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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In an Ames in vitro gene mutation study in bacteria and an in vitro gene mutation study in Chinese Hamster Ovary Cells the test substance was found to have no mutagenic activity.

The test substance also did not show evidence of causing an increase in the induction of micronuclei in in vitro cultured human lymphocytes and was therefore considered to be non-mutagenic in the assay.

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:

03 May 2016 to 15 May 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)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine for Salmonella
Tryptophan for E. Coli

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:

Phenobarbitone / 5,6 benzoflavone

Test concentrations with justification for top dose:

Strains TA100, TA1535, TA1537 and WP2 uvrA:
Experiment 1: 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 2: 5, 15, 50, 150, 500, 1500 and 5000 µg/plate

Strain TA98:
Experiment 1: 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 2 : 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Solubility of the test item was determined in a number of solvents and found to be most soluble in DMSO.

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of TA98

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

With metabolic activation

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of TA100

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

With metabolic activation

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

With metabolic activation

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

With metabolic activation

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of WP2uvrA

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

With metabolic activation

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of TA98

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Without metabolic activation

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of TA100

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without metabolic activation

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without metabolic activation

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Without metabolic activation

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of WP2uvrA

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Without metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation) for experiment 1 and preincubation for experiment 2.

DURATION
- Preincubation period: 10h
- Exposure duration: 48 - 72hrs

NUMBER OF REPLICATIONS: Triplicate for each treatment

DETERMINATION OF CYTOTOXICITY
- Method: Plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn.

Evaluation criteria:

If exposure to a test item produces a reproducible increase in mean revertant colony numbers of at least twice that of the concurrent vehicle controls, with some evidence of a positive concentration-response relationship, it is considered to exhibit mutagenic activity in this test system.
If exposure to a test item does not produce a reproducible increase in mean revertant colony numbers, it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed since additionally this is not required by the guideline. If the results obtained fail to satisfy the criteria for a clear "positive" or "negative" response, even after additional testing, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers. The statistical procedures used are those described by Mahon et al (1989) and are usually Dunnett's test followed, if appropriate, by trend analysis. Biological importance will be considered along with statistical significance. In general, treatment-associated increases in mean revertant colony numbers below two or three times those of the vehicle controls (as described above) are not considered biologically important. It should be noted that it is acceptable to conclude an equivocal response if no clear results can be obtained.
Occasionally, these criteria may not be appropriate to the test data and, in such cases, the Study Director would use his/her scientific judgment.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No test item precipitate was observed on the plates at any of the doses tested in either the presence of S9 mix.

Experiment 1 (Plate Incorporation Assay)

In the first experiment, toxicity, observed as reduction in the number of revertant colonies, was obtained in strains following exposure to the test item in strain TA98 at 150 μg/plate and above in the absence of S9 mix and in strain TA1535 at 1500 μg/plate in the absence of S9 mix. No precipitate was observed on plates following exposure to the test item. A maximum exposure concentration of 5000 μg/plate was, therefore, selected for use in the second experiment.

No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to test item at any concentration up to and including 5000 μg/plate in either the presence or absence of S9 mix.

Strain TA98, in the absence of S9 mix, did not fulfil the criteria for a valid experiment as set out in the study plan as only 3 non-toxic concentrations were obtained, therefore an additional experiment was performed.

 

Additional Plate Incorporation Assay

No evidence of toxicity was obtained following exposure to the test item in strain TA98. Previously toxic concentrations (150 μg/plate and above) produced 0.6 fold revertants of the vehicle control culture. A maximum exposure concentration of 5000 μg/plate was, therefore, selected for use in the second experiment. No substantial increases in revertant colony numbers over control counts were obtained with strain TA98 following exposure to the test item at any concentration up to and including5000 μg/plate in the absence of S9 mix.

