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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 August - 09 December 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well conducted and well described study in accordance with GLP and OECD Guideline 487 without any deviation.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: OECD Guideline 487 In Vitro Mammalian Cell Micronucleus Test
Deviations:
no
Qualifier:
according to
Guideline:
other: EU Method B.49 In Vitro Mammalian Cell Micronucleus Test
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity
Test material form:
liquid
Specific details on test material used for the study:
- Name of test material (as cited in study report): Dimetol
- Chemical name (IUPAC): 2,6-dimethylheptan-2-ol
- Physical state: Clear colourless liquid
- Analytical purity: 99.6 %
- Lot/batch No.: PE00090447
- Production date: 10 March 2014
- Expiration date of the lot/batch: 28 May 2016
- Storage condition of test material: Stored at refrigerator (2-8 °C) protected from light

Method

Target gene:
None
Species / strain
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
1.8 % (v/v) S9-fraction; S9 fraction prepared from male Sprague Dawley rats orally induced with phenobarbital (80 mg/kg bw) and β-naphthoflavone (100 mg/kg bw)
Test concentrations with justification for top dose:
Dose range finding test: 17, 52, 164, 512 and 1442 μg/mL culture medium, 3 and 24 h in the absence of S9-mix and for 3 h in the presence of S9-mix

First cytogenetic assay:
Without and with S9-mix: 150, 200, 250, 300, 350 and 400 μg/mL culture medium; 3 h exposure time, 27 h harvest time
Cytogenetic assay 1A:
With S9-mix: 150, 250, 350, 360, 370, 380, 390 and 400 μg/mL culture medium; 3 h exposure time, 27 h harvest time
Cytogenetic assay 1B:
With S9-mix: 100, 350, 360, 370, 380, 390, 400, 410, 420 and 430 μg/mL culture medium; 3 h exposure time, 27 h harvest time

Second cytogenetic assay:
Without S9-mix : 50, 100, 150, 200, 250, 300, 350 and 400 μg/mL culture medium; 24 h exposure time, 24 h harvest time
Cytogenetic assay 2A:
Without S9-mix : 50, 100, 150, 175, 200, 225, 250 and 275 μg/mL culture medium; 24 h exposure time, 24 h harvest time
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO)
- Test substance preparation: No correction was made for the purity/composition of the test substance. Dimetol was dissolved in DMSO of spectroscopic quality (SeccoSolv, Merck, Darmstadt, Germany). Dimetol concentrations were used within 2 h after preparation. The final concentration of the solvent in the culture medium was 1.0 % (v/v).
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
other: colchicine
Remarks:
Without metabolic activation: mitomycin C (0.25 and 0.38 μg/mL for a 3 h exposure period and 0.15 and 0.23 μg/mL for a 24 h exposure period); colchicine (0.1 μg/mL for a 3 h exposure period and 0.05 μg/mL for a 24 h exposure period)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With metabolic activation: cyclophosphamide (15 and 17.5 μg/mL for a 3 h exposure period)
Details on test system and experimental conditions:
PREPARATION OF CULTURE:
Cultured peripheral human lymphocytes were used as test system. Blood was collected from healthy adult, non-smoking, male volunteers (aged < 35 years). The Average Generation Time (AGT) of the cells and the age of the donor at the time the AGT was determined (December 2013) are presented below:
Dose range finding study: age 31, AGT = 13.5 h
First cytogenetic assay: age 34, AGT = 12.8 h; Cytogenetic assay 1A: age 31, AGT = 13.5 h; Cytogenetic assay 1B: age 22, AGT = 12.8 h
Second cytogenetic assay: age 22, AGT = 12.9 h; Cytogenetic assay 2A: age 31, AGT = 12.9 h

Lymphocyte cultures: Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 mL (9 mg/mL) phytohaemagglutinin was added.
Environmental conditions: Cultures were incubated in a humid atmosphere of 80 - 100 % (actual range 60 - 91 %), containing 5.0 ± 0.5 % CO2 in air in the dark at 37.0 ± 1.0 °C (actual range 35.0 - 37.4 °C) for 46 ± 2 h and thereafter exposed to test item.

