2-amino-6-methyl-4-propyl-1,2,4-triazolo[1,5-a]pyrimidin-5(4H)-one

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item is not mutagenic in bacteria, as determined in a GLP compliant OECD 471 study.

The test item is not mutagenic in mammalian cells, as determined in a GLP compliant OECD 476 study.

The test item is not cytogenetic in mammalian cells, as determined in a GLP compliant OECD 487 study.

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

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine revertants (for Salmonella typhimurium strains)
tryptophan revertants (for Escherichia coli strain)

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital/5,6-benzoflavone induced rat liver S9 fraction

Test concentrations with justification for top dose:

EXPERIMENT 1 (PLATE INCORPORATION ASSAY) and EXPERIMENT 2 (PRE-INCUBATION ASSAY)
5, 15, 50, 150, 500, 1500, 5000 µg/plate (concentrations were corrected for the purity)

JUSTIFICATION FOR TOP DOSE
The tope dose in EXPERIMENT 1 (5000 μg/plate) is the standard limit concentration recommended in OECD 471. The highest concentration in each test was diluted with DMSO to produce a series of lower concentrations, separated by approximately half-log10 intervals.
In the absence of any toxic effects, the maximum concentration selected for use in the Pre-incubation assay (EXPERIMENT 2) is the same as that used in the Plate incorporation assay. If toxic effects are observed, a lower concentration might be chosen, ensuring that signs of bacterial inhibition are present at this maximum concentration.

Vehicle / solvent:

- Solvent used: DMSO

Untreated negative controls:

yes

Remarks:

Sterility control, plates were prepared without the addition of bacteria.

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: See Table in 'Any other information on materials and methods incl. tables'

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation, EXPERIMENT 1) and preincubation (EXPERIMENT 2)

EXPERIMENTAL DESIGN
- Aliquots of 0.1 mL of the test item solutions, positive control or vehicle control were placed in glass tubes.
- S9 mix (0.5 mL) or 0.1 M pH 7.4 sodium phosphate buffer (0.5 mL) was added, followed by 0.1 mL of a 10-hour bacterial culture and 2 mL of agar containing histidine (0.05 mM), biotin (0.05 mM) and tryptophan (0.05 mM). The mixture was thoroughly shaken and overlaid onto previously prepared Petri dishes containing 25 mL minimal agar.

DURATION
- Preincubation period: 30 minutes with shaking (EXPERIMENT 2 only, preincubation before the addition of the agar overlay)
- Exposure duration: ca. 72 hours at 37°C

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: reduction in mean revertant colony counts; sparse or absent background bacterial lawn

ANALYSIS OF DATA
The mean number and standard deviation of revertant colonies were calculated for all groups. The "fold-increases" relative to the vehicle controls were calculated in order to compare the means for all treatment groups with those obtained for the vehicle control groups.

Evaluation criteria:

See 'Any other information on materials and methods incl. tables'

Statistics:

The statistical procedures used are those described by Mahon et al (1989) and are usually Dunnett's test followed, if appropriate, by trend analysis.

Key result

Species / strain:

S. typhimurium, other: TA98 and TA1537

Remarks:

EXPERIMENT 1

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium, other: TA98 and TA1537

Remarks:

EXPERIMENT 1

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium, other: TA100 and TA1535

Remarks:

EXPERIMENT 1

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Remarks:

EXPERIMENT 1

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535, TA1537

Remarks:

EXPERIMENT 2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Remarks:

EXPERIMENT 2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In EXPERIMENT 1 and 2, no precipitate was observed on plates following exposure to the test item.

ACCEPTENCE OF RESULTS
- criteria for the vehicle control were satisfied: The control mean revertants per plate for strains TA98 and TA1537 in the absence of metabolic activation in experiment 2 were just below the minimum HCD value (23.4 vs 24.0 and 7 vs 8) but were considered to be fully acceptable.
- criteria for the positive controls were satisfied
- criteria of analyzable concentrations were satisfied.

ADDITIONAL INFORMATION ON CYTOTOXICITY
Toxicity, observed as a reduction in the number of revertant colonies, was obtained in strains following exposure to the test substance in the absence of S9 mix at 50 μg/plate in strain TA98, and at 50 and 5000 μg/plate in strain TA1537 in EXPERIMENT 1.

