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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:
26 October 2018 to 20 March 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EU method B.49 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
2012
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
- Batch No.of test material: 180006P040

Method

Species / strain
Species / strain / cell type:
lymphocytes: primary human
Details on mammalian cell type (if applicable):
CELLS USED
- Type: primary human lymphocytes

For lymphocytes:
- Sex, age and number of blood donors: in the 1st Experiment a 30 year old female donor and in the 2nd Experiment a male (32 years old) donor were used.
- Whether whole blood or separated lymphocytes were used: separated lymphocytes
- Whether blood from different donors were pooled or not: not pooled
- Mitogen used for lymphocytes: yes, phytohemagglutinin (PHA)

MEDIA USED
- Type and composition of media,
All media were supplemented with:
- 1% [v/v] penicillin/streptomycin (final concentration 100 μg/mL)
- 1% [v/v] HEPES buffer (1 M)

For the stimulation the medium was supplemented with:
- 0.5% [v/v] Phytohemagglutinin (PHA, stock solution 0.6 mg/mL, final concentration 3 μg/mL)
- 0.5% [v/v] sodium heparin (25000 IE)
For the Cytochalasin B treatment the medium was supplemented with:
- 30 μL Cytochalasin B (Cyt B, stock solution: 2 mg/mL in DMSO, final concentration: 6 μg/mL)

Culture medium
DMEM / Ham's F12 (1:1) (DMEM/F12) medium containing stable glutamine supplemented with 10% [v/v] fetal calf serum (FCS).

Treatment medium (4 hours treatment without and with S9 mix)
DMEM/F12 medium containing stable glutamine

All incubations were performed at 37°C with a relative humidity of ≥ 90% in a 5% [v/v] CO2 atmosphere.
Cytokinesis block (if used):
Cyt B
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : The S9 fraction was prepared according to Ames et al. (1975) at BASF SE. At least 5 male Wistar rats [Crl:WI(Han)] (200 - 300 g) received 80 mg/kg b.w. phenobarbital i.p. and ß-naphthoflavone orally each on three consecutive
days. During this time, the animals were housed in polycarbonate cages: central air conditioning with a fixed range of temperature of 20 - 24°C and a fixed relative humidity of 45 - 65%. The day/night rhythm was 12 hours: light from 6 am to 6 pm and dark from 6 pm to 6 am. Standardized pelleted feed and drinking water from bottles were available ad libitum. 24 hours after the last administration, the rats were sacrificed, and the induced livers were prepared using sterile solvents and glassware at a temperature of +4°C. The livers were weighed and washed in a weight-equivalent volume of a 150 mM KCl solution (1 mL ≙ 1 g wet liver), then homogenized in three volumes of KCl solution. After centrifugation of the homogenate at 9000 x g for 10 minutes at +4°C, 5-mL portions of the supernatant (S9 fraction) was stored at -70°C to -80°C.
- method of preparation of S9 mix
The S9 mix was prepared freshly prior to each experiment. For this purpose, a sufficient amount of S9 fraction was thawn at room temperature; 1 part S9 fraction was mixed with 9 parts S9 supplement (cofactors). The mixture of both components (S9 mix) was kept cool until use.

Following the concentrations of the cofactors:
MgCl2 8 mM
KCl 33 mM
glucose-6-phosphate 5 mM
NADP 4 mM
phosphate buffer (pH 7.4) 15 mM
The phosphate buffer was prepared by mixing a Na2HPO4 solution with a NaH2PO4 solution in a ratio of about 4:1.

- concentration or volume of S9 mix in the final culture medium : 6 mL (20% v/v)
- quality controls of S9 (metabolic capability): Metabolic activation of Cyclophosphamide
Test concentrations with justification for top dose:
1st experiment
Without S9 mix, 4 hours exposure
5.6, 10.1, 18.1, 32.7, 58.8, 105.8, 190.5, 342.9 μg/mL

With S9 mix, 4 hours exposure
32.7, 58.8, 105.8, 190.5, 342.9, 617.3, 1111.1, 2000 µg/mL

2nd experiment
without S9 mix, 4 hours exposure
0.5, 0.9, 1.6, 2.9, 5.1, 9.3, 16.7, 30.0 µg/mL

Test substance precipitation in culture medium occurred at 125.0 μg/mL and above 4 hours after start of treatment in the absence and at 1000 µg/mL and above in the presence of S9 mix.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: used as vehicle, which has been demonstrated to be suitable in the in vitro cytogenetic assay and for which historical control data are available. The final concentration of the vehicle DMSO in culture medium was 1% (v/v).
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Stimulating time: 48 hours
- Exposure duration/duration of treatment: 4 hours
- Harvest time after the end of treatment: recovery time 16 hours, harvest time 20 hours

