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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:
2017-03-31 to 2017-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
2016
Deviations:
yes
Remarks:
recovery period and harvest time
Qualifier:
according to
Guideline:
other: Commission Regulation (EC) No 640/2012 of 06 July 2012 amending, for the purpose of its adaptation to technical progress, Regulation (EC) No 440/2008 of 30 May 2008: In vitro Mammalian Cell Micronucleus Test, No B.49; No L 193
Version / remarks:
2012
Deviations:
yes
Remarks:
recovery period and harvest time
Principles of method if other than guideline:
Deviation:
A series of in-house non-GLP validation experiments was performed to get distinct responses of statistical significance when using the specified positive controls. To achieve such response the test design, specifically for the treatment, the recovery phase and harvest time, was slightly modified comparing the current proposal given in the OECD Guideline 487. The optimum in responses was found with the time schedule stated in the Summary.
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 0013537161

Method

Species / strain
Species / strain:
lymphocytes:
Details on mammalian cell lines (if applicable):
CELLS USED
- Source of cells: Blood samples were drawn from healthy non-smoking donors not receiving medication
- Suitability of cells: The lymphocytes of the respective donors have been shown to respond well to stimulation of proliferation with PHA and to positive control substances. All donors had a previously established low incidence of micronuclei in their peripheral blood lymphocytes.
- Sex, age and number of blood donors: female donor (30 years old) Experiment I; female donor (32 years old) Experiment IIA; male donor (23 years old) for Experiment IIB
- Whole blood was used

MEDIA USED
- Type and identity of media including CO2 concentration:
Blood cultures were established by preparing an 11 % mixture of whole blood in medium within 30 hrs after blood collection. 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.
- Properly maintained: yes
Cytokinesis block (if used):
Cytochalasin B
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/beta-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Without S9
Experiment I 26; 45.5; 79.6; 139; 244; 426; 746; 1306; 2286; 4000 µg/mL
Experiment II A 244; 426; 746; 1306; 2286; 4000 µg/mL

With S9
Experiment I 26; 45.5; 79.6; 139; 244; 426; 746; 1306; 2286; 4000 µg/mL
Experiment II A 244; 426; 746; 1306; 2286; 4000 µg/mL
Experiment II B 426; 746; 1306; 2286; 4000 µg/mL

Concentrations are based on preliminary cytotoxicity test
Vehicle:
- Vehicle/solvent used: deionised water
- Justification for choice of solvent/vehicle: well solubility and non-toxic properties to the cell cultures.
Controls
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: Demecolcin without S9, 100 ng/mL in deionised water
Details on test system and conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: Exp I: 4 hrs; Exp IIA: without S9 20 hrs, with S9 4 hrs
- Expression time: 40 hrs
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hrs

SPINDLE INHIBITOR: Cytochalasin B

NUMBER OF CELLS EVALUATED: At least 1000 binucleate cells per culture were scored

STAIN: Giemsa

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: slides were prepared by dropping the cell suspension in fresh fixative onto a clean microscope slide

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: according to Countryman and Heddle, 1976

DETERMINATION OF CYTOTOXICITY
- Method: Cytokinesis-block proliferation index

Evaluation criteria:
A test item can be classified as non-clastogenic and non-aneugenic if:
- 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 control.

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:
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.

Results and discussion

Test results
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Evaporation from medium: no
- Water solubility: well soluble
- Precipitation: not observed

RANGE-FINDING/SCREENING STUDIES: yes

HISTORICAL CONTROL DATA: please refer to "any other information on result incl. tables"

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: CBPI

Any other information on results incl. tables

Table 1. Summary of results

Exp.

Preparation

Test item

Proliferation

Cytostasis

Micronucleated

 

interval

concentration

index

in %*

cells

 

 

in µg/mL

CBPI

 

in %**

Exposure period 4 hrs without S9 mix

I

40 hrs

Solvent control1

1.68

 

0.95

 

 

Positive control2

1.40

41.5

14.45S

 

 

1306

1.88

n.c.

0.30

 

 

2286

1.81

n.c.

0.60

 

 

4000

1.85

n.c.

0.55

Exposure period 20 hrs without S9 mix

IIA

40 hrs

Solvent control1

1.90

 

0.20

 

 

Positive control3

1.48

47.0

 4.15S

 

 

1306

1.87

2.8

0.45

 

 

2286

1.82

9.0

0.25

 

 

4000

1.68

23.9

0.40

Exposure period 4 hrs with S9 mix

I

40 hrs

Solvent control1

2.05

 

0.75

 

 

Positive control4

1.47

55.0

 9.35S

 

 

1306#

1.96

8.9

 1.38S

 

 

2286

2.03

2.6

0.95

 

 

4000

1.99

5.7

0.55

IIB

40 hrs

Solvent control1

2.11

 

0.40

 

 

Positive control5

1.80

28.0

 9.30S

 

 

1306

2.09

1.2

0.50

 

 

2286

2.15

n.c.

0.50

 

 

4000#

2.01

9.1

0.78

*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

#       The number of micronucleated cells was determined in a sample of 4000 binucleated cells

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         Deion. water10.0 % (v/v)
2
           MMC              0.8 µg/mL

3           Demecolcin     100 ng/mL

4           CPA              17.5 µg/mL

5          CPA       15.0 µg/mL

Table 2 Historical control data Solvent control and Positive control

 

Solvent Control without S9

Micronucleated cells in %

 

Pulse treatment (4/40)

Continuous treatment (20/40)

No. of experiments

78*

79**

Mean

0.60

0.57

95 % Ctrl limit

0.08 – 1.12

0.12 – 1.03

1x SD

0.26

0.23

Min – Max

0.15 – 1.65

0.05 – 1.35

 

 

Solvent Control with S9

Micronucleated cells in %

 

Pulse treatment (4/40)

No. of experiments

96*

Mean

0.62

95 % Ctrl limit

0.16 – 1.08

1x SD

0.23

Min – Max

0.15 – 1.30

 

 

Positive Control without S9

Micronucleated cells in %

 

Pulse treatment (4/40)

Continuous treatment (20/40)

MMC

Demecolcin

No. of experiments

78

81

Mean

12.48

3.72

95 % Ctrl limit

1.44 – 23.52

1.43 – 6.01

1x SD

5.52

1.15

Min – Max

4.15 – 30.30

2.10 – 7.25

 

 

Positive Control with S9

Micronucleated cells in %

 

Pulse treatment (4/40)

CPA

No. of experiments

165

Mean

5.16

95 % Ctrl limit

0.84 – 9.49

1x SD

2.16

Min – Max

2.10 – 11.90

 

Applicant's summary and conclusion