Conclusions:

In this ames test according to OECD 471, no evidence of mutagenic potential of the test item was found.

Executive summary:

In this GLP compliant in vitro study according to OECD 471, the mutagenic potential of the test item was assessed. Histidine-dependent auxotrophic mutants of Salmonella typhimurium, strains TA1535, TA1537, TA98 and TA100, and a tryptophan-dependent mutant of Escherichia coli, strain WP2 uvrA (pKM101), were exposed to the test item diluted in dimethyl sulfoxide (DMSO). DMSO was also used as a vehicle control. Two independent mutation tests were performed in the presence and absence of liver preparations (S9 mix) from rats treated with phenobarbital and 5,6-benzoflavone. The first experiment was a standard plate incorporation assay; the second included a pre-incubation stage. Concentrations of 5, 15, 50, 150, 500, 1500, 5000 µg/plate were tested. In the first experiment, toxicity, observed as a reduction in the number of revertant colonies, was obtained in strains following exposure to the test item in the absence of S9 mix at 50 μg/plate in strain TA98, and at 50 and 5000 μg/plate in strain TA1537. No precipitate was observed on any plates containing test item up to 5000 μg/plate. In the second experiment, no signs of toxicity towards the tester strains were observed in either mutation test following exposure. No precipitate was observed on any plates containing test item up to 5000 μg/plate. No evidence of mutagenic activity was seen at any concentration of test item in either experiment. The concurrent positive controls verified the sensitivity of the assay and the metabolizing activity of the liver preparations. The mean revertant colony counts for the vehicle controls were within or close to the current historical control range for the laboratory. It was concluded that the test item showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

2015

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro gene mutation study in mammalian cells

Target gene:

Hprt

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

CHO-K1

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: European Collection of Cell Cultures
- Methods for maintenance in cell culture: All cell cultures were maintained at between 34 and 39°C in an atmosphere of 5% CO2 in air.

MEDIA USED
- Type and identity of media: The following media, obtained from a suitable supplier, containing approximate concentrations of supplements, were used:
H0: Ham’s Nutrient Mixture F12, supplemented with 2 mM L glutamine and 50 µg/mL gentamicin;
H10: H0 medium supplemented with 10% heat inactivated foetal calf serum. H10 medium was used for cell culture.
Selective medium: H10 supplemented with 6-TG at a final concentration of 10 µg/mL
- Periodically checked for Mycoplasma contamination: yes
- Periodically 'cleansed' against high spontaneous background: no, Spontaneous mutants were eliminated from the cultures by 3-day incubation in the presence of methotrexate (0.3 μg/mL), thymidine (4 μg/mL), hypoxanthine (15 μg/mL) two days prior to storage at -196°C, in heat-inactivated foetal calf serum (HiFCS) containing 10% dimethyl sulphoxide (DMSO).

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/5,6-benzoflavone induced rat liver S9 mix

Test concentrations with justification for top dose:

JUSTIFICATION: 2000 μg/mL is the standard limit concentration within this test system as recommended by OECD Guideline 476 (2015).

PRELIMINARY TOXICITY TEST: 15.63; 31.25; 62.5; 125; 250; 500; 1000; 2000 µg/mL (concentrations were corrected for the purity)

MAIN TEST: 1000; 1200; 1400; 1600; 1800; 2000 µg/mL (concentrations were corrected for the purity)

Vehicle / solvent:

- Vehicle used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding: 4 x 10^5 cells/mL

DURATION
- Preincubation period: 20 hours
- Exposure duration: 3 hours
- Expression time (cells in growth medium): 7 days
- Selection time: 7 days (Main experiment only)
- Fixation time: 7 days and 3 hours

SELECTION AGENT: 6-thioguanine

STAIN: Giemsa

NUMBER OF REPLICATIONS
- Preliminary toxicity test: one per concentration of test item and two for the vehicle controls
- Main experiment: duplicates per concentration of test item and for the positive control, quadruplicates for the vehicle controls. For each culture, three flasks were seeded with 200 cells each, to determine cloning efficiency and five flasks with 5 x 105 cells each in selective medium to determine cloning efficiency.

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Colonies growing in the plate were fixed and stained in a methanol:Giemsa solution (4:1 v/v).

