction was made for the purity/composition of the test material. A solubility test was performed in as part of the chromosome aberration study. The test material was dissolved in dimethyl sulfoxide at a concentration of 16.4 mg/mL. The final concentration of the solvent in the exposure medium was 1 % (v/v).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

MUTAGENICITY TEST
All incubations were carried out in a humid atmosphere (80 - 100 %) containing 5.0 ± 0.5 % CO2 in air in the dark at 37.0 ± 1.0 °C.
The test material was tested in the presence of S9-mix with a 3 hour treatment period and in the absence of S9-mix with 3 and 24 hour treatment periods. Eight doses of the test material were tested in the mutation assay.

TREATMENT OF THE CELLS
Per culture 8 x 10^6 cells (10^6 cells/mL for 3 hour treatment) or 6 x 10^6 cells (1.25 x 10^5 cells/mL for 24 hour treatment) were used. The cell cultures for the 3 hour treatment were placed in sterile 30 mL centrifuge tubes and incubated in a shaking incubator at 37.0 ± 1.0 °C and 145 rpm. The cell cultures for the 24 hour treatment were placed in sterile 75 cm2 culture flasks at 37.0 ± 1.0 °C. Solvent and positive controls were included and the solvent control was tested in duplicate.
In the first experiment, cell cultures were exposed for 3 hours to the test material in exposure medium in the absence and presence of S9-mix. In the second experiment, cell cultures were exposed to the test material in exposure medium for 24 hours in the absence of S9-mix.
For the 3 hour treatment, cell cultures were exposed for to the test material in exposure medium in the absence as well as in the presence of S9-mix. After exposure, the cells were separated from the treatment solutions by 2 centrifugation steps (216 g, 5 min). The first centrifugation step was followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and finally resuspension in 50 mL growth medium (R10).
For the 24 hour treatment, cell cultures were exposed to the test material in exposure medium in the absence of S9-mix. After exposure, the cells were separated from the treatment solutions by 2 centrifugation steps (216 g, 5 min). The first centrifugation step was followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and after the second centrifugation step the cells were re-suspended in 20 mL growth medium (R10). The cells in the final suspension were counted with the coulter particle counter.

EXPRESSION PERIOD
For expression of the mutant phenotype, the remaining cells were cultured for 2 days after the treatment period. During this culture period at least 4 x 10^6 cells (where possible) were sub-cultured every day in order to maintain log phase growth. Two days after the end of the treatment with the test material the cells were plated for determination of the cloning efficiency (CEday2) and the mutation frequency (MF).

DETERMINATION OF THE MUTATION FREQUENCY
For determination of the CEday2 the cell suspensions were diluted and seeded in wells of a 96-well dish. One cell was added per well (2 x 96-well microtitre plates/concentration) in non-selective medium.
For determination of the mutation frequency (MF) a total number of 9.6 x 10^5 cells/concentration were plated in five 96-well microtitre plates, each well containing 2000 cells in selective medium (TFT-selection), with the exception of the positive control groups (MMS and CP) where a total number of 9.6 x 10^5 cells/concentration were plated in ten 96-well microtitre plates, each well containing 1000 cells in selective medium (TFT-selection). The microtitre plates for CEday2 and MF were incubated for 11 or 12 days. After the incubation period, the plates for the TFT-selection were stained for 2 hours, by adding 0.5 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide (MTT) to each well. The plates for the CEday2 and MF were scored with the naked eye or with the microscope.

DETERMINATION OF MUTANT COLONIES
The colonies were divided into small and large colonies. The small colonies are morphologically dense colonies with a sharp contour and with a diameter less than a quarter of a well. The large colonies are morphologically less dense colonies with a hazy contour and with a diameter larger than a quarter of a well. A well containing more than one small colony is classified as one small colony. A well containing more than one large colony is classified as one large colony. A well containing one small and one large colony is classified as one large colony.

