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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:
19/09/2017 - 05/02/2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
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: crystalline
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Chemring/ batch 0727/16
- Expiration date of the lot/batch: 24 April 2019
- Purity test date: 99%
- Physical state/Appearance: Off white powder

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark
- Stability under test conditions: The test item was formulated within two hours of it being applied to the test system; the test item formulations were assumed to be stable.
- Solubility and stability of the test substance in the solvent/vehicle: The test item was soluble in MEM at 8.5 mg/mL and in dimethyl sulphoxide (DMSO) at 85 mg/mL in solubility checks performed in-house. MEM was chosen as the solvent for the study.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Prior to each experiment, the test item was accurately weighed, formulated in MEM and appropriate serial dilutions prepared. There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm (Scott et al., 1991).
- Preliminary purification step (if any):
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid:

FORM AS APPLIED IN THE TEST (if different from that of starting material)

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)

OTHER SPECIFICS:

Method

Target gene:
Chromosome aberrations
Species / strain
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non-smoking volunteer (aged 18-35) 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. 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.
The details of the donors used are:
Preliminary Toxicity Test: male, aged 34 years
Main Experiment: female, aged 23 years
Main Experiment (repeat) female, aged 28 years

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 ºC with 5 % CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Microsomal Enzyne Fraction and S9-Mix
Test concentrations with justification for top dose:
Group Final concentration of Triazolone (µg/mL)
4(20)-hour without S9 0, 13.25, 26.56, 53.13, 106.25, 212.5, 425, 850, MMC 0.4
4(20)-hour wit S9 (2%) 0, 26.56, 53.13, 106.25, 212.5, 425, 850, CP 2
24-hour without S9 0, 13.25, 26.56, 53.13, 106.25, 212.5, 425, 850, MMC 0.2

Preliminary Toxicity Test
The dose range for the Preliminary Toxicity Test was 3.32 to 850 μg/mL. The maximum dose was the maximum recommended dose level, 10 mM concentration.No precipitate of the test item was observed in any of the parallel blood-free cultures at the end of the exposure.
Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 850 μg/mL in all three exposure groups. The mitotic index data are presented in Table 1 of the study report. The test item induced evidence of toxicity in all of the exposure groups.
The selection of the maximum dose level for the Main Experiment was based on the maximum recommended dose level 10 mM concentration for all three exposure groups
Vehicle / solvent:
Identity: Eagle's minimal essential medium with HEPES buffer (MEM)
Supplier: Sigma
Batch number (purity):
-Preliminary Toxicity Test: RNBF9655 (Not applicable) 09/2018
-Main test: 1892415 (Not applicable) 30/11/2017
-Main test repeat (4-hour+S9): 1928996 (Not applicable) 31/10/2018
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: 48hours
- Exposure duration:
4-hour exposure with Metabolic Activation (S9)
4-hour exposure without Metabolic Activation (S9)
24-Hour exposure without Metabolic Activation (S9)
- Expression time (cells in growth medium):
- Selection time (if incubation with a selection agent): 20-hour culture in treatment-free media prioor to cell harvest for the 2 4-hour exposure.
- Fixation time (start of exposure up to fixation or harvest of cells): 2.5 hours before the required harvest time.

SELECTION AGENT (mutation assays):

SPINDLE INHIBITOR (cytogenetic assays):
demecolcine
Mitosis was arrested by addition of demecolcine (Colcemid 0.1 μg/mL) 2.5 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 ºC to ensure complete fixation prior to slide preparation.

STAIN (for cytogenetic assays):


NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re-suspension 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.
When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

NUMBER OF CELLS EVALUATED:
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.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells):
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) (Appendix 1). 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. Endoreduplicated cells were recorded separately and are included in the polyploid cell total number. Many experiments with human lymphocytes have established a range of aberration frequencies acceptable for control cultures in normal volunteer donors. The current historical range is shown in Appendix 1.

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
- Any supplementary information relevant to cytotoxicity:

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: Yes
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): No

- OTHER:
Rationale for test conditions:
According to guideline
Evaluation criteria:
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.

Criteria for determining the Study Conclusion

Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly negative if, in any of the experimental conditions examined:
1) The number of cells with structural aberrations in all evaluated dose groups should be within the range of the laboratory historical control data.
2) No toxicologically or statistically significant increase of the number of cells with structural chromosome aberrations is observed following statistical analysis.
3) There is no concentration-related increase at any dose level

A test item can be classified as genotoxic if:
1) The number of cells with structural chromosome aberrations is outside the range of the laboratory historical control data.
2) At least one concentration exhibits a statistically significant increase in the number of cells with structural chromosome aberrations compared to the concurrent negative control.
3) The observed increase in the frequency of cells with structural aberrations is considered to be dose-related
When all of the above criteria are met, the test item can be considered able to induce chromosomal aberrations in human lymphocytes.
Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include numerical aberrations in the form of polyploidy and endoreduplicated cells.
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:
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
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.
Remarks on result:
other: NA

Any other information on results incl. tables

Preliminary Toxicity Test

The dose range for the Preliminary Toxicity Test was 3.32 to 850 μg/mL. The maximum dose was the maximum recommended dose level, 10 mM concentration.

No precipitate of the test item was observed in any of the parallel blood-free cultures at the end of the exposure.

Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 850 μg/mL in all three exposure groups. The mitotic index data are presented in Table 1 of the study report. The test item induced evidence of toxicity in all of the exposure groups.

The selection of the maximum dose level for the Main Experiment was based on the maximum recommended dose level 10 mM concentration for all three exposure groups.

Chromosome Aberration Test – Main Experiment

The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present up to 850 μg/mL in all three exposure groups.

No precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure in any of the exposure groups tested.

The mitotic index data for the Main Experiment are given in Table 2 and Table 3. They confirm the qualitative observations in that no marked dose-related inhibition of mitotic index was observed. In the 4(20)-hour exposure group in the absence of S9, 23% mitotic inhibition was achieved at 850 μg/mL. In the presence of S9, a inhibition of mitotic index was observed where 32% and 14% inhibition was observed at 425 and 850 μg/mL, respectively. An inhibition of mitotic index of 19% was noted at 850 μg/mL in the 24-hour continuous exposure group.

The maximum dose level selected for metaphase analysis of all three exposure groups was the maximum recommended dose level the 10 mM concentration dose level (850 μg/mL).

The chromosome aberration data are given in Table 4, Table 5 and Table 6 of the study report.

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

Applicant's summary and conclusion

Conclusions:
Triazolone 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:

A Chromosome aberration test in Human Lymphocytes in vitro, Lacey F.E., 2018, has been performed according to the 473 OECD guideline and in GLP compliance.

Duplicate culture of human lymphocytes, treated with the test item were evaluated for chormosome aberrations at 3 dose levels, together with vehicle and positive controls, at 3 exposure conditions : 4 hours exposure in the presence of an induce rat liver homogenate metabolizing system (S9), 4 hours expposure in the absence of metabolic activation (S9) both 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, depending on the exposure group 0, 13,28, 26,56, 53,13, 106,25, 212,5, 425 and 850 µg/mL : concentrations tested for the 4 -hour exposure without S9, 0, 26,56, 53,13, 106,25, 212,5, 425 and 850 µg/mL : concentrations tested for the 4 -hour exposure with S9 and 0, 26,56, 53,13, 106,25, 212,5, 425 and 850 µg/mL: concentrations used for the 24 -hour exposure without S9.

All vehicle 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 to human lymphocytes but did not induce any statistically significant increases in the frequency of cells with aberration either in the presence or absence of metabolic activation at any dose level in any of the exposure groups.

The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in all of the exposure groups.

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