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

OTHER EXAMINATIONS:
- The osmolality of the test item in medium was tested at 2000 µg/mL.
- The fluctuations in pH of the medium was tested at 2000 µg/mL.

Evaluation criteria:

ACCEPTANCE CRITERIA
Tests were accepted on the basis of the following criteria:
- Acceptance criteria for test item: The criteria for selection of the top dose concentration are:
a) Fluctuations in osmolality of the medium should not exceed 50 mOsm/kgcompared with the vehicle control.
b) Fluctuations in pH of the medium should not exceed 1.0 unit compared with the vehicle control.
- Acceptance criteria for vehicle controls: The mean vehicle control value for mutant frequency was between 1 and 20 x 10^-6; The mean cloning/plating efficiency was between 65 and 120%; Obvious outliers were excluded. However, there were at least 2 vehicle control cultures remaining; The vehicle controls of both parallel cultures remain within or close to the control limit of the laboratory historical control data range.
- Acceptance criteria for positive controls: Positive controls showed a statistically significant increase in mean total MF above the mean concurrent vehicle control MF and within, or close to, the range of the historical control data.
Assays that did not fulfil the required criteria were rejected and therefore not reported.

Statistics:

- The statistical significance of the data was analysed by weighted analysis of variance, weighting assuming a Poisson distribution following the methods described by Arlett et al. (1989).
- The computer systems used to acquire and quantify data for this main report include SAS and Xybion Pristima

Key result

Species / strain:

Chinese hamster Ovary (CHO)

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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No fluctuations in pH of the medium were observed at 2000 µg/mL of more than 1.0 unit compared with the vehicle control.
- Effects of osmolality: no fluctuations in osmolality of the medium of more than 50 mOsm/kg were observed at 2000 µg/mL compared with the vehicle control.
- Precipitation: No precipitate was seen by eye at the end of each treatment.
- Other confounding effects: There was an absence of confounding technical problems such as contamination, excessive numbers of outliers and excessive toxicity.

ADDITIONAL INFORMATION ON CYTOXICITY
Exposure for 3 hours at concentrations from 1000 to 2000 μg/mL in both the absence and presence of S9 mix resulted in RS values from 110 to 44% and 102 to 66% respectively.

PRELIMINARY TOXICITY EXPERIMENT
- Precipitation: No precipitate was seen by eye at the end of each treatment.
- The cell concentration was confirmed to be 1.4 x 10^6 cells/mL (i.e. 14 x 10^6 cells treated per concentration, 28 x 10^6 cells for the vehicle control).
- Cytotoxicity: Exposure for 3 hours at concentrations from 15.63 to 2000 μg/mL in both the absence and presence of S9 mix resulted in RS values from 114 to 26% and 136 to 17% respectively. Concentrations for the main test were based upon these data.

DATA EVALUATION
- Acceptance criteria of the study are met.
- Definition of acceptable cells for analysis: The average cell density across all flasks at the beginning of the treatment period was determined to ensure a minimum of 20 x 10^6 cells being present during treatment.
Main experiment without S9 mix: The cell concentration was confirmed to be 1.7 x 10^6 cells/mL (i.e. 34 x 10^6 cells treated per concentration, 68 x 10^6 cells for the vehicle control).
Main experiment with S9 mix: The cell concentration was confirmed to be 1.6 x 10^6 cells/mL (i.e. 32 x 10^6 cells treated per concentration, 64 x 10^6 cells for the vehicle control).

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: see ‘Any other information on results incl. tables’
- Negative (solvent/vehicle) historical control data: see ‘Any other information on results incl. tables’

Table 1. Historical Control Data in the absence of S9 mix

		Mean Day 1 Cloning Efficiency (%)	Mean Mutant Frequency (x 10^-6)
Vehicle Controls	Mean	73	7.2
	Maximum	95	19.3
	SD	0.1	5.9
Ethyl methanesulphonate 250 μg/mL	Mean	-	310.9
	Minimum	-	74.9
	SD	-	126
Data collection period:		27-Jul-12	09-Aug-16

Upper control limit for vehicle controls: 11.9 x 10^-6

Number of tests: 25

 

Table 2. Historical Control Data in the presence of S9 mix

		Mean Day 1 Cloning Efficiency (%)	Mean Mutant Frequency (x 10^-6)
Vehicle Controls	Mean	74	7.8
	Maximum	88	20.5
	SD	0.1	6.2
3-Methylcholanthrene 5 μg/mL	Mean	-	392.7
	Minimum	-	63.4
	SD	-	164
Data collection period:		20-Aug-12	11-Aug-16

Upper control limit for vehicle controls: 12.2 x 10^-6

Number of tests: 26

Conclusions:

In this HPRT test according to OECD 476, no evidence of mutagenic potential of the test item was found.

