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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 October 2013 to 12 February 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Conducted per OECD and EU test guidelines and in accordance to GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
The study director used the test data sheet to obtain the purity value (99.9 %), which was different to the COA (99 %). This was considered to not affect the purpose or the integrity of the study.
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
The study director used the test data sheet to obtain the purity value (99.9 %), which was different to the COA (99 %). This was considered to not affect the purpose or the integrity of the study.
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Physical State/ Appearance: Off-white powder

Method

Species / strain
Species / strain / cell type:
primary culture, other: human lymphocytes
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Group Final concentration of Boronic Acid (µg/mL)
4(20)-hour without S9 0, 29.38, 58.75, 117.5, 235, 470, 705, 940
4(20)-hour with S9 (2%) 0, 29.38, 58.75, 117.5, 235, 470, 705, 940
24-hour without S9 0, 3.67, 7.34, 14.69, 29.38, 58.75, 117.5
4(20)-hour with S9 (1 %) 0, 14.69, 58.75, 117.5, 235, 470, 705
Vehicle / solvent:
The vehicle control used was dimethyl sulphoxide (DMSO).
Controls
Untreated negative controls:
yes
Remarks:
DMSO
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Remarks:
In the absence of S9, mitomycin C was used dissolved in Minimal Essential Medium. In the presence of S9, cyclophosphamide was used dissolved in DMSO.
Details on test system and experimental conditions:
Cells
For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non-smoking volunteer who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. The cell-cycle time for the lymphocytes from the donors used in this study was determined using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells and so calculate the average generation time (AGT). The mean value of the AGT for the pool of regular donors used in this laboratory has been determined to be approximately 16 hours under typical experimental exposure conditions.

Cell Culture
Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented "in-house" with L-glutamine, penicillin/streptomycin, amphotericin B and 10% foetal bovine serum (FBS), at approximately 37 °C with 5% C02 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).

Culture conditions
Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture:
9.05 - 9.2 mL MEM, 10% (FBS)
0 .1 mL Li-heparin
0.1 mL phytohaemagglutinin
0.60 - 0.75 mL heparinized whole blood

With Metabolic Activation (S9) Treatment
After approximately 48 hours incubation at approximately 37 °C, 5% C02 in humidified air, the cultures were transferred to tubes and centrifuged. Approximately 9 mL of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and 0.1 mL of the appropriate solution of vehicle control or test item was added to each culture. For the positive control, 0.1 mL of the appropriate solution was added to the cultures. lmL of 20% S9-mix (i.e. 2% final concentration of S9 in standard co-factors) was added to the cultures of the Preliminary Toxicity Test and of Experiment 1.

In Experiment 2, 1 mL of 10% S9-mix (i.e. 1 % final concentration of S9 in standard co-factors), was added. All cultures were then returned to the incubator. The nominal final volume of each culture was 10 mL.

After 4 hours at approximately 37 °C, 5% C02 in humidified air the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the original culture medium. The cells were then re-incubated for a further 20 hours at approximately 37 °C in 5% C02 in humidified air.

Without Metabolic Activation (S9) Treatment
In Experiment 1, after approximately 48 hours incubation at approximately 37 °C with 5% C02 in humidified air the cultures were decanted into tubes and centrifuged. Approximately 9 mL of the culture medium was removed and reserved. The cells were then resuspended in the required volume of fresh MEM (including serum) and dosed with 0.1 mL of the appropriate vehicle control, test item solution or 0.1 mL of positive control solution. The total volume for each
culture was a nominal 10 mL. After 4 hours at approximately 37 °C, 5% C02 in humidified air the cultures were centrifuged the treatment medium was removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced
with the reserved original culture medium. The cells were then returned to the incubator for a further 20 hours.

In Experiment 2, in the absence of metabolic activation, the exposure was continuous for 24 hours. Therefore, when the cultures were established the culture volume was a nominal 9.9 mL. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 0.1 mL of vehicle control, test item dose solution or 0.1 mL of positive control solution. The nominal final volume of each culture was 10 mL. The cultures were then incubated at approximately 37 °C, 5% C02 in humidified air for 24 hours. The preliminary toxicity test was performed using both of the exposure conditions as described for Experiment 1 and for Experiment 2 in the absence of metabolic activation only.