Experiment 2 (Pre-incubation Assay)

Toxicity, observed as a reduction in the number of revertant colonies, was obtained in strain TA98 following exposure to the test item at 500 ug/plate and above in the absence and presence of S9 mix. Thinning of the background lawn was observed at 1500 μg/plate and above in both the absence and presence of S9 mix in strain TA98.

Toxicity, observed as reduction in the number of revertant colonies, was obtained in strain TA1535 following exposure to the test item at 5, 500 and 5000 ug/plate in the absence of S9 mix and 5000 μg/plate in the presence of S9 mix. The reduction to 0.5 fold at 5 ug/plate in the absence of S9 mix is considered to be an anomaly and therefore considered not to be of significance.

Toxicity, observed as a reduction in the number of revertant colonies, was obtained in strain TA1537 following exposure to the test item at 500 and 5000 μg/plate in the absence of S9 mix and 50 μg/plate in the presence of S9 mix.

Toxicity, observed as a reduction in the number of revertant colonies, was obtained in strain WP2 uvrA (pKM101) following exposure to the test item at 5000 μg/plate in the presence of S9 mix.

No precipitate was observed on plates following exposure to the test item. No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to the test item at any concentration up to and including 5000 μg/plate in either the presence or absence of S9 mix.

Conclusions:

The mutagenic potential of the test item was assessed according to the OECD Test Guideline 471. It was concluded that the test item showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.

Executive summary:

The mutagenic potential of the test item to 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 assessed according to the OECD Guidelines for the Testing of Chemicals (1997) Genetic Toxicology: Bacterial Reverse Mutation Test, Guideline 471. Two independent mutation tests were performed in the presence and absence of liver preparations (S9 mix).

The first experiment was a standard plate incorporation assay; the second included a pre-incubation stage. Concentrations of the test item up to 5000 μg/plate were tested. Other concentrations used were a series of ca half-log10 dilutions of the highest concentration. In the first experiment, toxicity, observed as reduction in the number of revertant colonies, was obtained in strains following exposure to the test item in strain TA98 at 150 μg/plate and above in the absence of S9 mix and in strain TA1535 at 1500 μg/plate in the absence of S9 mix. No precipitate was observed on plates following exposure to test item. Strain TA98, in the absence of S9 mix, did not fulfil the criteria for a valid experiment as set out in the study plan, therefore an additional experiment was performed. No evidence of toxicity was obtained in the additional experiment following exposure to the test item in strain TA98. Previously toxic concentrations (150 μg/plate and above) produced 0.6 fold revertants of the vehicle control culture.

In the second experiment, toxicity, observed as a reduction in the number of revertant colonies, was obtained in strain TA98 following exposure to the test item at 500 ug/plate and above in the absence and presence of S9 mix. Thinning of the background lawn was observed at 1500 μg/plate and above in both the absence and presence of S9 mix in strain TA98. Toxicity, observed as reduction in the number of revertant colonies, was obtained in strain TA1535 following exposure to the test item at 5, 500 and 5000 ug/plate in the absence of S9 mix and 5000 μg/plate in the presence of S9 mix. The reduction to 0.5 fold at 5 ug/plate in the absence of S9 mix is considered to be an anomaly and therefore considered not to be of significance. Toxicity, observed as a reduction in the number of revertant colonies, was obtained in strain TA1537 following exposure to the test item at 500 and 5000 μg/plate in the absence of S9 mix and 50 μg/plate in the presence of S9 mix. Toxicity, observed as a reduction in the number of revertant colonies, was obtained in strain WP2 uvrA (pKM101) following exposure to the test item at 5000 μg/plate in the presence of S9 mix. No evidence of mutagenic activity was seen at any concentration of the test item in either experiment.

The concurrent positive controls verified the sensitivity of the assay and the metabolising 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

Study period:

02 August 2016 to 09 September 2016

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:

The study plan stated that; “For each culture, three culture flasks will be seeded with 200 cells in H10, to determine cloning efficiency (CE) in non-selective medium, and five flasks with 2 x 105 cells in selective medium will be seeded to determine CE in selective medium.” However, to determine CE in selective medium, five flasks with 5 x 105 cells (in selective medium) were in fact seeded.