METHOD OF APPLICATION: in medium (RPMI 1640 medium)
- Culture medium consisted of RPMI 1640 medium (Invitrogen Corporation), supplemented with 20 % (v/v) heat-inactivated (56 °C; 30 min) foetal calf serum (Invitrogen Corporation), L-glutamine (2 mM) (Invitrogen Corporation), penicillin/streptomycin (50 U/mL and 50 μg/mL respectively) (Invitrogen Corporation) and 30 U/mL heparin (Sigma, Zwijndrecht, The Netherlands).

DURATION
- Exposure duration: Dose range finding test: 3 and 24 h (- S9-mix); 3 h (+ S9-mix); First cytogenetic assay: 3 h (± S9 mix); Second cytogenetic assay: 24 h (- S9 mix)
- Fixation time (start of exposure up to fixation or harvest of cells): Dose range finding test: 24 h (± S9 mix); First cytogenetic assay: 27 h (± S9 mix); Second cytogenetic assay: 24 h (- S9 mix)
SPINDLE INHIBITOR (cytogenetic assays): Prior to the mitosis (during or after exposure of the test substance) the chemical cytochalasin B (5 μg/mL) was added to the cultures and incubated for 24 h.
STAIN (for cytogenetic assays): 5 % (v/v) Giemsa solution (in Sorensen buffer pH 6.8) for 10 - 30 min

NUMBER OF REPLICATIONS:
- Duplicate cultures per dose for test item, vehicle and positive controls

NUMBER OF CELLS EVALUATED:
- Cytotoxicity: A minimum of 500 cells per culture was counted, scoring cells with one, two or more nuclei (multinucleated cells).
- At least 1000 (with a maximum deviation of 5 %) binucleated cells per culture were examined by light microscopy for micronuclei. In addition, at least 1000 (with a maximum deviation of 5 %) mononucleated cells per culture were scored for micronuclei separately. Due to cytotoxicity the number of examined bi- or mononucleated cells in the positive control groups might be <1000.

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity/cytostasis of test item in the lymphocyte cultures was determined using the cytokinesis-block proliferation index (CBPI index).
%Cytostasis = 100-100{(CBPIt – 1)/(CBPIc –1)}
CBPI = [(No. mononucleate cells) + (2 x No. binucleate cells) + (3 x No. multinucleate cells)] / [Total number of cells]
t = test substance or control treatment culture
c = vehicle control culture
Three analysable concentrations were scored for micronuclei. The number of micronuclei per cell was not recorded. The highest dose level examined for micronuclei were the cultures that produced 55 ± 5 % cytotoxicity. The lowest dose level had little or no cytotoxicity (approximately the same as solvent control). Also cultures treated with an intermediate dose level were examined.

OTHER:
The following criteria for scoring of binucleated cells were used:
- Main nuclei that were separate and of approximately equal size.
- Main nuclei that touch and even overlap as long as nuclear boundaries are able to be distinguished.
- Main nuclei that were linked by nucleoplasmic bridges.

The following cells were not scored:
- Trinucleated, quadranucleated, or multinucleated cells.
- Cells where main nuclei were undergoing apoptosis (because micronuclei may be gone already or may be caused by apoptotic process).

The following criteria for scoring micronuclei were adapted from Fenech, 1996:
- The diameter of micronuclei should be less than one-third of the main nucleus.
- Micronuclei should be separate from or marginally overlap with the main nucleus as long as there is clear identification of the nuclear boundary.
- Micronuclei should have similar staining as the main nucleus.
Evaluation criteria:
A test substance was considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if:
a) It induces a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of mono or binucleated cells with micronuclei.
b) A statistically significant and biologically relevant increase is observed in the number of mono or binucleated cells with micronuclei in the absence of a clear dose-response relationship.
A test substance was considered negative (not clastogenic or aneugenic) in the in vitro micronucleus test if:
a) none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of mono and binucleated cells with micronuclei.
b) The number of mono and binucleated cells with micronuclei was within the laboratory historical control data range.
Statistics:
The incidence of micronucleated cells (cells with one or more micronuclei) for each exposure group was compared to that of the solvent control using Chi-square statistics.