CALCULATION OF THE SURVIVAL OR VIABILITY
The suspension growth (SG) for the 3 hour treatment = [Day 1 cell count/1.6 x 10^5] x [Day 2 cell count/1.25 x 10^5]

The suspension growth (SG) for the 24 hour treatment = [Day 0 cell count/1.25 x 10^5] x [Day 1 cell count/1.25 x 10^5] x [Day 2 cell count/1.25 x 10^5]

Relative Suspension Growth (RSG) = SG (test) / SG (controls) x 100

The cloning efficiency was determined by dividing the number of empty wells by the total number of wells. The value obtained is the P(0), the zero term of the Poisson distribution:
P(0) = number of empty wells/total number of wells

The cloning efficiency (CE) was then calculated as follows:
CE = -ln P(0)/number of cells plated per well

The relative cloning efficiency (RCE) at the time of mutant selection = CE (test) / CE (controls) x 100

The Relative Total Growth (RTG) was also calculated as the product of the cumulative relative suspension growth (RSG) and the relative survival for each culture:
RTG = RSG x RCE/100

CALCULATION OF THE MUTATION FREQUENCY
The mutation frequency was expressed as the number of mutants per 10^6 viable cells. The plating efficiencies of both mutant and viable cells (CEday2) in the same culture were determined and the mutation frequency (MF) was calculated as follows:
MF = {-ln P(0)/number of cells plated per well}/ CEday2 x 10^6

Small and large colony mutation frequencies were calculated in an identical manner.

Evaluation criteria:

ACCEPTABILITY OF THE ASSAY
A mutation assay was considered acceptable if it met these criteria:
a) The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120 % in order to have an acceptable number of surviving cells analysed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 10^6 survivors and ≤ 170 per 10^6 survivors.
c) The suspension growth (SG) over the 2-day expression period for the solvent controls should be between 8 and 32 for the 3 hour treatment, and between 32 and 180 for the 24 hour treatment.
d) The positive control should demonstrate an absolute increase in the total mutation frequency above the spontaneous background MF (an induced MF (IMF) of at least 300 x 10^-6). At least 40 % of the IMF should be reflected in the small colony MF. Furthermore, the positive control should have an increase in the small colony MF of at least 150 x 10^-6 above that seen in the concurrent solvent/control (a small colony IMF of at least 150 x 10^-6).

DATA EVALUATION
Any increase of the mutation frequency should be evaluated for its biological relevance including comparison of the results with the historical control. The GEF has been defined as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.
A test material is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
A test material is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
A test material is considered negative (not mutagenic) in the mutation assay if: none of the tested concentrations reaches a mutation frequency of MF(controls) + 126.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test material precipitated in the exposure medium at the test material concentration of 100 μg/mL after 3 and 24 hours of treatment.

RANGE-FINDING/SCREENING STUDIES:
Both in the absence and presence of S9-mix, no toxicity in the relative suspension growth was observed up to and including the highest test material concentration of 164 μg/mL compared to the suspension growth of the solvent control. The test material precipitated in the exposure medium at the two highest tested concentrations of 52 and 164 μg/mL. The relative suspension growth was 36 % at the test material concentration of 164 μg/mL compared to the relative suspension growth of the solvent control.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: In the 3 hour treatment period, no toxicity was observed up to and including the highest tested dose level of 100 μg/mL. Therefore, the dose level of 0.39 μg/mL was not regarded relevant for mutation frequency measurement.
In the 24 hour treatment period, the dose levels of 0.39 to 1.57 μg/mL showed no cytotoxicity. Therefore, the dose level of 0.39 μg/mL was not regarded relevant for mutation frequency measurement. The relative total growth of the highest test material concentration was 34 % compared to the total growth of the concurrent solvent controls.

MUTATION EXPERIMENT
In the first mutation experiment (3 hour treatment), no significant increase in the mutation frequency at the TK locus was observed after treatment with the test material either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the test material treated cultures were comparable to the numbers of small and large colonies of the solvent controls.
In the second mutation experiment (24 hour treatment), no significant increase in the mutation frequency at the TK locus was observed after treatment with the test material. The numbers of small and large colonies in the test material treated cultures were comparable to the numbers of small and large colonies of the solvent controls. Increase above the 95 % upper control limit were observed in the first mutation experiment in the absence and presence of S9-mix. Although these increases are above the 95 % upper control limit, the highest increases in the mutation frequencies (161 and 152 per 10^6 survivors in the absence and presence of S9-mix, respectively) are below the GEF: MF(controls) + 126 (228 and 199 per 10^6 survivors in the absence and presence of S9-mix, respectively). Therefore, these increases are considered to be not biologically relevant. In addition, these limits are 95 % control limits and a slightly higher response is within the expected response ranges.

DISCUSSION
The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95 % control limits of the distribution of the historical negative control database.
Positive control chemicals both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95 % control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.
The growth rate over the two-day expression period for cultures treated with DMSO was between 17 and 21 (3 hour treatment) and 72 and 74 (24 hour treatment).
In the absence of S9-mix, the test material did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in a repeat experiment with modification in the duration of treatment.
In the presence of S9-mix, the test material did not induce a significant increase in the mutation frequency.

