Boron carbide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

December 2009 - Septembert 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 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Boron carbide

EC Number:

235-111-5

EC Name:

Boron carbide

Cas Number:

12069-32-8

Molecular formula:

CB4

IUPAC Name:

2,3,4,5-tetraboratetracyclo[2.1.0.0¹,³.0²,⁵]pentane

Details on test material:

- Name of test material (as cited in study report): Boron carbide, B4C
- Tradename: TETRABOR
- Source: ESK Ceramics GmbH & Co. KG
- Substance type: Inorganic
- Physical state: Solid
- Analytical purity: Technical grade, purity 97%
- Lot/batch No.: 908M1300

Method

Species / strain

Species / strain / cell type:

mammalian cell line, other: human lymphocytes

Details on mammalian cell type (if applicable):

- Cell type:

Human peripheral blood lymphocytes from healthy and non-smoking donors with no known recent exposure to genotoxic chemicals and radiation. For this study (in each experiment) blood was collected only from a single donor to reduce inter-individual variability.

- Type and identity of media:

Complete Culture Medium: RPMI 1640 medium supplemented with:
15% foetal bovine serum (FBS)
100U/100 µg/mL penicillin/streptomycin solution
2 mM L-glutamine
2.4 µg/ml phytohaemagglutinin (PHA)

Treatment Medium (short-time exposure):
Complete culture medium without FBS.

After Treatment Medium / Treatment Medium (long-time exposure): Complete culture medium with FBS (15%) and cytochalasin B (6 µg/ml)

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

liver microsome preparations (S9 mix)

Test concentrations with justification for top dose:

The test item was extracted in cell culture medium at a weight/volume ratio of 0.2 g/mL for 72h (±2h) at 37°C (± 1°C) according to ISO 10993-3, 2003 and ISO 10993-12, 2007. The extract was centrifuged at room temperature for 5 minutes at 1000 g and processed by sterile filtration (manufacturer: Whatman, batch: 8255139, material: cellulose acetate, pore size: 0.2 µm).

Duplicate cultures were treated at each concentration. The following concentrations were used in the main experiments:

Experiment I: with metabolic activation: 10, 20, 40, 60, 80, 100% Extract
Experiment I: without metabolic activation: 10, 20, 40, 60,80, 100% Extract.
Experiment II: without metabolic activation: 10, 20, 40, 60, 80, 100% Extract

The following concentrations were selected for the microscopic analyses:
Experiment I with short exposure (4 h): with metabolic activation: 60, 80, 100% Extract
Experiment I with short exposure (4 h): without metabolic activation: 60, 80, 100% Extract
Experiment II with extended exposure (44h): without metabolic activation: 10, 20, 40 and 60% Extract

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Negative controls (cell culture medium) are treated in the same way as all dose groups.

Controls 2

Positive controls:

yes

Remarks:

Clastogenic control substance

Positive control substance:

ethylmethanesulphonate

Remarks:

Dissolved in RPMI (Roswell Park Memorial Institute medium). Final concentration: 600 µg/mL

Controls 3

Positive controls:

yes

Remarks:

Clastogenic control substance

Positive control substance:

cyclophosphamide

Remarks:

Dissolved in RPMI (Roswell Park Memorial Institute medium). Final concentration: 7.5 µg/mL.

Controls 4

Positive controls:

yes

Remarks:

Aneugenic control substance

Positive control substance:

other: Colcemide

Remarks:

Dissolved in RPMI (Roswell Park Memorial Institute medium). Final concentration: 0.08 - 0.8 µg/mL

Details on test system and experimental conditions:

Cells:
Human peripheral blood lymphocytes from healthy and non-smoking donors with no known recent exposure to genotoxic chemicals and radiation are used to examine the ability of chemicals to induce cytogenetic damage. Blood samples were drawn by venous puncture and collected in heparinized tubes. Before use the blood was stored under sterile conditions at 4°C for a maximum of 4 h. Whole blood samples treated with an anti-coagulant (e. g. heparin) are pre-cultured in the presence of mitogen (phyto-haematogglutinin, PHA).

Mammalian Microsomal Fraction S9 Homogenate:
The 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. A stock of the supernatant containing the microsomes was frozen in aliquots of 2 and 4.5 mL and stored at -75°C. The protein concentration in the S9 preparation was 33 mg/mL.

Evaluation criteria:

There are several criteria for determining a positive result:
- a concentration-related increase or reproducible increase in the number of cells containing micronuclei
- a biologically relevant increase in the number of cells containing micronuclei for at least one of the dose groups, which is higher than the laboratory negative
control range.

A test item is considered to be negative if there is no biologically relevant increase in the percentages of cells with micronuclei above concurrent control levels, at any dose group.