Executive summary:

The test item was tested for mutagenic potential in an in vitro mammalian cell mutation assay according to OECD 476 and GLP. This test system is based on detection and quantitation of forward mutation at the functionally hemizygous hypoxanthine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO-K1) cells. The results of two independent tests, one in the absence of exogenous metabolic activation (S9 mix) and one in the presence of S9 mix, are reported. The vehicle was DMSO (dimethyl sulfoxide), in which the test item dissolved at up to 200 mg/mL. The highest final concentration used in the preliminary toxicity experiment was 2000 μg/mL. No precipitate was seen by eye at the end of treatment. Cytotoxicity was measured as Day 1 relative survival (RS). After exposure at concentrations from 15.63 to 2000 µg/mL RS values ranged from 114 to 26% and from 136 to 17%, in the absence and presence of S9 mix respectively. In the main mutation experiment in the absence of S9 mix, cells were exposed to concentrations from 1000 to 2000 μg/mL of test item. No precipitate was observed by eye at the end of treatment. RS values ranged from 110 to 44% relative to the vehicle control. The test item did not induce a statistically significant increase in mutant frequency. The positive control, ethyl methanesulphonate, induced a significant increase in mutant frequency demonstrating the correct functioning of the assay. In the main mutation experiment in the presence of S9 mix, cells were exposed to concentrations from 1000 to 2000 μg/mL of test item. No precipitate was observed by eye at the end of treatment. RS values ranged from 102 to 66% relative to the vehicle control. The test item did not induce a statistically significant increase in mutant frequency. The positive control, 3-methylcholanthrene, induced a significant increase in mutant frequency demonstrating the correct functioning of the assay. It was concluded that the test item did not demonstrate mutagenic potential in this in vitro HPRT cell mutation assay, under the experimental conditions described.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

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:

2014

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes: Human

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Human blood was collected aseptically from two healthy, non-smoking, adult donors, pooled (in equal volumes from each donor) and diluted with HML media. Separate batches were made for the preliminary and main tests.
- Suitability of cells: In this test system, human lymphocytes are cultured In vitro and are stimulated to divide by adding phytohaemagglutinin (PHA) to the culture, resulting in a high mitotic yield (Nowell 1960).
- Cell cycle length, doubling time or proliferation index: Historically the average donor doubling time for this laboratory was ca. 13 hours and the study design is based upon this.
- Sex, age and number of blood donors: female; between 18-35 years of age; two donors
- Whether whole blood or separated lymphocytes were used: Whole (diluted) blood
- Methods for maintenance in cell culture: Cultures were established from the prepared (pooled) sample and dispensed as 5 mL aliquots (in sterile universal containers) so that each culture contained blood (0.4 mL), HML media (4.5 mL) and PHA solution (0.1 mL). All cultures were then incubated at 37°C, and the cells were resuspended (twice daily) by gentle inversion.

MEDIA USED
- Type and identity of media: HML media, obtained from a suitable supplier, was used (RPMI 1640, supplemented with 10% fetal calf serum, 0.2 IU/mL sodium heparin, 20 IU/mL penicillin / 20 μg/mL streptomycin and 2.0 mM L-glutamine)

Cytokinesis block (if used):

Cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and 5,6-benzoflavone induced rat liver S9 mix

Test concentrations with justification for top dose:

PRELIMINARY TOXICITY TEST
- Concentrations were corrected for the purity
- Justification of top dose: 2000 µg/mL is the standard limit concentration within this test system as recommended in the regulatory guideline.
- Concentrations without/with S9 mix: 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µg/mL

MAIN TESTS
- Concentrations were corrected for the purity
- Without/with S9 mix, 3 hours, concentrations for exposure: 250, 500, 1000 and 2000 µg/mL.
- Without/with S9 mix, 3 hours, concentrations for micronucleus frequency analysis: 250, 1000 and 2000 µg/mL
- Without S9 mix, 20 hours, concentrations for exposure: 12.5, 125, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475 and 500 µg/mL
- Without S9 mix, 20 hours, concentrations for micronucleus frequency analysis: 12.5, 350, and 500 µg/mL