Experiment 1: Cytotoxic and mutagenic response of the test material in the mouse lymphoma L5178Y test system

Dose (μg/mL)	RSG (%)	CEday2 (%)	RCE (%)	RTG (%)	Mutation frequency per 10^6 survivors
					Total	Small	Large
Without metabolic activation - 3 hours of treatment
SC1	100	77	100	100	107	48	55
SC2		89			97	35	58
0.78	103	88	106	103	116	46	64
1.57	101	91	110	105	93	37	53
3.13	102	102	124	119	90	36	50
6.25	97	71	86	79	144	63	74
12.5	99	93	112	104	109	48	56
25	95	83	100	89	161	68	82
50	82	90	109	84	117	48	62
100*	81	118	142	109	96	39	52
MMS	73	58	69	48	738	287	374
With metabolic activation - 3 hours of treatment
SC1	100	105	100	100	67	14	51
SC2		79			78	35	41
0.78	106	99	108	115	64	16	46
1.57	98	79	86	93	68	24	42
3.13	96	68	74	79	112	25	84
6.25	100	94	102	113	94	32	59
12.5	100	85	92	103	150	34	109
25	95	88	95	101	152	41	103
50	80	89	96	86	107	40	62
100*	86	98	106	102	93	30	60
CP	40	32	34	15	1092	367	637

RSG = Relative Suspension Growth

CE = Cloning Efficiency

RCE = Relative Cloning Efficiency

RTG = Relative Total Growth

SC = Solvent control (DMSO)

MMS = Methylmethanesulfonate

CP = Cyclophosphamide

*The test material precipitated in the exposure medium

 

Experiment 2: Cytotoxic and mutagenic response of the test material in the mouse lymphoma L5178Y test system

Dose (μg/mL)	RSG (%)	CEday2 (%)	RCE (%)	RTG (%)	Mutation frequency per 10^6 survivors
					Total	Small	Large
Without metabolic activation - 24 hours of treatment
SC1	100	69	100	100	107	20	84
SC2		108			55	17	37
0.78	83	71	80	67	69	10	58
1.57	86	97	109	94	47	8	39
3.13	64	81	92	59	67	16	51
6.25	42	72	82	34	79	19	58
12.5	52	66	75	39	55	19	35
25	65	72	82	53	94	23	68
50	53	99	112	60	58	20	36
100*	35	86	97	34	50	27	22
MMS	73	61	69	50	543	306	195

RSG = Relative Suspension Growth

CE = Cloning Efficiency

RCE = Relative Cloning Efficiency

RTG = Relative Total Growth

SC = Solvent control (DMSO)

MMS = Methylmethanesulfonate

*The test material precipitated in the exposure medium

Conclusions:

Under the conditions of this study, it is concluded that the test material is not mutagenic in the TK mutation test system.

Executive summary:

The potential of the test material to cause mutagenic effects in mammalian cells was assessed in accordance with the standardised guideline OECD 490 under GLP conditions.

The mutagenicity evaluation of the test material was carried out in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells. The test was performed in the absence of S9-mix with 3 and 24 hour treatment periods and in the presence of S9-mix with a 3 hour treatment period. The test material was dissolved in dimethyl sulfoxide at a concentration of 16.4 mg/mL.

In the first experiment, the test material was tested up to concentrations of 100 μg/mL in the absence and presence of S9-mix. The incubation time was 3 hours. No toxicity was observed at this dose level in the absence and presence of S9-mix. The test material precipitated in the culture medium at this dose level.

In the second experiment, the test material was again tested up to concentrations of 100 μg/mL in the absence of S9-mix. The incubation time was 24 hours. The relative total growth was 34 %. The test material precipitated in the culture medium at this dose level.

The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95 % control limits of the distribution of the historical negative control database.

Positive control chemicals both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95 % control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

In the absence of S9-mix, the test material did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modification in the duration of treatment.

In the presence of S9-mix, the test material did not induce a significant increase in the mutation frequency.

Under the conditions of this study, it is concluded that the test material is not mutagenic in the TK mutation test system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The following studies were assigned a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

Ames Test

The potential of the test material to cause mutagenic effects in bacteria was assessed in accordance with the standardised guidelines OECD 471 and EU Method B.13/14 under GLP conditions.

The test material was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by Aroclor 1254).

