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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Deviations:
not applicable
Principles of method if other than guideline:
The in vitro micronucleus test provides a relatively rapid method to investigate the ability of chemicals to induce chromosomal damage or damage to the mitotic apparatus. Lymphocytes in whole blood cultures are stimulated to divide by exposure to phytohaemagglutinin (PHA). After approximately 48 hours, cells are treated with the test item or control solutions. One treatment series was performed where cells were continuously exposed without metabolic activation until sampling at a time corresponding to approximately 1.5-2.0 cell cycle lengths. At this time cells have completed one cell division after chemical treatment. Treatment of cultures with the inhibitor of actin polymerisationCytochalasin B blocks cytokinesis and cells that have completed one cell cycle after treatment can be distinguished from non dividing cells by their binucleate appearance thus allowing the detection of micronuclei.
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

Method

Species / strain
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
Humans Lymphocites
Metabolic activation:
without
Test concentrations with justification for top dose:
0,0; 6,10; 9,77; 15,6; 25,0; 40,0 ng/mL
Vehicle / solvent:
steril distilled water of injectable grade
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
water
Positive controls:
yes
Positive control substance:
colchicine
Details on test system and experimental conditions:
Preparation of the test item and reference item.

Solutions of the test item and reference item, as received, were prepared immediately before use in sterile water of injectable grade. Solutions were prepared on a weight/volume basis without correction for the displacement due to the volume occupied by the test item.
Concentrations of solutions were expressed in terms of chemicals as received. All dose levels in this report are expressed to three significant figures. All test item and reference item manipulations were performed under safe light conditions.
All solutions were used within 28 minutes from the initial preparation.
Evaluation criteria:
Statistical analysis
For the statistical analysis, a modified χ2 test was used to compare the number of cells with micronuclei in control and treated cultures.

Acceptance criteria
The assay is considered valid if the following criteria are met:
– The incidence of micronucleated cells of the negative control is within the distribution range of our historical control values.
– Adequate cell proliferation is observed in solvent control cultures.

Criteria for outcome
There is general consensus that genotoxic agents acting by some non-DNA-reactive mechanisms including mitotic spindle disturbance, are expected to exhibit a threshold level below which no biological effect is expected. The No Observed Genotoxic Effect Level (NOGEL) represents a point on the dose-response curve where the response is indistinguishable frombackground (Gollapudi et al. 2013). The NOGEL is directly analogous the No Observed Adverse Effect Level (NOAEL), widely used in regulatory settings, and is defined as the highest tested dose for which no statistically significant increase in the incidence of the genotoxic effect is observed compared to the concurrent negative control, and below which there are no statistically significant increases in the genotoxic effect (MacGregor et al. 2015).

Protocol deviations
Based on the results obtained from the preliminary scoring of cytotoxicity, it was considered appropriate to examine for the presence of micronuclei the highest five dose levels selected for treatment instead of all the dose levels.

Results and discussion

Test results
Key result
Species / strain:
lymphocytes: Human
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

 Dose levels(ng/mL)  % Mn cells BiN  Sig  % Mn cells MonoN  Sig  % Cytotoxicity
 0  0.25    -    0
 6.10  0.20  NS  -    9
 9.77  0.4  NS  0.40  NS  75
15.6  -  -  2.15  ***  95
25  -  -  2.30  ***  99
40  -  2.75  ***  100

Applicant's summary and conclusion

Conclusions:
Results show that the incidence of micronucleated cells of the negative controls was within the distribution range of our historical control values.
Adequate cell proliferation was observed in negative control cultures. The study was accepted as valid.
Statistically significant increases in the incidence of micronucleated cells were observed following treatments with the test item Thiocolchicine at the dose levels of 40.0, 25.0 and 15.6 ng/mL and the reference item Colchicine at the dose level of 40.0 ng/mL.
All incidences were outside the normal distribution of historical control data (95% confidence limits). In addition the incidence observed for the reference item Colchicine at 40 ng/mL was compatible with those generated in our historical positive control database. A summary of the results is presented in Table 4, giving the incidence of micronucleated cells and the cytotoxicity for each test point. It is concluded that, using the in vitro micronucleus test in human lymphocytes as genotoxicity endpoint, the NOGEL for Thiocolchicine is 9.77 ng/mL, while the NOGEL for Colchicine is 25.0 ng/mL.