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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:
from May 30th to August 11th, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
other: chromosome aberrations in human lymphocytes

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Species / strain
Species / strain / cell type:
lymphocytes: Human
Details on mammalian cell type (if applicable):
Peripheral blood for lymphocyte cultures
For the study, blood samples obtained from two healthy female volunteer donors, one of 19
years old and one of 28 years old , were pooled for use. The volunteers were non-smoker and were not receiving any medication or radiation exposure to the time of sampling.
Metabolic activation:
with and without
Metabolic activation system:
One batch of S9 tissue fraction, provided by Trinova Biochem GmbH, was used in this study and had the following characteristics: Species: Rat Strain: Sprague Dawley Tissue: Liver Inducing Agents: Phenobarbital – 5,6-Benzoflavone
Test concentrations with justification for top dose:
Based on the preliminary solubility assay, dose levels of 10.0, 5.00, 2.50, 1.25, 0.625, 0.313, 0.156 and 0.0781mM (corresponding to 1160, 580, 290, 145, 72.5, 36.3, 18.2, and 9.08 µg/ml) were used.
Vehicle / solvent:
DMSO
This solvent was selected since it is compatible with the survival of the cells and the S9 metabolic activity.
Controls
Untreated negative controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
PREPARATION OF THE TEST CULTURES AND TREATMENT
Three treatment series were performed (short termtreatment with and without S9 metabolism and long termtreatment), including negative and positive controls. Two cultures were prepared at each test point.
Lymphocyte cultures were treated 48 after they were initiated. Before treatment, cultures were centrifuged at 1000 rpm for 10 minutes and the culture medium was decanted and replaced with treatment medium.

The composition of the treatment media was as follows:
- Treatment medium in the presence of S9 metabolism
Test item or control solution: 0.05 ml
S9 mix: 1.00 ml
Culture medium (without PHA): 3.95 ml
- Treatment medium in the absence of S9 metabolism
Test item or control solution: 0.05 ml
Culture medium (without PHA): 4.95 ml
The final percentage of solvent/vehicle (DMSO) in treatment medium was 1 %.

For the short termtreatment, the treatment media were added to the tubes and the cultures were incubated for 3 hours at 37 °C. At the end of treatment time, the cell cultures were centrifuged and washed twice with Phosphate Buffered Saline (PBS). Fresh medium was added and the cultures were incubated for a further 21 hours (Recovery Period) before harvesting.
For the long termtreatment, the treatment media were added to the tubes and the cultures were incubated for 24 hours.
Colcemid was added (0.2 µg/ml final concentration) for the last 3 hours, leading up to harvesting.

HARVESTING AND SLIDE PREPARATION
The lymphocyte cultures were centrifuged for 10 minutes at 1000 rpm and the supernatant was removed up to approximately 5 mm from the pellet.
The cells were resuspended in hypotonic solution and then centrifuged. The pellet was fixed in fresh methanol/acetic acid fixative and washed two times with fixative. A few drops of the cell suspension obtained in this way were dropped onto clean, wet, grease-free, glass slides and air-dried to produce metaphase chromosome spreads. Three slides were prepared for each test point. The slides were stained in 3 % Giemsa in tap water and rinsed twice in tap and distilled water. The slides were made permanent with Eukitt.
Evaluation criteria:
the test item is considered as clearly positive if the following criteria are met:
– Any dose level shows a statistically significant increase in aberration-bearing cells (excluding gaps)
– The incidence of cells bearing aberrations is outside the normal distribution of historical control values
– The increase of cells bearing aberration is dose-related when evaluated with an appropriate trend test
The test item is considered clearly negative in this assay if none of the above criteria is met.
Statistics:
Fisher’s Exact Test was used to compare the number of cells bearing aberrations (assumed to be Poisson distributed) in control and treated cultures. Bonferroni’s corrections were applied for multiple comparisons. The analysis was performed using sets of data either including or excluding gaps. Cochran-Armitage trend test (one-sided) was performed to aid determination of concentration response relationship.
The percentage of cells bearing aberrations excluding gaps was considered for the evaluation of the outcome of the study.

Results and discussion

Test results
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- No opacity nor precipitation of the medium was observed at the beginning or end of treatment, in the absence or presence of S9 metabolism at any dose level.
- Following treatment with the test item, for all treatment series, a dose-related reduction of pH was observed at higher dose levels. However, pH values at the dose levels selected for metaphase analysis were deemed adeguate since only pH changes higher than one unit are considered culture conditions leading to artifactual positive results ( Scott et al., 1991).
- No remarkable variation of osmolality was observed at any dose level, in the absence or presence of S9 metabolism.

SOLUBILITY TEST
A preliminary solubility trial was performed using dimethylsulfoxide (DMSO).
The test item was found soluble in DMSO at the concentration of 116 mg/ml. An aliquot of this solution, added to culture medium in the ratio of 1:100, gave a clear solution. Based on these results, the final concentration of 1160 µg/ml in DMSO was selected as the highest dose level to be used in the cytogenetic assay.

SELECTION OF DOSE LEVELS FOR SCORING
For short term treatment, no toxicity was observed at any dose level in the absence or presence of S9 metabolic activation.
For continuous treatment in the absence of S9 metabolism, dose related toxicity was observed at higher dose levels reducing the mitotic index to 45 % of the concurrent negative control at 5 mM concentration.
On the basis of these results, the dose levels selected for scoring of aberrationswere as follows:
10.0, 5.00 and 2.50 with and without S9 in case of 3 h treatment
5.00, 1.25 and 0.313 without S9 in case of 24 h treatment

Applicant's summary and conclusion

Conclusions:
The test substance does not induce chromosome aberrations in human lymphocytes after in vitro treatment
Executive summary:

The test item was assayed for the ability to induce chromosomal damage in cultured human lymphocytes, following in vitro treatment in the absence and presence of S9 metabolic activation, according to OECD guideline 473. Three treatment series were included in the study. A short term treatment was performed where the cells were treated for 3 hours in the presence and absence of S9 metabolism. The harvest time of 24 hours corresponding to approximately 1.5 cell cycle was used. A long term(continuous) treatment was also performed, only in the absence of S9 metabolism, until harvest at 24 hours. Solutions of the test item were prepared in dimethylsulfoxide (DMSO).

Dose levels of 1160, 580, 290, 145, 72.5, 36.3, 18.2, and 9.08 µg/mL) were used for all treatment series. Appropriate negative and positive control were included. Two replicate cell cultures were prepared at each test point. For all treatment series, dose levels were selected for the scoring of chromosomal aberrations on the basis of the cytotoxicity of the test item treatments (as determined by the reduction in mitotic index). Where no toxicity was observed, the highest dose level was selected for scoring chromosomal aberrations.

Dose levels selected for scoring were as follows:

10.0, 5.00 and 2.50 with and without S9 in case of 3 h treatment and

5.00, 1.25 and 0.313 without S9 in case of 24 h treatment.

For each replicate culture, 150 well spread metaphases were scored to assess the frequency of aberrant cells.

Following treatment with the test item, no statistically significant increase in the incidence of cells bearing aberrations, including or excluding gaps, was observed at any dose level and treatment condition. It is concluded that the substance does not induce chromosomal aberrations in human lymphocytes after in vitro treatment, under the reported experimental conditions.