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of vehicle: Prior to commencing testing, the solubility of the test item in a vehicle compatible with this test system was assessed. The test item was found to form a dosable suspension at 400 mg/mL in dimethyl sulphoxide (DMSO).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Colchicine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hours (following stimulation with PHA)
- Exposure duration: 3 hours (with/without S9 mix) and 20 hours (without S9 mix)
- Expression time: 17 hours (with exposure duration 3 hours), 0 hours (with exposure duration 20 hours)
- Fixation time: 20 hours

SPINDLE INHIBITOR: Cytochalasin B (6 μg/mL)

STAIN: acridine orange (0.0125 mg/mL using purified water)

NUMBER OF REPLICATIONS:
- Preliminary toxicity test: Single cultures were prepared for each treatment level and duplicate cultures were prepared for vehicle controls.
- Main test: duplicate cultures were prepared for each treatment level and positive control cultures; quadruplicate cultures were prepared for vehicle controls; two slides were prepared from each culture.

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
- Harvesting and fixation: The cells were harvested by centrifugation at 500 g for 5 minutes. The supernatant was removed and the cell pellet re-suspended and treated with a 4 mL hypotonic solution (0.075M KCl) at 37°C, cultures were then incubated for 3 minutes at 37°C to cause swelling. Cultures were agitated, 4 mL of ice-cold fixative (3:1 v/v methanol: acetic acid) was added slowly onto the culture surface and the cultures were slowly inverted to mix. The cultures were centrifuged at 500 g for five minutes. The supernatant was removed, and the cell pellet re-suspended. A further 4 mL of fresh fixative was then added and the cells stored at 4°C until slide preparation.
- Slide preparation: The cultures were centrifuged at 500 g for 5 minutes and the supernatant removed. A homogeneous cell suspension was prepared. Pre-cleaned microscope slides were prepared for each culture by aliquoting the re-suspended cells onto the slides, and allowing the slides to air-dry. One slide was prepared from each culture. The remaining cell cultures were stored at approximately 4°C until slide analysis was complete.
- Slide staining: The following steps were performed to stain the prepared slides
1. Rinsed in purified water
2. Stained in acridine orange solution (0.0125 mg/mL using purified water) for 4 minutes
3. Washed in purified water for 5 minutes
4. Rinsed in cold tap water for 2 minutes
5. Stored at room temperature protected from light until required
6. Immediately prior to scoring, slides are wet mounted with glass coverslips using purified water

MICROSCOPIC EXAMINATION
The prepared slides were examined by fluorescence microscopy. The incidences of mononucleate, binucleate and polynucleate cells were assessed per culture. The presence of an unusual number of, for example, cells undergoing mitosis, polyploid cells, necrotic cells and debris, if any, was also noted.

SELECTION OF CONCENTRATIONS
- Selection of concentrations in main experiment: The highest concentration was based on the preliminary toxicity test and was intended to be that which caused a depression in the cytokinesis-block proliferative index (CBPI) equivalent to 55±5% cytostasis (approximately) when compared with the concurrent vehicle control or, where no cytostasis was observed, the maximum concentration as recommended in the test guidelines or the limit of solubility.
- Selection of concentrations for micronucleus analysis: At least three concentrations were selected for micronucleus analysis. The highest concentration was intended to be that which caused a depression in the cytokinesis block proliferative index (CBPI) equivalent to 55±5% cytostasis (approximately) when compared with the concurrent vehicle control or, where no cytostasis was observed, the maximum concentration as recommended in the test guidelines or the limit of solubility.

NUMBER OF CELLS EVALUATED
Cytotoxicity: to calculate the CBPI at least 500 cells were assessed per culture.
Genotoxicity: At least 2000 binucleate cells per concentration (4000 for vehicle controls).