Cytotoxicity:
As an assessment of the cytotoxicity, a cytokinesis block proliferation index (CBPI) was determined from 500 cells. At least three analysable concentrations of the test item extract with no more than half log spacing between the concentrations should be tested. If toxicity is found, the highest concentration evaluated should induce approximately 60% toxicity. If possible, the concentration tested should exhibit substantial toxicity, intermediate toxicity and/or no toxicity.
Assessment of cytotoxicity and/or cytostasis should be quantified from Cytokinesis-Block Proliferation Index (CBPI) in both treated and control cultures.

Statistics:

Statistical methods may be used as an aid in evaluating the test results. Statistical significance should not be the only determination of a positive response. No statistics performed as no effect occured.

Results and discussion

Test results

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: In experiment II without metabolic activation at an extract concentration of 60% a relative CBPI of 45% was noted.

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Toxicity
In experiment I without and with metabolic activation no decrease of the relative cytokinesis block proliferation index (CBPI) below 70% was noted.
In experiment II without metabolic activation no decrease of the relative CBPI below 70% was noted up to an extract concentration of 40%. At an extract concentration of 60% a relative CBPI of 45% was noted.

Clastogenicity / Aneugenicity
In experiment I without metabolic activation the micronucleated cell frequencies of the negative control (0.80%) were within the range of the historical control data of the negative control (0.4% - 2.3%). The numbers of micronucleated cells found after treatment with the test item were within the historical control data range of the negative control. The mean values noted were 0.70% (60%), 0.85% (80%) and 0.80% (100%). The number of micronucleated cells found in the groups treated with the test item did not show a biologically relevant increase compared to the corresponding negative control.

In experiment I with metabolic activation the micronucleated cell frequency of the negative control (0.80%) was within the historical control data of the negative control (0.6% - 2.1%). The numbers of micronucleated cells found after treatment with the test item were within the historical control data range of the negative control. The mean values of micronucleated cell frequencies found after treatment with the test item noted were 0.65% (60%), 0.85% (80%) and 0.65% (100%). The number of micronucleated cells found in the groups treated with the test item did not show a biologically relevant increase compared to the corresponding negative control.

In experiment II without metabolic activation the micronucleated cell frequency of the negative control (0.75%) was within the historical control data of the negative control (0.4% - 2.3%,). The numbers of micronucleated cells found after treatment with the test item were within the historical control data range of the negative control. The mean values noted were 1.25% (10%), 1.20% (20%) ,0.65% (40%) and 1.0% (60%). The number of micronucleated cells found in the groups treated with the test item did not show a biologically relevant increase compared to the corresponding negative control.

EMS (600 µg/ml-) and CPA (7.5 µg/mL) were used as clastogenic controls and colcemid as aneugenic control (0.08 and 0.8 µg/ml). They induced distinct and biologically relevant increases of micronucleus frequency. This demonstrates the validity of the assay.

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item boron carbide B4C did not induce structural and/or numerical chromosomal damage in human lymphocytes. Therefore, boron carbide B4C is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in the in vitro Mammalian Cell Micronucleus Test.

Executive summary:

In order to investigate an extract of boron carbide for a possible potential to induce micronuclei in human lymphocytes in vitro a Micronucleus assay was carried out. The test item was extracted in cell culture medium at a weight/volume ratio of 0.2 g/mL. The extract was centrifuged and processed by sterile filtration. The test item extract was diluted in cell culture medium prior to treatment.

In experiment I with and without metabolic activation 100% Extract was selected as highest dose group for the microscopic analysis of micronuclei. In experiment II without metabolic activation 60% Extract was selected as highest dose group for the microscopic analysis of micronuclei. No precipitate of the test item was noted in all dose groups evaluated in experiment I and II. In experiment I without and with metabolic activation no decrease of the relative CBPI below 70% was noted. In experiment II without metabolic activation no decrease of the relative CBPI below 70% was noted up to an extract concentration of 40%. At an extract concentration of 60% a relative CBPI of 45% was noted. In the main experiment I with and without metabolic activation no biologically relevant increase of the micronucleus frequency was noted after treatment with an extract of the test item. In the main experiment II without metabolic activation no biologically relevant increase of the micronucleus frequency was noted after treatment with an extract of the test item. In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item boron carbide B4C did not induce structural and/or numerical chromosomal damage in human lymphocytes. Therefore, boron carbide B4C is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in the in vitro Mammalian Cell Micronucleus Test. Ethylmethanesulfonate and Cyclophosphamide were used as clastogenic controls. Colcemide was used as aneugenic control. All induced distinct and biologically relevant increases of micronucleus frequency. This demonstrates the validity of the assay.