FOR MICRONUCLEUS:
- If cytokinesis blocked method was used for micronucleus assay: cytB, 6 µg/mL for 20 hours
- Number of cells spread and analysed per concentration: 1000 binucleated cells per culture, in total at least 2000 binucleated cells per test group, were evaluated for the occurrence of micronuclei .
- Criteria for scoring micronucleated cells: The analysis of micronuclei was carried out the following criteria of Countryman and Heddle.
> The diameter of the micronucleus was less than 1/3 of the main nucleus
> The micronucleus was not linked to the main nucleus and was located within the cytoplasm of the cell.
> Only binucleated cells were scored.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cytokinesis-block proliferation index

METHODS FOR MEASUREMENTS OF GENOTOXICIY
The cells were transferred into tubes, centrifugated at 900 g for 5 min and washed with HBSS. After washing, the cells were centrifuged (900 g, 5 min) and suspended in 0.0375 M KCl (37°C), incubation for 20 minutes at 37°C. After the hypotonic treatment, the cells were fixed by adding of fixative (19 parts methanol and 1 part acetic acid). The cells were centrifuged (900 g, 5 min, 4°C) and suspended in fresh fixative and incubated for 20 min at 4°C. The fixation step will be repeated twice. After the last fixation step, the cells can be centrifugated directly (900 g, 5 min, 4°C), suspended in 1-2 mL fresh fixative and spread on slides. The slides were dipped in deionized water, the cells were pipetted on the slide and fixed by passing through a flame. The cells were stained with May-Grünwald (3 min) and 10% [v/v] Giemsa (in Titrisol, pH 7.2, 10 min) and mounted. The slides were scored microscopically.
Evaluation criteria:
Acceptance criteria
The in vitro micronucleus assay is considered valid if the following criteria are met:
- The quality of the slides allowed the evaluation of a sufficient number of analyzable cells in the control groups (vehicle/positive) and in at least three exposed test groups.
- Sufficient cell proliferation was demonstrated in the vehicle control.
- The number of cells containing micronuclei in the vehicle control was within the range of our laboratory’s historical negative control data (95% control limit). Weak outliers can be judged acceptable if there is no evidence that the test system is not “under control”.
- The positive controls both with and without S9 mix induced a distinct, statistically significant increase in the number of micronucleated cells in the expected range.

Assessment criteria
A test substance is considered to be clearly positive if all following criteria are met:
- A statistically significant increase in the number of micronucleated cells was obtained.
- A dose-related increase in the number of cells containing micronuclei was observed.
- The number of micronucleated cells exceeded both the concurrent vehicle control value and the range of our laboratory’s historical negative control data (95% control limit).

A test substance is considered to be clearly negative if the following criteria are met:
- Neither a statistically significantly nor dose-related increase in the number of cells containing micronuclei was observed under any experimental condition.
- The number of micronucleated cells in all treated test groups was close to the concurrent vehicle control value and within the range of our laboratory’s historical negative control data (95% control limit).
Statistics:
An appropriate statistical analysis was performed. The proportion of cells containing micronuclei was calculated for each test group. A comparison of the micronucleus rates of each
test group with the concurrent vehicle control group was carried out for the hypothesis of equal proportions (i.e. one-sided Fisher's exact test, BASF SE). If the results of this test were statistically significant compared with the respective vehicle control (p ≤ 0.05), labels (s) were printed in the tables.
In addition, a statistical trend test (SAS procedure REG (15)) was performed to assess a possible dose-related increase of micronucleated cells. The used model is one of the proposed
models of the International Workshop on Genotoxicity Test procedures Workgroup Report. The dependent variable was the number of micronucleated cells and the independent variable
was the concentration. The trend was judged as statistically significant whenever the one-sided p-value (probability value) was below 0.05. However, both, biological and statistical significance were considered together.

Results and discussion

Test resultsopen allclose all
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:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: pH was not influenced
- Data on osmolality: osmolality was not influenced
- Precipitation and time of the determination: in the absence of S9 mix, test substance precipitation in culture medium was observed at the end of treatment at 58.8 μg/mL and above in the 1st Experiment. In the 2nd Experiment precipitation was not observed up to the highest concentration applied (30.0 μg/mL). In the presence of S9 mix (1st Experiment) precipitation occurred at the end of treatment at the concentrations 342.9 μg/mL and above.

STUDY RESULTS
Micronucleus test in mammalian cells:
- Results from cytotoxicity measurements:
Reduced proliferation was observed in the 1st Experiment in the absence of S9 mix at all concentrations evaluated for cytotoxicity. The cytostasis at 10.1 μg/mL and 18.1 μg/mL was 31.4% and 69.2%, respectively; higher concentrations were not evaluable for cytotoxicity. Thus, in this experimental part only two cultures (treated with 5.6 and 10.1 μg/mL) could have been evaluated for the occurance of micronuclei, therefore this experimental part did not fulfill the acceptance criteria of the current OECD guidline and was repeated in the 2nd Experiment. In the 2nd Experiment without S9 mix relevantly reduced cell proliferation was observed at 16.7 μg/mL (CBPI cytostasis: 46.7%); higher concentrations were not evaluable for cytotoxicity. In the presence of S9 mix a cytostasis of 20.7% was observed at 105.8 μg/mL. Treatment with higher concentrations induced too strong cytotoxic effects. The cytostasis at 190.5 μg/mL was 67.9% and cultures treated with higher concentrations were not scorable.