This deviation was considered to have not affected the integrity or validity of the study

Deviations:

yes

Remarks:

See 'Remarks' for details of deviation from study plan.

GLP compliance:

yes

Type of assay:

other: in vitro mammalian cell gene mutation test using the Hprt and xprt genes (migrated information)

Target gene:

Hypoxanthine phosphoribosyl tranferase (HPRT) locus

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: European Collection of Cell Cultures.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:

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 unless otherwise specified.

The selective medium, in which only HPRT deficient cells will grow, consists of H10 supplemented with 6-TG at a final concentration of 10 µg/mL.

All cell cultures were maintained at between 34 and 39°C in an atmosphere of 5% CO2 in air.

- Periodically checked for Mycoplasma contamination: Yes
- Periodically 'cleansed' against high spontaneous background: Yes. 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:

S9 fraction, prepared from male Sprague-Dawley derived rats dosed with phenobarbital and 5,6-benzoflavone to stimulate mixed-function oxidases in the liver and stored at ca -80°C.

Test concentrations with justification for top dose:

Preliminary toxicity experiment: 12.91, 25.81, 51.63, 103.25, 206.5, 413, 826 and 1652 µg/mL.
Main experiments:
-S9 mix (3 h): 51.63, 103.25, 206.5, 413, 826, and 1652 µg/mL.
+S9 mix (3 h): 51.63, 103.25, 206.5, 413, 826, and 1652 µg/mL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: H10 media

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

H10 cell culture media

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; in suspension
- Cell density at seeding (if applicable): 2 x 10E6 cells in 40 mL medium

DURATION
- Preincubation period: Approximatlely 20 hours prior to exposure to test item
- Exposure duration: 3 hours
- Expression time (cells in growth medium): 7 days

NUMBER OF REPLICATIONS: Duplicate culture for all culture expect for vehicle controls whch were quadruplicate cultures.

STAINING TECHNIQUE USED: At the end of the incubation period, colonies growing in the flasks were fixed and stained in a methanol:Giemsa solution (4:1 v/v) and counted.

- OTHER:
The osmolality of the test item in medium was tested at 1652 µg/mL; no fluctuations in osmolality of the medium of more than 50 mOsm/kg were observed compared with the vehicle control. No fluctuations in pH of the medium were observed at 1652 µg/mL of more than 1.0 unit compared with the vehicle control. The maximum final concentration tested in the preliminary toxicity experiment was 1652 µg/mL.

Evaluation criteria:

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). Tests were conducted for a linear concentration-response relationship of the test item, for non-linearity and for the comparison of positive control and treated groups to solvent control.

Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly positive if, in any of the experimental conditions examined:

a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control

b) the increase is concentration-related when evaluated with an appropriate trend test

c) any of the results are outside the distribution of the historical negative control data.

When all of these criteria are met, the test item is then considered able to induce gene mutations in cultured mammalian cells in this test system. Providing that all acceptability criteria are fulfilled, a test item is considered clearly negative if, in all experimental conditions examined:

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 an appropriate trend test

c) all results are inside the distribution of the historical negative control data.

The test item is then considered unable to induce gene mutations in cultured mammalian cells in this test system. There is no requirement for verification of a clearly positive or negative response.

In cases when the response is neither clearly negative nor clearly positive as described above, or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations.

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).

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Remarks:

tested up to 1652 µg/mL (10mM)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Preliminary toxicity experiment

Prior to treatment with the test item, cultures established concurrently to those used in this experiment were assessed to ascertain the cell density. This cell concentration was used in the calculation of the adjusted cloning efficiency.

The test item was initially dosed at concentrations up to 1652 μg/mL (10mM). No precipitate was observed by eye at the end of treatment at any concentration tested.

Exposure to the test item for 3 hours at concentrations from 12.91 to 1652 μg/mL in both the absence and presence of S9 mix resulted in relative survival (RS) values from 137 to 82% and from 103 to 72% respectively. Concentrations for the main experiment were based upon these data.

