boron nitride (cubic)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 2009 - January 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

boron nitride (cubic)

EC Number:

701-307-9

Cas Number:

10043-11-5

Molecular formula:

BN

IUPAC Name:

boron nitride (cubic)

Test material form:

solid: crystalline

Method

Target gene:

The S. typhimurium strains are constructed to differentiate between base pair (TA 1535, TA 100, TA 102) and Frameshift (TA 1537, TA 98) mutations.

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9 Homogenate The 59 liver microsomal fraction was prepared at BSL BIOSERVICE GmbH. The S9 mix preparation was performed according to Ames et al..The protein concentration in the S9 preparation (Lot: 091009) was 33 mg/ml.

Test concentrations with justification for top dose:

The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item extracts were prepared and used in the experiment: 10, 20, 40, 60, 80 and 100%The 100% extract concentration corresponds to a weight/volume-ratio of 0.2 g/mL 0.9% NaCl resp. DMSO.

Vehicle / solvent:

0.9% NaCl Lot No.: 25811-1C, DeltaSelect (physiological solution) DMSO Lot No.: 9Q003865, Applichem

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Details on test system and experimental conditions:

Test System1. BacteriaFive strains of S. typhimurium with the following characteristics were used:TA 98: his D 3052; rfa-; uvrB-; R-factor: frame shift mutationsTA 100: his G 46; rfa-; uvrB-; R-factor: base-pair substitutionsTA 1535:his G 46; rfa-; uvrB-: base-pair substitutionsTA 1537: his C 3076; rla-; uvrB-; frame shift mutationsTA 102: his G 428 (pAQ1); rfa-; R-factor: base-pair substitutionsTester strains TA 98, TA 100, TA 1535, TA 1537 and TA 102 were obtained from MOLTOX, INC., NC 28607, USA. They are stored as stock cultures in ampoules with nutrient broth (OXOID) supplemented with DMSO (approx. 80% v/v) over liquid nitrogen.All Salmonella strains contain mutations in the histidine operon, thereby imposing a requirement for histidine in the growth medium. They contain the deep rough (rfa) mutation, which deletes the polysaccharide side chain of the lipopolysaccharides of the bacterial cell surface. This increases cell permeability of larger substances. The other mutation is a deletion of the uvrB gene coding for a protein of the DNA nucleotide excision repair system resulting in an increased sensitivity in detecting many mutagens. This deletion also includes the nitrate reductase (chl) and biotin (bio) genes (bacteria require biotin for growth).The tester strains TA 98, TA 100 and TA 102 contain the R-factor plasmide, pkM101 . These strains are reverted by a number of mutagens that are detected weakly or not at all with the non R-factor parent strains. pkM101 increases chemical and spontaneous mutagenesis by enhancing an error-prone DNA repair system which is normally present in these organisms.The properties of the S typhimurium strains with regard to membrane permeability, ampicillin- and tetracycline-resistance as well as normal spontaneous mutation rates are checked regularly according to Ames et al. In this way it is ensured that the experimental conditions set up by Ames are fulfilled.2. Preparation of BacteriaSamples of each tester strain were grown by culturing for 12 h at 38.5 °C in nutrient broth to the late exponential or early stationary phase of growth. The nutrient medium consists per litre:8 g Nutrient Broth5 g NaClA solution of ampicillin (125 µL, 10 mg/ml) (TA 98, TA 100 and TA 102) was added in order to retain the phenotypic characteristics of the strain.3. Agar PlatesThe Vogel-Bonner Medium E agar plates with 2Vo glucose used in the Ames Test were prepared by BSL BIOSERVICE GmbH or provided by an appropriate supplier. Quality controls were performed. Vogel-Bonner-salts contain per litre:10 g MgSO4 x 7H2O100 g Citric acid175 g NaNH4HPO4 x 4H2O500 g K2HPO4Sterilisation was performed at 121°C in an autoclave.Vogel-Bonner Medium E agar plates contain per litre:15 g Agar Agar20 mL Vogel-Bonner salts50 mL Glucose-solvent (40%)Sterilisation was performed at 121°C in an autoclave.4. Overlay AgarThe overlay agar contains per litre:7 .0 g Agar Agar6.0 g NaCl10.5 mg L-histidine x HCI x H2O12.2 mg BiotinSterilisation was performed at 121°C in an autoclave.5. Mammalian Microsomal Fraction 59 MixThe bacteria most commonly used in these reverse mutation assays do not possess the enzyme system which, in mammals, is known to convert promutagens into active DNA damaging metabolites. In order to overcome this major drawback an exogenous metabolic system is added in form of mammalian microsome enzyme activation mixture.6. S9 HomogenateThe S9 liver microsomal fraction was prepared at BSL BIOSERVICE GmbH. Male Wistar rats were induced with Phenobarbital (80 mg/kg bw) and ß-Naphthoflavone (100 mg/kg bw) for three consecutive days by oral route. The following quality control determinations are performed:a) Biological activity in the Salmonella typhimurium assayb) Sterility TestA stock of the supernatant containing the microsomes was frozen in ampoules of 2 and 4.5 mL and stored at <-75 °C.The protein concentration in the 59 preparation (Lot: 091009) was 33 mg/ml. The S9 mix preparation was performed according to Ames et al..7. Preparation of S9 MixThe S9 mix preparation was performed according to Ames et al (l). 100 mM of ice-cold sodium-ortho-phosphate-buffer, pH 7.4, was added to the following preweighed sterilised reagents to give final concentrations in the S9 mix of:8 mM MgCl233 mM KCI5 mM Glucose-6-phosphate4 mM NADPThis solution was mixed with the liver 9000 x g supernatant fluid in the following proportion:co-factor solution 9.5 partsliver preparation 0.5 partsDuring the experiment the S9 mix was stored on ice.8. Exposure ConcentrationsThe test item was tested with the following extract concentrations in theexperiments: 10,20,40,60, 80 and 100%9. Experimental Performance:The following materials were mixed in a test tube and incubated for 60 min. at 37°C (pre-incubation method):100 µL Test extract at each dose level, extract vehicle control, negative control or reference mutagen solution (positive control),500 µL S9 mix (for testing with metabolic activation) or S9 mix substitution buffer (for testing without metabolic activation),100 µL Bacteria suspension (cf. Preparation of Bacteria, preculture of the strain),After the incubation period (60 min.), the overlay agar (2000 µL) was added and poured onto the surface of a minimal agar plate.For each strain and dose level, including the controls, three plates were used.After solidification the plates were inverted and incubated at 37°C for at least 48 h in the dark.10. Data RecordingThe colonies were counted using a ProtoCOL counter (Meintrup DWS Laborgeräte GmbH). If precipitation of the lest item precludes automatic counting the revertant colonies were counted by hand. In addition, tester strains with a low spontaneous mutation frequency like TA 1535 and TA 1537 were counted manually.11. Evaluation of CytotoxicityCytotoxicity can be detected by a clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately < 0.5 in relation to the extract vehicle control. 12. Criteria of ValidityA test is considered acceptable if for each strain:- the bacteria demonstrate their typical responses to ampicillin (TA 98, TA 100, TA 102)- the control plates without 59 mix are within the following ranges (mean values of the spontaneous reversion frequency are within the historical control data range (-s9 / +s9)):TA98: 18-46 / 18-61TA i00: 75 - 159 / 81 - 165TA1535: 5-29 / 5-31T4 1537: 5-30 / 5-36TA 102: 165 - 390 / 163 - 541- corresponding background growth on negative control, extract vehicle control and test plates is observed.- the positive controls show a distinct enhancement of revevant rates over the control plate.13 Extraction of test itemThe test item was extracted in a polar extraction medium [0.9% NaCl Lot No.: 25811-1C, DeltaSelect (physiological solution)l and in a non-polar extraction medium (DMSO Lot No.: 9Q003865, Applichem) for 72 h at 37°C at a weight/volume ratio of 0.2 g/ml- according to ISO 10993-3, 2003 and ISO 10993-12,2007 . After extraction the polar and non polar extracts were centrifuged at room temperature for 5 minutes at 1000 g. The polar extract was further processed by sterile filtration using a syringe filter (Manufacturer: Whatman, Batch: 8479109, Material: cellulose acetate, Pore size: 0.2 pm).14. ControlPositive and negative controls were included in the experiment. Strain specific positive controls were included in the assay, which demonstrated the effective performance of the test.Negative ControlsVehicle controls, consisting of vehicle alone, as well as untreated controls were treated in the same way as the treatment groups.Positive ControlsStrain specific positive controls. The stability of the controls in solution is unknown but a mutagenic is sufficient evidence of biological stability.