Results and discussion

Test results
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At a concentration of 1442 μg/mL Dimetol precipitated in the culture medium.

DOSE RANGE FINDING TEST:
- Without metabolic activation (- S9-mix); 3 h exposure time, 27 h harvest time: 0, 0, 7 and 11 % cytostasis was observed at concentrations of 0, 17, 52 and 164 μg/mL. At 512 and 1442 μg/mL, cell lysis was observed.
- Without metabolic activation (- S9-mix); 24 h exposure time, 24 h harvest time: 0, 9, 11 and 43 % cytostasis was observed at concentrations of 0, 17, 52 and 164 μg/mL. At 512 and 1442 μg/mL, cell lysis was observed.
- With metabolic activation (+ S9-mix); 3 h exposure time, 27 h harvest time: 0, 8, 5 and 8 % cytostasis was observed at concentrations of 0, 17, 52 and 164 μg/mL. At 512 and 1442 μg/mL, cell lysis was observed.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The number of mono- and binucleated cells with micronuclei found in the solvent control cultures was within the laboratory historical control data range. Historical control data generated from experiments performed between September 2010 and September 2013.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- In the first cytogenetic assay, Dimetol was tested up to 350 μg/mL for a 3 h exposure time with a 27 h harvest time in the absence of S9- mix and up to 390 μg/mL for a 3 h exposure time with a 27 h harvest time presence of S9-fraction. Appropriate toxicity was reached at these dose levels.
The following dose levels were selected for scoring of micronuclei:
Without S9-mix: 150, 250 and 350 μg/mL culture medium (3 h exposure time, 27 h harvest time).
With S9-mix: 100, 360 and 390 μg/mL culture medium (3 h exposure time, 27 h harvest time).

- In the second cytogenetic assay, Dimetol was tested up to 150 μg/mL for a 24 h exposure time with a 24 h harvest time in the absence of S9-mix. Appropriate toxicity was reached at this dose level.
The following dose levels were selected for the scoring of micronuclei:
Without S9-mix: 50, 100 and 150 μg/mL culture medium (24 h exposure time, 24 h harvest time).
Remarks on result:
other: all strains/cell types tested

Any other information on results incl. tables

Table 7.6.1/1: Number of mononucleated or binucleated cells with micronuclei of human lymphocyte cultures treated with Dimetol in the first and second cytogenetic assay

Concentration (μg/mL)

 

Cytostasis (%)

 

Number of mononucleated cells with micronuclei 1)

 

Number of binucleated cells with micronuclei 1)

 

1000

1000

2000

1000

1000

2000

A

B

A+B

A

B

A+B

First cytogenetic assay - without metabolic activation (- S9-mix); 3 h exposure time, 27 h harvest time

0

0

1

1

2

3

9

12

150

14

2

3

5

4

1

5

250

30

1

1

2

1

2

3

350

60

1

0

1

1

2

3

0.25 MMC-C 

49

1

2

3

32

36

68***

0.1 Colch 

91

19

22

41****

12

16

28**

First cytogenetic assay - with metabolic activation (+ S9-mix); 3 h exposure time, 27 h harvest time

0

0

0

0

0

3

4

7

100

10

2

0

2

2

1

3

360

33

1

0

1

5

5

10

390

53

3

1

4

5

6

11

15 CP 

58

1

1

2

25

41

66***

Second cytogenetic assay - without metabolic activation (- S9-mix); 24 h exposure time, 24 h harvest time

0

0

1

0

1

1

1

2

50

20

0

1

1

2

3

5

100

40

0

0

0

2

1

3

150

61

1

1

2

3

1

4

0.15 MMC-C 

50

1

1

2

17

19

36***

0.05 Colch 

94

42

37

79***

12)

02)

12)

 

*) Significantly different from control group (Chi-square test), * P < 0.05, ** P < 0.01 or *** P < 0.001.