Main experiment: 3-hour Treatment in the absence of S9 mix

Prior to treatment with test item, cultures established concurrently to those used in this experiment were assessed to ascertain the cell density. This cell concentration was used in the calculation of the adjusted cloning efficiency.

Cultures were exposed to the test item at concentrations from 51.63 to 1652 μg/mL. No precipitate was seen by eye at the end of treatment. Exposure to the test item resulted in RS values from 102 to 91%. All cultures were plated out for determination of cloning efficiency and mutant frequency. No significant increases in mutant frequency were observed after exposure to the test item.

The cloning efficiency, used in the assessment of the relative survival, observed in the vehicle control cultures were below the range as detailed in the acceptance criteria. There was no sign of confounding technical problems such as contamination, excessive numbers of outliers and excessive toxicity within the assay. No individual culture within the vehicle control group was considered to be an outlying value. Therefore, the cloning efficiency was considered to be acceptable as it lies close to the acceptance limit and the assay is considered to be valid.

EMS, the positive control, induced a significant increase in mutant frequency.

 

Main experiment: 3-hour treatment in the presence of S9 mix

Prior to treatment with test item, cultures established concurrently to those used in this experiment were assessed to ascertain the cell density. This cell concentration was used in the calculation of the adjusted cloning efficiency.

Cultures were exposed to test item at concentrations from 51.63 to 1652 μg/mL. No precipitate was seen by eye at the end of treatment. Exposure to the test item resulted in RS values from 105 to 67%. All cultures were plated out for determination of cloning efficiency and mutant frequency. No significant increases in mutant frequency were observed after exposure to the test item.

3MC, the positive control, induced a significant increase in mutant frequency.

 

Conclusions:

The purpose of the study was to test for the mutagenic potential of the test item in an in vitro mammalian cell mutation assay, according to the OECD Guideline 476. 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.

Executive summary:

The purpose of the study was to test for the mutagenic potential of the test item in an in vitro mammalian cell mutation assay, according to the OECD Guideline 476.

The 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. Two independent experiments were conducted, one in the absence of exogenous metabolic activation (S9 mix) and one in the presence of S9 mix.

The vehicle was H10 media, in which the test item formed a fine doseable suspension at up to 3.304 mg/mL. The highest final concentration used in the preliminary toxicity experiment was 1652 μg/mL (10 mM). The concentration was chosen with regard to the molecular weight and the purity of the test item and with respect to the current OECD Guideline 476 (2015). No precipitate was observed by eye at the end of treatment at any concentration tested. Cytotoxicity was measured as Day 1 relative survival (RS). After exposure to the test item at concentrations from 12.91 to 1652 µg/mL RS values ranged from 137 to 82% and from 103 to 72%, 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 51.63 to 1652 μg/mL of test item. No precipitate was observed by eye at the end of treatment. RS values ranged from 102 to 91% compared 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 51.63 to 1652 μg/mL of the test item. No precipitate was observed by eye at the end of treatment. RS values ranged from 105 to 67% compared 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 thisin vitroHPRT cell mutation assay, under the experimental conditions described.

 

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 May 2016 to 25 May 2016

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)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Human volunteers, healthy, non-smoking adults between 18 and 35 years of age.
- Cell cycle length, doubling time or proliferation index: Historically the average donor doubling time for the laboratory is ca. 13 hours.
- Sex, age and number of blood donors if applicable: Blood was pooled (equal volumes from each donor) from two female donors for the preliminary test and from one male and one female donor for the main test.
- Whether whole blood or separated lymphocytes were used if applicable: Whole blood diluted in HML media.

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 phytohaemagglutinin (PHA) solution (0.1 mL). All cultures were then incubated at 37°C, and the cells were resuspended (twice daily) by gentle inversion.