- Genotoxicity results
Please refer to table 1 - 3 in the section "any other information on results".

HISTORICAL CONTROL DATA
- Positive historical control data: please refer to table 5 in the section "any other information on results".
- Negative (solvent/vehicle) historical control data: please refer to table 4 in the section "any other information on results".

Any other information on results incl. tables

Table 1: Analysis of micronuclei – 1st Experiment; 4 hours exposure, without S9 mix

 

Test group

[µg/mL]

Culture

No. of

evaluated cells

Cells containing Micronuclei

[n]

[n]

[%]

Vehicle control*

A

1000

6

12

0.6

B

1000

6

5.6

A

n.d.

B

10.1

A

1000

19

33

1.7s

B

1000

14

18.1

A

n.s.

B

32.7

A

n.s.

B

58.8

A

n.s.

B

105.8

A

n.p.

B

190.5

A

n.p.

B

342.9

A

n.p.

B

MMC 0.31

A

n.d.

B

 

Table 2: Analysis of micronuclei – 1st Experiment; 4 hours exposure, with S9 mix

 

Test group

[µg/mL]

Culture

No. of

evaluated cells

Cells containing Micronuclei

[n]

[n]

[%]

Vehicle control*

A

1000

13

17

0.9

B

1000

4

32.7

A

1000

6

10

0.5

B

1000

4

58.8

A

1000

6

13

0.7

B

1000

7

105.8

A

1000

8

12

0.6

B

1000

4

190.5

A

n.s.

B

342.9

A

n.s.

B

617.3

A

n.p.

B

1111.1

A

n.p.

B

2000.0

A

n.p.

B

CPA 2.50

A

1000

94

137

6.9s

B

1000

43

 

 

Table 3:Analysis of micronuclei – 2nd Experiment; 4 hours exposure, without S9 mix

 

Test group

[µg/mL]

Culture

No. of

evaluated cells

Cells containing Micronuclei

[n]

[n]

[%]

Vehicle control*

A

1000

3

9

0.5

B

1000

6

0.5

A

n.d.

B

0.9

A

n.d.

B

1.6

A

n.d.

B

2.9

A

n.d.

B

5.1

A

1000

6

14

0.7

B

1000

8

9.3

A

1000

11

29

1.5s

B

1000

18

16.7

A

1000

14

31

1.6s

B

1000

17

30.0

A

n.s.

B

MMC 0.31

A

1000

128

236

11.8s

B

1000

108

 

* DMSO 1% (v/v)

s = Frequency statistically significantly higher than corresponding control values

n.s. = not scorable due to strong cytotoxicity

n.p. = not scorable due to strong cytotoxicity

n.d. = not determined

 

Table 4

Historical negative control data

 

Summary – Without S9 Mix, All Vehicles*

Period: April 2018 – February 2019

Micronucleated cells [%]

Exposure period

4 hrs

Mean

0.6

Minimum

0.4

Maximum

1.0

Standard Deviation

0.15

95% Lower Control Limit

0.3

95% Upper Control Limit

0.9

No. of Experiments

23

*culture medium, DMSO 1%, Aceton 1%, Ethanol 1%

 

Summary – With S9 Mix, All Vehicles*

Period: April 2018 – February 2019

Micronucleated cells [%]

Exposure period

4 hrs

Mean

0.7

Minimum

0.3

Maximum

1.3

Standard Deviation

0.26

95% Lower Control Limit

0.2

95% Upper Control Limit

1.3

No. of Experiments

23

* culture medium, DMSO 1%, Aceton 1%, Ethanol 1%

 

Table 5 Historical positive control data

 

Summary – without S9 Mix, Mitomycin C and Colchicin

Period: April 2018 – February 2019

Micronucleated cells [%]

Exposure period

4 hrs

Substance and Concentration

MMC 0.31 µg/mL

Mean

8.1

Minimum

3.8

Maximum

12.4

Standard Deviation

2.91

No. of Experiments

21

 

Summary – with S9 Mix, Cychlophosphamid

Period: April 2018 – February 2019

Micronucleated cells [%]

Exposure period

4 hrs

Substance and Concentration

CPA 2.50 µg/mL

Mean

3.7

Minimum

1.9

Maximum

5.8

Standard Deviation

1.12

No. of Experiments

19

 

Applicant's summary and conclusion