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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:
03 May 2017 - 09 July 2017
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)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes
Type of assay:
other: in vitro mammalian chromosome aberration test (migrated information)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Physical appearance: pale yellow liquid
- Storage conditions: at room temperature

Method

Species / strain
Species / strain / cell type:
lymphocytes: cultured peripheral human lymphocytes
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: whole blood treated with heparin, after being collected from healthy, adult, non-smoking volunteers
- Method of sampling: blood samples were collected by venepuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started.
- Suitability of cells: cells from healthy, adult, non smoking humans were used as recommended in international guidelines (e.g. OECD).
- Average Generation Time: 13.1 h (dose-range finding and first assay) and 13.2 (second assay)
- Sex, age and number of blood donors: 29 years old (dose-range finding and first assay) and 31 years old (second assay)

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI 1640 medium supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/ml and 50 μg/ml respectively) and 30 U/ml heparin.
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital (80 mg/kg bw) and ß-naphthoflavone (100 mg/kg bw).
Test concentrations with justification for top dose:
The highest concentration analyzed was selected based on the solubility of the test item in the culture medium.

- Dose range finding study and first cytogenetic assay:
3 h exposure, with and without S9: 4.9, 9.8, 19.5 and 39 μg/mL
24 h exposure, 24 h fixation, without S9: 4.9, 9.8, 19.5, 39, 78 and 156 μg/mL
48 h exposure, 48 h fixation, without S9: 4.9, 9.8,19.5, 39, 78 and 156 μg/mL

- Second cytogenetic assay (24 h exposure, 24 h fixation and 48 h exposure, 48 h fixation; without S9): 5, 20 and 80 μg/mL
Vehicle / solvent:
- Solvent used: ethanol
- Justification for choice of solvent: A solubility test was performed based on visual assessment. The test item was dissolved in ethanol, which is recommended by internation guidelines as solvent.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9
Details on test system and experimental conditions:
Two independent cytogenetic assays were performed, preceeded by a dose-range finding assay.

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hr
- Exposure duration experiment 1: 3 h (with and without S9)
- Exposure duration experiment 2: 24, 48 h (without S9)
- Fixation time: 24 h (for 3 or 24 hour exposure period) and 48 h (for 48 hour exposure period)

ENVIRONMENTAL CONDITIONS:
- Humidity: 35-90%
- Temperature: 34.7-37.1 °C

SPINDLE INHIBITOR (cytogenetic assays): colchicine

STAIN (for cytogenetic assays): Giemsa

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96% (v/v) ethanol (Merck)/ether (Merck) and cleaned with a tissue. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10 - 30 min with 5% (v/v) Giemsa (Merck) solution in Sörensen buffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry.

NUMBER OF REPLICATIONS: duplicates in two independent experiments

NUMBER OF CELLS EVALUATED: 1000

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE: 150 in each replicate

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (the highest concentration analyzed was determined by the solubility in the culture medium)

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
Evaluation criteria:
EVALUATION CRITERIA:
A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) At least one of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose related when evaluated with an Cochran Armitage trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.

A test substance was considered negative (not clastogenic) in the chromosome aberration test if:
a) None of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with an Cochran Armitage trend test.
c) All results are inside the 95% control limits of the negative historical control data range.

ACCEPTABILITY CRITERIA:
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b) The concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database.
c) The positive control item induces a statistically significant increase in the number of cells with chromosome aberrations. The positive control data will be analysed by the Fisher’s exact test (one-sided, p < 0.05).
Statistics:
Fisher's exact test, one-sided, was used to compare the incidence of abberant cells cells with the concurrent negative control. In case the Fisher’s exact test showed statistically significant differences between one or more of the test item groups and the vehicle control group, a Cochran Armitage trend test (p < 0.05) was performed to test whether there was a significant trend in the induction.
Graphpad Prism version 4.03 (Graphpad Software, San Diego, USA) was used for statistical analysis of the data.

Results and discussion

Test results
Key result
Species / strain:
lymphocytes: cultured peripheral human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No

DOSE LEVELS SELECTED FOR SCORING:
- First cytogenetic assay: 9.8, 19.5 and 39 μg/mL
- Second cytogenetic assay: all dose levels tested were selected for scoring

RANGE-FINDING/SCREENING STUDIES:
- The test item precipitated in culture medium at a concentration of 39 μg/mL
- The test item did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations

FIRST CYTOGENETIC ASSAY
- Precipitation in the main tests: In the first cytogenetic test, the test item precipitated in the culture medium from 250 µg/mL and upwards. In the second cytogenetic test, in the 24 h exposure time, the test item precipitated in the culture medium at 250 µg/mL. No precipitation was observed in the additional second test.
- The test item did not increase the number of polyploid cells and cells with endoreduplicated chromosomes.

SECOND CYTOGENETIC ASSAY
- The test item did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations at the 48 h continuous exposure time
- At the 24 h exposure time, the test item induced a statistically significant increase in the number of cells with chromosome aberrations at the highest tested concentration of 80 μg/ml only. Although a significant trend was observed, the increase was within the 95% control limits of the negative control historical data range. Therefore this increase is not considered biologically relevant.
- The test item did not increase the number of polyploid cells and cells with endoreduplicated chromosomes


COMPARISON WITH HISTORICAL CONTROL DATA:
- The number of cells with chromosome aberrations and the number of polyploid cells and cells with endoreduplicated chromosomes found in the solvent control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide induced appropriate responses. In addition, the number of cells with chromosome aberrations found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database (see attached illustration for details).

Applicant's summary and conclusion

Conclusions:
A chromosome aberration study with X-19657 was performed according to OECD 473 guideline and GLP principles, in cultured peripheral human lymphocytes in two independent experiments. It is concluded that X-19657 is not clastogenic in human lymphocytes under the experimental conditions described in this report.