1) 1000 bi- and mononucleated cells were scored for the presence of micronuclei (second cytogenetic assay);1000-1027 bi- and mononucleated cells were scored for the presence of micronuclei (first cytogenetic assay). 

Duplicate cultures are indicated by A and B.

2) 46 and 41 binucleated cells were scored in the A and B culture respectively.

Applicant's summary and conclusion

Conclusions:
Under the test conditions, Dimetol is not clastogenic or aneugenic in human lymphocytes with and without metabolic activation.
Executive summary:

In an in vitro mammalian cell micronucleus test performed according to OECD Guideline 487 and in compliance with GLP, cultured human lymphocytes were exposed to Dimetol at the following concentrations:

Dose range finding test: 17, 52, 164, 512 and 1442 μg/mL culture medium, 3 and 24 h in the absence of S9-mix and for 3 h in the presence of S9-mix

First cytogenetic assay:

Without and with S9-mix: 150, 200, 250, 300, 350 and 400 μg/mL culture medium; 3 h exposure time, 27 h harvest time

Cytogenetic assay 1A: With S9-mix: 150, 250, 350, 360, 370, 380, 390 and 400 μg/mL culture medium; 3 h exposure time, 27 h harvest time

Cytogenetic assay 1B: With S9-mix: 100, 350, 360, 370, 380, 390, 400, 410, 420 and 430 μg/mL culture medium; 3 h exposure time, 27 h harvest time

Second cytogenetic assay:

Without S9-mix : 50, 100, 150, 200, 250, 300, 350 and 400 μg/mL culture medium; 24 h exposure time, 24 h harvest time

Cytogenetic assay 2A: Without S9-mix : 50, 100, 150, 175, 200, 225, 250 and 275 μg/mL culture medium; 24 h exposure time, 24 h harvest time

Prior to the mitosis (during or after exposure of the test substance) the chemical cytochalasin B (5 μg/mL) was added to the cultures and incubated for 24 h. After harvesting, the cells were then treated with a hypotonic solution, fixed, stained and examined for micronuclei.

In the first cytogenetic assay, Dimetol was tested up to 350 μg/mL for a 3 h exposure time with a 27 h harvest time in the absence of S9- mix and up to 390 μg/mL for a 3 h exposure time with a 27 h harvest time presence of S9-fraction. Appropriate toxicity was reached at these dose levels.

The following dose levels were selected for scoring of micronuclei:

Without S9-mix: 150, 250 and 350 μg/mL culture medium (3 h exposure time, 27 h harvest time).

With S9-mix: 100, 360 and 390 μg/mL culture medium (3 h exposure time, 27 h harvest time).

In the second cytogenetic assay, Dimetol was tested up to 150 μg/mL for a 24 h exposure time with a 24 h harvest time in the absence of S9-mix. Appropriate toxicity was reached at this dose level.

The following dose levels were selected for the scoring of micronuclei:

Without S9-mix: 50, 100 and 150 μg/mL culture medium (24 h exposure time, 24 h harvest time).

 

Test item did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two independently repeated experiments. The number of mono- and binucleated cells with micronuclei found in the solvent control cultures was within the laboratory historical control data range. The positive control chemicals produced a statistically significant increase in the number of binucleated cells with micronuclei (mitomycin C and cyclophosphamide) and mononucleated cells with micronuclei (colchicine) indicating the validity of the study.

 

Under the test conditions, Dimetol is not clastogenic or aneugenic in human lymphocytes with and without metabolic activation.