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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
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-07-05 - 2016-09-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
Microsomal Enzyme Fraction: the S9 fraction made for this study was pre-prepared using the methodology outlined in the Study Plan. This unplanned deviation is thought not to affect the validity, integrity or the result of the study.
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
yes
Remarks:
Microsomal Enzyme Fraction: the S9 fraction made for this study was pre-prepared using the methodology outlined in the Study Plan. This unplanned deviation is thought not to affect the validity, integrity or the result of the study.
GLP compliance:
yes (incl. certificate)
Remarks:
The Department of Health of the Government of the United Kingdom
Type of assay:
other: in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
- State of aggregation: Brown/black solid
- Storage conditions: Room temperature in the dark

Method

Species / strain
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: blood of non-smoking volunteers
- Cell cycle length, doubling time or proliferation index: and had not knowingly recently suffered from a viral infection. Based on over 20 years in-house data for cell cycle times for lymphocytes using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT) for human lymphocytes it is considered to be approximately 16 hours. Therefore using this average the in-house exposure time for the experiments for 1.5 x AGT is 24 hours.
- Sex, age and number of blood donors if applicable: Preliminary Toxicity Test: male, aged 28 years
Main Experiment: female, aged 23 years
- Whether whole blood or separated lymphocytes were used if applicable: whole blood
- Number of passages if applicable:
- Methods for maintenance in cell culture if applicable: 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 % CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: 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 % CO2 in humidified air
- Properly maintained: yes
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
preliminary test: 0, 4.88, 9.77, 19.53, 39.06, 78.13,156.25, 312.5, 625 and 1250 µg/mL (maximal possible dose)
main test: 0, 2.5, 5, 10, 20, 40, 80, 160 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was insoluble in DMSO and acetone at 250 and 500 mg/mL but was doseable suspension in DMSO at 125 mg/mL in solubility checks performed in-house. Therefore, the maximum practical dose level achieved in the cultures was 1250 µg/mL.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hrs
- Exposure duration: 4 hrs
- Recovery period after washing: 20 hrs
- Expression time (cells in growth medium):
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells):

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

FIXATION METHOD
- Mitosis was arrested by addition of demecolcine (Colcemid 0.1 pg/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 KC1. 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 KC1 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.
- Staining: 5% Giemsa for 5 minutes
Evaluation criteria:
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.

Where possible, 300 consecutive well-spread metaphases from each concentration were counted (150 per duplicate), where there were at least 15 cells with aberrations (excluding gaps), slide evaluation was terminated. 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 (%) (including 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.

The following criteria were used to determine a valid assay:
• The frequency of cells with structural chromosome aberrations (excluding gaps) in the vehicle control cultures was within the laboratory historical control data range.
• All the positive control chemicals induced a positive response (p<0.01) and demonstrated the validity of the experiment and the integrity of the S9-mix.
• The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline.
• The required number of cells and concentrations were analyzed.
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).
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.

Results and discussion

Test results
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
some evidence of toxicity in all of the exposure groups in high doses
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
The dose range for the Preliminary Toxicity Test was 4.88 to 1250 µg/mL. The maximum dose was the maximum achievable dose level due to the solubility issues with the test item. A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure at and above 78.13 µg/mL, in the 4(20)-hour exposure groups and at and above 312.5 µg/mL in the 24-hour continuous exposure group. Hemolysis was observed following exposure to the test item at and above 625 µg/mL in the 4(20)-hour exposure groups and at and above 312.5 µg/mL in the 24-hour continuous exposure group. Hemolysis is an indication of a toxic response to the erythrocytes and not indicative of any genotoxic response to the lymphocytes.
Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 625 pg/mL in the 4(20)-hour exposures in the presence and absence of metabolic activation (S9). The maximum dose with metaphases present in the 24-hour continuous exposure was 312.5 µg/mL. The test item induced some evidence of toxicity in all of the exposure groups. The selection of the maximum dose level for the Main Experiment was based on the lowest precipitating dose level for all three exposure groups in the main test.