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction was prepared from male Sprague-Dawley derived rats, dosed with phenobarbital/5,6-benzoflavone to stimulate mixed-function oxidases in the liver.

Test concentrations with justification for top dose:

Main Test: 206.5, 413, 826 and 1652 μg/mL top dose was 10 mM the maximum concentration recommended in the test guidelines.
413, 826 and 1652 μg/mL were analysed for micronucleus frequency.
Due to the short-term nature of the study, no analysis was carried out to determine the homogeneity, concentration or stability of the test formulation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: HML Media (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).

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 in sterile universal containers

DURATION
- Pre-incubation: cultures were incubated for approximately 48 hours following stimulation with phytohaemagglutinin, before addition of the test item.
- Exposure duration: 3 hours in the absence and presence of exogenous metabolic activation (S9 mix) and for 20 hours in the absence of S9 mix.

STAIN (for cytogenetic assays): Stained in acridine orange solution (0.0125 mg/mL using purified water) for 4 minutes

NUMBER OF REPLICATIONS: Main test- deplicate cultures for each treatment level and each positive controls, quadeuplicate cultures were prepared for vehicle controls. Two slides were prepared from each culture.

NUMBER OF CELLS EVALUATED:
Prior to micronucleus analysis, all slides were randomly coded. Interphase cells were examined by fluorescence microscopy and the incidence of micronucleated cells per 1000 binucleate cells per culture were scored where possible from 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.

Evaluation criteria:

The test item was considered to be clearly positive if, in any of the experimental conditions examined:
1) At least one of the test concentrations exhibits a statistically significant increase in the frequency of micronucleated cells compared with the concurrent negative control.
2) The increase in the frequency of micronucleated cells is dose-related when evaluated with an appropriate trend test.
3) Any of the results are outside the distribution of the historical negative control data.
If all of these criteria are met, the test item was considered able to induce chromosome breaks and/or gain or loss in the test system.
A negative response will be claimed if, in all of the experimental conditions examined:
a) None of the test concentrations exhibits a statistically significant increase in the frequency of micronucleated cells compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with an appropriate trend test.
c) All results are inside the distribution of the historical negative control data.
If all of these criteria are met, the test item was considered unable to induce chromosome breaks and/or gain or loss in the test system.

Statistics:

The analysis assumed that the replicate was the experimental unit. An arcsine square-root transformation was used to transform the data. Test substance 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 analysed 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:

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
- Effects of pH: No fluctuations in pH of the medium were observed at 1652 µg/mL of more than 1.0 unit compared with the control sample.
- Effects of osmolality: At 1652 µg/mL; no fluctuations in osmolality of the medium of more than 50 mOsm/kg were observed compared with the control sample.

Preliminary Toxicity Test

A preliminary toxicity test was performed using water as a vehicle. The test substance was dosed at concentrations up to 400μg/mL. There were no precipitate observed and no significant reductions in the Cytokinesis-block proliferative index (CBPI) at any concentration tested. Following this test, it was considered possible to increase the maximum concentration tested to that recommended in the test guidelines (10mM) using HML media as a vehicle and dosing at 50% v/v. Therefore an additional preliminary toxicity test was performed and the results from the initial preliminary test using water are not reported.

In all treatment conditions the highest concentration tested was 1652 μg/mL (10mM) and precipitate, assessed by eye at the end of treatment, was observed at 1652 μg/mL.

Following 3-hour treatments no significant reductions in CBPI were observed at any level when compared with the vehicle controls. Concentrations for the main micronucleus test were based upon these results.

Following a 20-hour treatment a reduction in CBPI compared to vehicle control values, equivalent to 32.7% cytostasis, was obtained with test substance at 1652 μg/mL. Concentrations for the main micronucleus test were based upon these results.

 

Main Test

3-Hour treatment in the absence of S9 mix

Cytostasis

Concentrations of test substance used for the main micronucleus tests were 206.5, 413, 826 and 1652 μg/mL. Precipitate was observed by eye at the end of treatment at 1652 μg/mL. No significant reduction in CBPI compared with the vehicle control was obtained with the test substance at any concentration tested. Concentrations of the test substance selected for micronucleus analysis were 413, 826 and 1652 μg/mL.

