Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-2018
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 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
issued by Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Mainzer Straße 80, D-65189 Wiesbaden
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
100 g/100 g (UVCB substance)


Specific details on test material used for the study:
Liqiude (viscous), yellowish to pink
Expiry date: 28 September 2018
Batch: 0016046440

Method

Species / strain
Species / strain / cell type:
lymphocytes: human lymphocytes, primary culture
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Blood samples were drawn from healthy non-smoking donors not receiving medication.
- Suitability of cells: yes
- Sex, age and number of blood donors if applicable: Blood was collected from a female donor (32 years old) for Experiment I and from a male donor (25 years old) for Experiment II.
- Whether whole blood or separated lymphocytes were used if applicable: whole blood
- Method of maintenace: The culture medium was Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) already supplemented with 200 mM GlutaMAX™. Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 µg/mL), the mitogen PHA (3 µg/mL), 10 % FBS (fetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL). All incubations were done at 37 °C with 5.5 % CO2 in humidified air.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) already supplemented with 200 mM GlutaMAX™. All incubations were done at 37 °C with 5.5 % CO2 in humidified air.
- Properly maintained: yes
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
Cytochalasin B
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/Beta-naphthoflavone induced rat liver microsomal fraction S9 Mix
Vehicle / solvent:
- Vehicle(s)/solvent(s) used:
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: Demecolcine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hours
- Exposure duration: 4 hours in Experiment I, 20 hours in Experiment II (without S9 Mix), 4 hours in Experiment II (with S9 Mix)

SPINDLE INHIBITOR (cytogenetic assays): Cytochalasin B

STAIN (for cytogenetic assays): Giemsa

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The harvested cells were spun down by gentle centrifugation, re-suspended in "saline G", spun down once again by centrifugation and resuspended in 5 mL KCl solution and incubated at 37 °C. Ice-cold fixative mixture of methanol and glacial acetic acid was added to the hypotonic solution and the cells were resuspended carefully. After removal of the solution by centrifugation the cells were resuspended for 2 x 20 minutes in fixative and kept cold. The slides were prepared by dropping the cell suspension in fresh fixative onto a clean microscope slide. The cells were stained with Giemsa.

NUMBER OF CELLS EVALUATED: At least 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells):
- the number of micronucleated cells in all evaluated dose groups is in the range of the historical laboratory control data and
- no statistically significant or concentration-related increase of the number of micronucleated cells is observed in comparison to the respective solvent contrl

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
- The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle (1976)
- The micronuclei have to be stained in the same way as the main nucleus
- The area of the micronucleus should not extend the third part of the area of the main nucleus.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: To describe a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity is expressed as % cytostasis. A CBPI of 1 (all cells are mononucleate) is equivalent to 100 % cytostasis.
Evaluation criteria:
The micronucleus assay will be considered acceptable if it meets the following criteria:

a) The rate of micronuclei in the solvent controls falls within the historical laboratory control data range.
b) The rate of micronuclei in the positive controls is statistically significant increased.
c) The quality of the slides must allow the evaluation of a sufficient number of analyzable cells.

A test item can be classified as clastogenic and aneugenic if:
- the number of micronucleated cells is not in the range of the historical laboratory control data and
- either a concentration-related increase in three test groups or a statistically significant increase in the number of micronucleated cells is observed.

Statistics:
Chi square test (α < 0.05)

Results and discussion

Test results
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
tested up to phase separation
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
In both experiments in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which showed phase separation.
In both experiments inthe absence and presence of S9 mix, no relevant increases in the number of micronucleated cells were observed after treatment with the test item.

A preliminary cytotoxicity test was performed to determine the concentrations to be used in the main experiment. Cytotoxicity is characterized by the percentages of reduction in the CBPI in comparison with the controls (% cytostasis) by counting 500 cells per culture in duplicate. The experimental conditions in this pre-experimental phase were identical to those required and described below for the mutagenicity assay.
The pre-test was performed with 11 concentrations of the test item separated by no more than a factor of √10 and a solvent and positive control. All cell cultures were set up in duplicate. Exposure time was 4 hrs (with and without S9 mix). The preparation interval was 40 hrs after start of the exposure.

Any other information on results incl. tables

Table:: Summary of results of thein vitromicronucleus test in human lymphocytes

Exp.

Preparation

Test item

Proliferation

Cytostasis

Micronucleated

 

 

interval

concentration

index

in %*

cells

95% Ctrl limit

 

 

in µg/mL

CBPI

 

in %**

 

Exposure period 4 hrs without S9 mix

I

40 hrs

Solvent control1

1.88

 

0.65

0.06 – 1.19

 

 

Positive control2

1.68

22.8

13.65S

3.92 – 25.34

 

 

11.3

1.93

n.c.

0.65

 

 

 

19.7

1.87

1.7

0.85

 

 

 

34.6PS

1.79

10.0

0.90

 

Exposure period 20 hrs without S9 mix

II

40 hrs

Solvent control1

1.82

 

0.85

0.00 – 1.11

 

 

Positive control3

1.62

25.3

 4.75S

1.47 – 5.89

 

 

18.8

1.70

15.3

0.40

 

 

 

37.5

1.66

19.6

0.35

 

 

 

75.0PS

1.60

27.5

0.50

 

Exposure period 4 hrs with S9 mix

I

40 hrs

Solvent control1

1.77

 

0.50

0.08 – 1.38

 

 

Positive control4

1.70

9.6

 3.00S

0.70 – 10.20

 

 

19.7

1.71

8.9

0.75

 

 

 

34.6

1.72

6.8

0.30

 

 

 

60.5PS

1.79

n.c.

0.45

 

*      For the positive control groups and the test item treatment groups the values are related to the solvent controls

**    The number of micronucleated cells was determined in a sample of 2000 binucleated cells

PS     Phase separation occurred at the end of treatment

S       The number of micronucleated cells is statistically significantly higher than corresponding control values

n.c.  Not calculated as the CBPI is equal or higher than the solvent control value

1        DMSO                 1.0 % (v/v)
2
        MMC                    0.8 µg/mL
3
        Demecolcine         50 ng/mL
4
        CPA                    17.5 µg/mL

Statistical significance was confirmed by the Chi square test (α < 0.05), using a validated test script of “R”, a language and environment for statistical computing and graphics. Within this test script a statistical analysis was conducted for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control.

Applicant's summary and conclusion

Conclusions:
The substance did not cause genotoxicity in the in-vitro micronucleus test.