Evaluation criteria:

Evaluation of MutagenicityThe Mutation Factor is calculated by dividing the mean value of the revertant counts through the mean values of the extract vehicle control (the exact and not the rounded values are used for calculation).A test item is considered as mutagenic if:- a clear and dose-related increase in the number of revertants occurs and/or- a biologically relevant positive response for at least one of the dose groups occursin at least one tester strain with or without metabolic activation.A biologically relevant increase is described as follows:- if in tester strains TA 100 and TA 102 the number of reversions is at least twice as high- if in tester strains TA 98, TA 1535 and TA 1537 the number of reversions is at least three times higher than the reversion rate of the extract vehicle control.A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system. Evaluation of CytotoxicityCytotoxicity can be detected by a clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately < 0.5 in relation to the extract vehicle control.

Statistics:

According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No toxic effects of the test item extracts were observed in any of the five tester strains used up to the highest extract concentrations evaluated (with and without metabolic activation) in experiment I and II. No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with extracts (polar and non-polar) of Synthetic cubic Boron Nitride Grade 2 (2 micron) Powder at any concentration level, neither in the presence nor absence of metabolic activation in both experiments.The reference mutagens induced a distinct increase of revertant colonies indicating the validity of the experiments.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):negative with metabolic activationnegative without metabolic activationIn conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item extracts did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.Therefore, extracts (polar and non-polar) of Synthetic cubic Boron Nitride Grade 2 (2 micron) Powder are considered to be non-mutagenic in this bacterial reverse mutation assay.