Any other information on results incl. tables

The qualitative assessment of the slides determined that precipitate was similar to thatvobserved in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present up to the maximum dose level in all three exposure groups. Precipitate observations were made at the end of exposure in blood-free cultures and was noted at and above 40 pg/mL in all exposure groups. No haemolysis was observed in the 4(20)-hour exposure groups but was observed at and above 160 µg/mL in the 24-hour exposure group only.

The mitotic index data for the Main Experiment are given in Table 1 and Table 2. They confirm the qualitative observations in that no dose-related inhibition of mitotic index was observed in any of the exposure groups. Therefore, the maximum dose level selected for metaphase analysis was the lowest precipitating dose level (40 µg/mL) in all three exposure groups.

The chromosome aberration data are given in attached Table 4, Table 5 and Table 6. The assay was considered valid as it met all of the following criteria:

The frequency of cells with chromosome aberrations (excluding gaps) in the vehicle control cultures were within the current historical control data range.

All the positive control chemicals induced a demonstrable positive response (p<0.01) and confirmed the validity and sensitivity of the assay and the integrity of the S9-mix.

The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline.

The required number of cells and concentrations were analyzed.

The test item did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation.

The test item did not induce any polyploid cells at any dose level in any of the exposure groups.

Table 1: Mitotoc Index - Main experiment (4(20)-hour Exposure Groups)

DoseLevel (µg/mL) 4(20)-Hour Without S9 4(20)-Hour With S9
A B Mean % of Control A B Mean % of Control
0 2.95 5.25 4.1 100 6.2 4.75 5.48 100
2.5 - - - - - - - -
5 2.15 4.6 3.38 82 4.75 5.5 5.13 94
10 3.15 3.4 3.28 80 7.10 4.8 5.95 109
20 5.1 2.25 3.68 90 5.05 3.10 4.08 74
40 3.00 P 3.85 P 3.43 84 6.20 P 4.00 P 5.10 93
80 -P -P - - -P -P - -
160 -P -P - - -P -P - -
MMC 0.4 2.95 1.5 2.23 54 NA NA NA NA
CP 2 NA NA NA NA 3.1 3.5 3.3 60

MMC = Mitomycin C

CP = Cyclophosphamide

P = Precipitate

NA = Not applicable

- = Not assessed for mitotoc index

Table 2: Mitotic Index - Main Experiment (24 -hour Exposure Group)

Dose Level (µg/mL) 24-Hour Without S9
A B Mean % of Control
0 9.05 9.25 9.15 100
5 7.65 6.55 7.10 78
10 8.90 10.75 9.83 107
20 5.20 7.20 6.20 68
40 10.05 P 10.30 P 10.18 111
80 -P -P H - -
160 -P H -P H - -
320 -P H -P H - -
MMC 0.2 1.90 2.10 2.00 22

MMC = Mitomycin C

P = Precipitate

NA = Not applicable

- = Not assessed for mitotoc index

Applicant's summary and conclusion

Conclusions:
The test item did not induce a statistically significant increase in the frequency of cells with chromosome aberrations in either the absence or presence of a liver enzyme metabolizing system. The test item was, therefore, considered to be non-clastogenic to human lymphocytes in vitro.
Executive summary:

The test item has been assed in an in vitro GLP-study in accordance with OECD Guideline 473 for structural chromosomal aberrations in cultured mammalian cells. It supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations (Scott et al., 1991).

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at four dose levels, together with vehicle and positive controls. In this study, three exposure conditions were investigated; 4 hours exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period, 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period and a 24-hour exposure in the absence of metabolic activation.

The dose levels used in the Main Experiment were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited on precipitate. The dose levels selected for the Main Test were as follows:

Group Final concentration of test item (µg/mL)

4(20)-hour without S9 0, 2.5, 5, 10, 20, 40, 80, 160

4(20)-hour with S9 (2%) 0, 2.5, 5, 10, 20, 40, 80, 160

24-hour without S9 0, 5,10, 20, 40, 80,160, 320

All vehicle (dimethyl sulphoxide (DMSO)) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control items induced statistically significant increases in the frequency of cells with aberrations. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item was toxic at high doses but it did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that was the lowest precipitating dose level.

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