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
The analysis for micronucleated cells was based on the following criteria (Fenech and Morley 1985 and Fenech, 1993): Cells were included in the analysis provided the cytoplasm remained essentially intact and any micronuclei present were separate in the cytoplasm or only just touching the main nucleus (not connected to the nucleus by a nucleoplasmic bridge). Micronuclei should lie in the same focal plane as the cell, and should possess a generally rounded shape with a clearly defined outline. The main nuclei of the binucleate cells scored for micronuclei should be of approximately equal size. The diameter of the micronucleus should be between 1/16 and 1/3 that of the main nucleus. The colour of the micronuclei should be the same or lighter than the main nucleus. There should be no micronucleus-like debris in the surrounding area.

DETERMINATION OF CYTOTOXICITY
- Method: Cytostasis, based on depression in the cytokinesis block proliferative index (CBPI)
- Calculation of CBPI: (No. mononucleate cells +2 x No. binucleate cells + 3 x No. multinucleate cells)/ (Total number of cells)
- Calculation of Cytostasis: 100-100{(CBPI test chemical treatment culture _ 1)/(CBPI solvent control culture_ 1)}

OTHER EXAMINATIONS:
- The osmolality of the test item in medium was tested at 2000 µg/mL.
- The fluctuations in pH of the medium was tested at 2000 µg/mL

Evaluation criteria:

ACCEPTANCE CRITERIA
The following criteria were applied for assessment of assay acceptability:
- The concurrent negative control must be considered acceptable for addition to the laboratories historical negative control database.
- Concurrent positive controls must induce responses that are compatible with the laboratories historical positive control database and produce statistically significant increases compared with the concurrent negative control.
- Acceptance criteria for for selection of the top dose concentration are:
a) Fluctuations in osmolality of the medium should not exceed 50 mOsm/kgcompared with the vehicle control.
b) Fluctuations in pH of the medium should not exceed 1.0 unit compared with the vehicle control.

Statistics:

- The analysis assumed that the replicate was the experimental unit.
- An arcsine square root transformation was used to transform the data. Test item treated groups were then compared to control using Williams' tests (Williams 1971, 1972). Positive controls were compared to control using t tests.
- Trend tests have also been carried out using linear contrasts by group number. These were repeated, removing the top dose group, until there were only 3 groups.
- Statistical significance was declared at the 5% level for all tests.
- Data were analyzed using SAS 9.1.3 (SAS Institute 2002) and Quasar 1.4.1 (Quasar 1.4.1 2016).

Key result

Species / strain:

lymphocytes: Human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

20 hour treatment without S9 mix, 500 μg/mL.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No fluctuations in pH of the medium were observed at 2000 µg/mL of more than 1.0 unit compared with the vehicle control.
- Effects of osmolality: no fluctuations in osmolality of the medium of more than 50 mOsm/kg were observed at 2000 µg/mL compared with the vehicle control.
- Precipitation: Precipitate was observed by eye at the end of treatment at 2000 μg/mL.

PRELIMINARY TOXICITY TEST:
- Precipitation: Precipitate was observed by eye at the end of treatment at 2000 μg/mL.
- Cytotoxicity, 3 hour treatment: no significant reductions in CBPI were observed at any level when compared with the vehicle controls.
- Cytotoxicity, 20 hour treatment: a reduction in CBPI compared to vehicle control values, equivalent to 65.8% cytostasis, was obtained at 500 μg/mL. Overt toxicity was observed at higher concentrations.

MAIN EXPERIMENT
- Cytotoxicity, 3 hour treatment: No significant reductions in CBPI were obtained at any concentration tested.
- Cytotoxicity, 20 hour treatment: A reduction in CBPI compared to vehicle control values, equivalent to 51.5% cytostasis, was obtained at 500 μg/mL.
- Genotoxicity: The test item did not cause any statistically significant increases in the number of binucleate cells containing micronuclei when compared with the vehicle controls. At 3 hour exposure without S9 mix, a statistically significant linear trend (p=0.034) was observed following the removal of the top dose group. This was a negative trend resulting from the two-tailed statistical model and was therefore disregarded.

DATA EVALUATION
- Acceptance criteria of the study are met.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: see ‘Any other information on results incl. tables’
- Negative (solvent/vehicle) historical control data: see ‘Any other information on results incl. tables’

Presented below are the historical negative and positive control data from the period 30 April 2014 to 29 February 2016. The positive control data presented here are pooled from two (Mitomycin C and Cyclophosphamide) or three (Colchicine) dose concentrations. The most suitable dose concentration was selected for micronucleus analysis based on the cytostasis profile achieved in each test at the Study Directors discretion.