Micronucleus Analysis

The test substance did not cause any statistically significant increases in the number of binucleate cells containing micronuclei when compared with the vehicle controls. Mean micronucleus induction in the vehicle control was within the historical control range. The positive control compounds (mitomycin C and colchicine) caused statistically significant increases in the number of binucleate cells containing micronuclei, demonstrating the sensitivity of the test system.

3-Hour treatment in the presence of S9 mix

Cytostasis

Concentrations of test substance used for the main micronucleus tests were 206.5, 413, 826 and 6512 μg/mL. Precipitate was observed by eye at the end of treatment at 1652 μg/mL. No significant reduction in CBPI compared with the vehicle control was obtained with test substance at any concentration tested. Concentrations of the test substance selected for micronucleus analysis were 413, 826 and 1652 μg/mL.

Micronucleus Analysis

The test substance did not cause any statistically significant increases in the number of binucleate cells containing micronuclei when compared with the vehicle controls. Mean micronucleus induction in the vehicle control was within the historical control range. The positive control compound (cyclophosphamide) caused a statistically significant increase in the number of binucleate cells containing micronuclei, demonstrating the efficacy of the S9 mix and the sensitivity of the test system.

20-Hour treatment in the absence of S9 mix

Cytostasis

Concentrations of test substance used for the main micronucleus tests were 206.5, 413, 826 and 16512 μg/mL. Precipitate was observed by eye at the end of treatment at 1652 μg/mL. No significant reduction in CBPI compared with the vehicle control was obtained with the test substance at any concentration tested. Concentrations of the test substance selected for micronucleus analysis were 413, 826 and 1652 μg/mL.

Micronucleus Analysis

The test susbtance did not cause any statistically significant increases in the number of binucleate cells containing micronuclei when compared with the vehicle controls. Mean micronucleus induction in the vehicle control was within the historical control range. The positive control compounds (mitomycin C and colchicine) caused statistically significant increases in the number of binucleate cells containing micronuclei, demonstrating the sensitivity of the test system.

Conclusions:

This study was designed to assess the potential of the test substance to cause an increase in the induction of micronuclei in cultured human peripheral blood lymphocytes in vitro, according to the OECD Test Guideline 487. It is concluded that the test substance did not show evidence of causing an increase in the induction of micronuclei in cultured human lymphocytes, in this in vitro test system.

Executive summary:

This study was designed to assess the potential of the test substance to cause an increase in the induction of micronuclei in cultured human peripheral blood lymphocytes in vitro, according to the OECD Test Guideline 487.

The study consisted of a preliminary toxicity test and a main micronucleus test. Human lymphocytes in whole blood culture were exposed to the test substance for 3 hours in 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 1652 μg/mL (10mM). The concentration was chosen with regard to the molecular weight and the purity of the test substance and with respect to the current OECD Guideline 487 at the time the test was performed (2014). Vehicle (HML media) and positive control cultures were included in all appropriate test conditions.

Three test substance concentrations were assessed for determination of induction of micronuclei. The highest concentration selected (1652 μg/mL) was the highest concentration tested. In all treatment conditions there were no reductions in Cytokinesis-block proliferative index (CBPI) obtained with the test substance at any concentration tested and the concentrations selected for micronucleus analysis were 413, 826 and 1652 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 substance 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 substance did not show evidence of causing an increase in the induction of micronuclei in cultured human lymphocytes, in this in vitro test system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The substance was found to be non-mutagenic in three reliable, valid in vitro studies on bacterial gene mutation (OECD TG 471), mammalian cell gene mutation (OECD TG 476) and clastogenic effects in mammalian cells (OECD TG 487). It can be concluded that the substance does not need to be classified for mutagenicity in accordance with Regulation (EC) No. 1272/2008.