In the dose range finding test, the test material was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. The test material was tested up to or beyond a precipitating dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. Results of this dose range finding test were reported as part of the first mutation assay.

Based on the results of the dose range finding test, the test material was tested in the first mutation assay at a concentration range of 5.4 to 1600 μg/plate in the absence and presence of 5 % (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. The test material was precipitated on the plates at concentrations of 512 and 1600 μg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

In a follow-up experiment of the assay with additional parameters, the test material was tested at a concentration range of 86 to 1568 μg/plate in the absence and presence of 10 % (v/v) S9-mix in all strains. Precipitation of the test material on the plates was observed at 1568 μg/plate in the absence of S9-mix, except in tester strain TA100 and in the presence of S9-mix in tester strain TA1535 only. In the presence of S9-mix, the bacterial background lawn was reduced in all tester strains at the highest tested concentration. In the absence of S9-mix, a reduction in the bacterial background lawn was observed in tester strain TA100 and a biologically relevant decrease in the number of revertants was observed in tester strain TA1537.

In the presence of S9-mix, the test material induced dose related increases in tester strain TA98. The increases observed were above the laboratory historical control data range, in two independently repeated experiments and were up to 4.4-fold the concurrent vehicle controls and therefore considered to be biologically relevant. In the other tester strains (TA1535, TA1537, TA100 and WP2uvrA), the test material did not induce a significant dose-related increase in the number of revertant colonies in any experiment.

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Under the conditions of the study, it is concluded that the test material is mutagenic in the Salmonella typhimurium reverse mutation assay and not mutagenic in the Escherichia coli reverse mutation assay. The mutagenicity was confined only to incubations with metabolic activation in one strain.

Given that the positive result was only observed in a single strain in only one test, the total weight of evidence suggests that the test material is not classified for mutagenicity.

Chromosome Aberration

A study was conducted to evaluate the test material for its ability to induce structural chromosome aberrations in cultured human lymphocytes in accordance with the standardised guideline OECD 473 under GLP conditions.

The test material in DMSO was investigated in both the presence and absence of a metabolic activation system (phenobarbital and β-naphthoflavone induced rat liver S9-mix). The possible clastogenicity of was tested in two independent experiments.

In the first cytogenetic assay, the test material was tested up to 52 μg/mL for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8 % (v/v) S9-fraction. The test material precipitated in the culture medium at this dose level.

In the second cytogenetic assay, the test material was tested up to the precipitating dose level of 52 μg/mL for a 24 h continuous exposure time with a 24 h fixation time and up to 150 μg/mL for a 48 h continuous exposure time with a 48 h fixation time in the absence of S9-mix. Appropriate toxicity was reached at this dose level.

The test material did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently performed experiments. No effects on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix.

Therefore it can be concluded that the test material does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions of this study.

The number of cells with chromosome aberrations found in the solvent control cultures was within the 95 % control limits of the distribution of the historical negative control database. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations. In addition, the number of cells with chromosome aberrations found in the positive control cultures was within the 95 % control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. The test is therefore considered to be valid.

Under the conditions of this study, the test material is not clastogenic in human lymphocytes in the presence or absence of metabolic activation.

Mouse Lymphoma Assay

The potential of the test material to cause mutagenic effects in mammalian cells was assessed in accordance with the standardised guideline OECD 490 under GLP conditions.

The mutagenicity evaluation of the test material was carried out in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells. The test was performed in the absence of S9-mix with 3 and 24 hour treatment periods and in the presence of S9-mix with a 3 hour treatment period. The test material was dissolved in dimethyl sulfoxide at a concentration of 16.4 mg/mL.

In the first experiment, the test material was tested up to concentrations of 100 μg/mL in the absence and presence of S9-mix. The incubation time was 3 hours. No toxicity was observed at this dose level in the absence and presence of S9-mix. The test material precipitated in the culture medium at this dose level.

In the second experiment, the test material was again tested up to concentrations of 100 μg/mL in the absence of S9-mix. The incubation time was 24 hours. The relative total growth was 34 %. The test material precipitated in the culture medium at this dose level.

The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95 % control limits of the distribution of the historical negative control database.

Positive control chemicals both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95 % control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

In the absence of S9-mix, the test material did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modification in the duration of treatment.

In the presence of S9-mix, the test material did not induce a significant increase in the mutation frequency.

Under the conditions of this study, it is concluded that the test material is not mutagenic in the TK mutation test system.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to genetic toxicity.