Table 1. Historical control data –S9 3hr (Number of tests: 22)

	Vehicle control		Mitomycin C (0.2 or 0.3 µg/mL)		Colchicine (0.05, 0.06 or 0.07 µg/mL)
	Binucleate Individual MN/1000 cells	Binucleate Group MN	Binucleate Individual MN/1000 cells	Binucleate Group MN	Binucleate Individual MN/1000 cells	Binucleate Group MN
Minimum	1.0	2.5	18.0	20.5	16.0	17.5
Maximum	11.0	9.8	67.0	64.5	43.0	38.0
Mean	6.4	6.4	33.1	33.1	24.0	24.0
Standard Deviation	2.4	1.7	9.8	9.4	6.3	5.9
Upper Control Limit		9.2

 

Table 2. Historical control data +S9 3hr (Number of tests: 23)

	Vehicle control		Cyclophosphamide (5 or 10 µg/mL)
	Binucleate Individual MN/1000 cells	Binucleate Group MN	Binucleate Individual MN/1000 cells	Binucleate Group MN
Minimum	0.0	2.8	12.0	13.0
Maximum	13.0	10.5	28.0	27.0
Mean	6.4	6.4	18.8	18.8
Standard Deviation	2.7	1.9	3.7	3.4
Upper Control Limit		9.7

 

Table 3. Historical control data –S9 20hr (Number of tests: 23)

	Vehicle control		Mitomycin C (0.05 or 0.1 µg/mL)		Colchicine (0.01, 0.02 or 0.03 µg/mL)
	Binucleate Individual MN/1000 cells	Binucleate Group MN	Binucleate Individual MN/1000 cells	Binucleate Group MN	Binucleate Individual MN/1000 cells	Binucleate Group MN
Minimum	2.0	3.3	15.0	16.0	14.0	14.0
Maximum	12.0	10.5	41.0	27.5	23.0	21.5
Mean	7.4	7.4	23.2	23.2	18.1	18.1
Standard Deviation	2.6	2.2	6.6	6.1	2.2	1.9
Upper Control Limit		9.6

 

Conclusions:

In this micronucleus test according to OECD 487, no evidence of cytogenetic potential of the test item was found.

Executive summary:

The potential of the test item to cause an increase in the induction of micronuclei in cultured human peripheral blood lymphocytes in vitro was assessed in a GLP compliant study according to OECD 487. The study consisted of a preliminary toxicity test and a main micronucleus test. Human lymphocytes in whole blood culture were exposed for 3 hours in both the absence and presence of exogenous metabolic activation (S9 mix) and for 20 hours in the absence of S9 mix. The maximum final concentration to which the cells were exposed was 2000 μg/mL. Vehicle (dimethyl sulfoxide [DMSO]) and positive control cultures were included in all appropriate test conditions. Three concentrations were assessed for determination of induction of micronuclei. For the 3-hour treatments, the highest concentration selected (2000 μg/mL) was the highest concentration tested. For the 20-hour treatment, the highest concentration selected (500 μg/mL) was that which caused a reduction in CBPI equivalent to 55±5% cytostasis. Following 3-hour treatment in both the absence and presence of S9 mix, no reductions in CBPI were obtained at any concentration tested. Concentrations of test item selected for micronucleus analysis were 250, 1000 and 2000 μg/mL. In the absence of S9 mix following 20-hour treatment, a reduction in CBPI equivalent to 51.5% cytostasis was obtained at 500 μg/mL. Concentrations of test item selected for micronucleus analysis were 12.5, 350 and 500 μg/mL. In both the absence and presence of S9 mix, following 3-hour treatment, and in the absence of S9 mix, following-20 hour treatment, the test item did not cause any statistically significant increases in the number of binucleate cells containing micronuclei when compared with the vehicle controls. The positive control compounds caused statistically significant increases in the number of binucleate cells containing micronuclei under appropriate conditions, demonstrating the efficacy of the S9 mix and the sensitivity of the test system. It is concluded that the test item did not show evidence of causing an increase in the induction of micronuclei in cultured human lymphocytes, in this in vitro test system under the experimental conditions described.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Mutagenicity in bacteria