Preliminary Toxicity Test
Three exposure groups were used:
i) 4 hours exposure to the test item without S9-mix, followed by a 20-hour recovery period in treatment-free media, 4(20)-hour exposure.
ii) 4 hours exposure to the test item with S9-mix (2%), followed by a 20-hour recovery period in treatment-free media, 4(20)-hour exposure.
iii) 24-hour continuous exposure to the test item without S9-mix.
The dose range of test item used was 7 .34 to 1880 µg/mL. Parallel flasks, containing culture medium without whole blood, were established for the three
exposure conditions so that test item precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods.

Using a qualitative microscopic evaluation of the microscope slide preparations from each treatment culture, appropriate dose levels were selected for mitotic index evaluation. Mitotic index data was used to estimate test item toxicity and for selection of the dose levels for the main
test.

Experiment 1
Two exposure groups were used for Experiment 1 :
i) 4-hour exposure to the test item without S9-mix, followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 29.38 to 940 µg/mL.
ii) 4-hour exposure to the test item with S9-mix (2% ), followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 29.38 to 940 µg/mL.

Experiment 2
Two exposure groups were used for Experiment 2:
i) 24-hour continuous exposure to the test item without S9-mix prior to cell harvest. The dose range oftest item used was 3.67 to 117.5 µg/mL.
ii) 4-hour exposure to the test item with S9-mix (1 %) followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 14.69 to 705 µg/mL.

Cell Harvest
Mitosis was arrested by addition of demecolcine (Colcemid 0.1 µg/mL) two hours before the required harvest time. After incubation with demecolcine, the cells were centrifuged, the culture medium was drawn off and discarded, and the cells re-suspended in 0.075M hypotonic KCL. After approximately fourteen minutes (including centrifugation), most of the hypotonic solution was drawn off and discarded. The cells were re-suspended and then fixed by dropping the KCl
cell suspension into fresh methanol/glacial acetic acid (3:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 °C to ensure complete fixation prior to slide preparation.

Preparation of Metaphase Spreads
The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re­suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Each slide was permanently labeled with the appropriate identification data.

Staining
When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

Qualitative Slide Assessment
The slides were checked microscopically to determine the quality of the metaphases and also the toxicity and extent of precipitation, if any, of the test item. These observations were used to select the dose levels for mitotic index evaluation.

Coding
The slides were coded using a computerized random number generator. Supplementary slides were coded manually.

Mitotic Index
A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

Scoring of Chromosome Damage
Where possible the first 100 consecutive well-spread metaphases from each culture were counted, where there were at least 30 to 50% of cells with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing and the ISCN (1985). Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.

In addition, cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) reported. Due to a weak polyploidy effect being observed additional independent evaluation of the frequency of polyploidy cells was performed. In addition, the incidence of cells with endoreduplicated chromosomes was also reported. Many experiments with human lymphocytes have established a range of aberration frequencies acceptable for control cultures in normal volunteer donors.
Evaluation criteria:
Negative Control
The frequency of cells with chromosome aberrations ( excluding gaps) in the vehicle control cultures will normally be within the laboratory historical control data range.

Positive Control
All the positive control chemicals must induce a clear positive response (psO.O 1 ). Acceptable positive responses demonstrate the validity of the experiment and the integrity of the S9-mix.

Criteria for determining the Study Conclusion
A test item can be classified as non-genotoxic if:
1. The number of induced chromosome aberrations in all evaluated dose groups is within the range of laboratory historical control data.
2. No toxicologically or statistically significant increase of the number of structural chromosome aberrations is observed following statistical analysis.
A test item can be classified as genotoxic if:
1. The number of induced structural chromosome aberrations is not in the range of laboratory historical control data.
And
2. Either a concentration-related or a statistically significant increase of the number of structural chromosome aberrations is observed. Marked increases only observed in one dose level will be assessed on a case by case basis.
Biological relevance of the results will be considered first. Statistical methods will be used to analyze the increases in aberration data as recommended in the OECD 4 73 guideline. However, statistical significance will not be the only determining factor for a positive response.

A toxicologically significant response is recorded when the p value calculated from the statistical analysis of the frequency of cells with aberrations excluding gaps is less than 0.05 when compared to its concurrent control and there is a dose-related increase in the frequency of cells with aberrations which is reproducible. Incidences where marked statistically significant increases are observed only with gap-type aberrations will be assessed on a case by case basis.
Statistics:
The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test. (Richardson et al. 1989).

Results and discussion

Test results
Species / strain:
lymphocytes:
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:
All the positive control items induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.

The test item did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range that included a dose level that induced approximately 50% mitotic inhibition or greater.
Remarks on result:
other: other: S9
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

The test item, Boronic Acid was considered to be non-clastogenic to human lymphocytes in vitro.