The mutagenic potential of the test substance was investigated in an OECD 471 test with GLP, using Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2 uvrA (pKM101). Two independent mutation tests were performed in the presence and absence of liver preparations (S9 mix) from rats treated with phenobarbital and 5,6-benzoflavone. The first experiment was a standard plate incorporation assay; the second included a pre-incubation stage. The test substance was dissolved in DMSO and was tested at concentrations of 5, 15, 50, 150, 500, 1500, 5000 µg/plate. No precipitation was observed. Toxicity was only observed in the first experiment following exposure to the test substance in the absence of S9 mix at 50 μg/plate in strain TA98, and at 50 and 5000 μg/plate in strain TA1537. No mutagenicity was observed at any dose level with and without metabolic activation. It was concluded that the test item showed no evidence of mutagenic activity in this bacterial system under the test conditions employed (Woods, 2016).

The test substance was tested for its gene mutation potential according to the method described by Ames et al., 1975. The test substance was tested with and without Aroclor 1254 induced rat liver S-9 mix in S. typhimurium strains TA 1535, TA 1538, TA 98, and TA 100. The test was performed in duplicate. The vehicle and positive controls were performed in parallel. The test substance was tested up to maximum solubility in water (about 5 mg/L) resulting in doses of 0, 0.8, 4, 20, 100, and 500 µg/plate. No cytotoxicity was observed. No mutagenicity was observed at any dose level with and without metabolic activation. Under the conditions of the test the substance was negative for genotoxicity (Longstaff, 1977).

Mutagenicity in mammalian cells

The test item was tested for mutagenic potential in an in vitro mammalian cell mutation assay according to OECD 476 and GLP. Tests were performed in the absence of exogenous metabolic activation (S9 mix) and in the presence of S9 mix. The vehicle was DMSO (dimethyl sulfoxide). The highest final concentration used in the preliminary toxicity experiment was 2000 μg/mL. No precipitate was seen by eye at the end of treatment. Cytotoxicity was measured as Day 1 relative survival (RS). After exposure at concentrations from 15.63 to 2000 µg/mL RS values ranged from 114 to 26% and from 136 to 17%, in the absence and presence of S9 mix respectively. In the main mutation experiment cells were exposed to concentrations from 1000 to 2000 μg/mL of test item. No precipitate was seen by eye at the end of treatment. After exposure at concentrations from 15.63 to 2000 µg/mL RS values ranged from 110 to 44% and from 102 to 66%, in the absence and presence of S9 mix respectively. The positive controls induced a significant increase in mutant frequency demonstrating the correct functioning of the assay. The test item did not induce a statistically significant increase in mutant frequency. It was concluded that the test item did not demonstrate mutagenic potential in this in vitro HPRT cell mutation assay, under the experimental conditions described (Woods, 2017).

Cytogenetic toxicity in mammalian cells

The potential of the test item to cause an increase in the induction of micronuclei in cultured human peripheral blood lymphocytes in vitro was assessed in a study according to OECD 487 and GLP. The study consisted of a preliminary toxicity test and a main micronucleus test. Human lymphocytes in whole blood culture were exposed for 3 hours in both the absence and presence of exogenous metabolic activation (S9 mix) and for 20 hours in the absence of S9 mix. The maximum final concentration to which the cells were exposed was 2000 μg/mL. Vehicle (DMSO) and positive control cultures were included in all appropriate test conditions. Three concentrations were assessed for determination of induction of micronuclei. Following 3-hour treatment in both the absence and presence of S9 mix, no reductions in CBPI were obtained at any concentration tested. Concentrations of test item selected for micronucleus analysis were 250, 1000 and 2000 μg/mL. In the absence of S9 mix following 20-hour treatment, a reduction in CBPI equivalent to 51.5% cytostasis was obtained at 500 μg/mL. Concentrations of test item selected for micronucleus analysis were 12.5, 350 and 500 μg/mL. The test item did not cause any statistically significant increases in the number of binucleate cells containing micronuclei when compared with the vehicle controls under each condition tested. The positive control compounds caused statistically significant increases in the number of binucleate cells containing micronuclei under appropriate conditions. It is concluded that the test item did not show evidence of causing an increase in the induction of micronuclei in cultured human lymphocytes, in this in vitro test system under the experimental conditions described (Gilby, 2016).

Justification for classification or non-classification

Based on the available data on genetic toxicity, the substance is not classified according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.