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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:
2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: OECD test guideline 487
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Commission Regulation (EC) No 640/2012; B.49
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Constituent 1
Reference substance name:
XBP 115
IUPAC Name:
XBP 115
Constituent 2
Reference substance name:
hexanedioic acid, bis(2-propylheptyl) ester
IUPAC Name:
hexanedioic acid, bis(2-propylheptyl) ester
Constituent 3
Chemical structure
Reference substance name:
Bis(2-propylheptyl) hexanedioate
EC Number:
940-510-9
Cas Number:
103043-58-9
Molecular formula:
C26 H50 O4
IUPAC Name:
Bis(2-propylheptyl) hexanedioate
Test material form:
other: liquid
Details on test material:
refer to confidential test material details

Method

Test concentrations with justification for top dose:
1st experiment: 6.3, 12.5, 25, 50, 100 and 200 µg/ml (with and without S9 mix)
2nd experiment: 12.5, 25, 50, 100 and 200 µg/ml (with and without S9 mix)
Vehicle / solvent:
acetone; test substance is insoluble in water
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4h with and without S9 (1st experiment), 24h without and 4h with S9 (2nd experiment)
- Expression time (cells in growth medium): 20h (+/- S9; 1st experiment); 0 / 40h (with / without S9, 2nd experiment)
- Fixation time (start of exposure up to fixation or harvest of cells): 24h (+/- S9, 1st experiment); 24 / 44h (with / without S9, 2nd experiment)

SPINDLE INHIBITOR (cytogenetic assays): cytochalasin B
STAIN (for cytogenetic assays): 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI) and propidium iodide in Fluoroshield™

NUMBER OF REPLICATIONS: harvest time of 24 hours is about 2 times the normal cell cycle length

NUMBER OF CELLS EVALUATED: at least 1 000 cells per culture, means at least 2 000 cells per test group

DETERMINATION OF CYTOTOXICITY
- Method: cell count, proliferation index, microscopical examination of cell morphology

OTHER EXAMINATIONS:
- Other: pH value, osmolarity, solubility

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.
• The number of cells containing micronuclei in the vehicle control was within the range of
our laboratory’s historical negative control data
• The positive control substances both with and without S9 mix induced a distinct increase
in the number of micronucleated cells

Assessment criteria
A test substance is considered "positive" if the following criteria are met:
• A significant, dose-related and reproducible increase in the number of cells containing
micronuclei was observed.
• The number of micronucleated cells exceeded both the value of the concurrent vehicle
control and the range of our laboratory’s historical negative control data.
A test substance generally is considered "negative" if the following criteria are met:
• The number of micronucleated cells in the test groups is not distinctly increased above the
concurrent vehicle control and is within our laboratory’s historical negative control data
range.
Statistics:
The statistical evaluation of the data was carried out using the MUVIKE program system
(BASF SE). The proportion of cells containing micronuclei was calculated for each group. A
comparison of each dose group with the concurrent vehicle control group was carried out
using Fisher's exact test for the hypothesis of equal proportions. This test is Bonferroni-Holm
corrected versus the dose groups separately for each time and was performed one-sided.
If the results of this test were statistically significant compared with the respective vehicle
control, labels (* p ≤ 0.05, ** p ≤ 0.01) have been printed in the tables.

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not influenced by test substance
- Effects of osmolality: not influenced by test substance
- Precipitation: Test substance precipitation (macroscopical assessment) in culture medium at the end of exposure period was observed from 50 μg/mL onward in the 1st Experiment with and without S9 mix and from 100 μg/mL onward in the 2nd Experiment with and without S9 mix.


RANGE-FINDING/SCREENING STUDIES: performed with concentrations up to 10 mM (= 4400 µg/ml) with (4h) and without (4h and 24h) S9 mix

COMPARISON WITH HISTORICAL CONTROL DATA: performed as part of the acceptance and assessment criteria

Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

In this study, no biologically relevant increase in the number of micronucleated cells was observed either without S9 mix or after the addition of a metabolizing system. In both experiments in the absence and presence of metabolic activation after 4 and 24 hours treatment with the test substance the values (0.3 – 0.8% micronucleated cells) were close to the concurrent vehicle control values (0.2 – 0.6% micronucleated cells) and clearly within our historical negative control data range (0.1 - 1.8% micronucleated cells). Besides, in the 1st Experiment in the absence of S9 mix a single statistically significant value (0.8% micronucleated cells) compared to the respective vehicle control value was obtained at an intermediate concentration of 50 μg/mL. However, this observation occurred due to the low rate of micronucleated cells in the concurrent vehicle control group. This value was clearly within our historical negative control data range and, therefore, this finding has to be regarded as biologically irrelevant. The positive control substances EMS (without S9 mix; 400 and 500 μg/mL) and CPP (with S9 mix; 0.5 μg/mL) induced statistically significant increased micronucleus frequencies in both independently performed experiments. However, in the experimental part without S9 mix of the 1st Experiment the positive control EMS 400 μg/mL did not lead to the expected clear increase of micronucleated cells (2.0%). Therefore, for this experimental part the positive control EMS 500 μg/mL was evaluated additionally. Finally, in this study, in at least one positive control group per experimental part in the absence and presence of metabolic activation the frequency of micronucleated cells (2.6 – 4.9% micronucleated cells) was clearly above the range of our historical negative control data range (0.1 - 1.8% micronucleated cells) and within our historical positive control data range (2.3 – 26.6% micronucleated cells).

In addition, in both main experiments in the absence and the presence of S9 mix no growth inhibition indicated by reduced cell counts was observed and no relevantly reduced proliferative activity was observed either after 4 hours exposure interval in the absence and presence of S9 mix or after 24 hours continuous test substance treatment in the test groups scored for cytogenetic damage. Cell morphology/attachment was not adversely influenced at any test group tested for the occurrence of micronuclei. Osmolarity and pH values were not influenced by test substance treatment. Test substance precipitation (macroscopical assessment) in culture medium at the end of exposure period was observed from 50 μg/mL onward in the 1st Experiment with and without S9 mix and from 100 μg/mL onward in the 2nd Experiment with and without S9 mix .

Summary result table: experiments without S9 mix

Experiment

Exposure /Preparation

interval

Test group

Genotoxicity

Cytotoxicity

Relative number of binucleated cells with micronuclei per 2 000 cells scored per test group

Proliferation

Index cytostasis

(CBPI) in %

 

Cell count

1

4/24h

Vehicle control

0.2

0.0

100.0

 

 

6.3 μg/mL

Not determined

Not determined

92.6

 

 

12.5 μg/mL

Not determined

Not determined

91.5

 

 

25.0 μg/mL

0.5

0.4

78.3

 

 

50.0 μg/mL

0.8*

-0.8

83.4

 

 

100.0 μg/mL

0.5

0.6

88.9

 

 

200.0 μg/mL

Not determined

Not determined

73.6

 

 

Positive control(EMS 400µg/ml)

2.0*

4.7

90.1

 

 

Positive control(EMS 500µg/ml)

2.6*

8.4

90.4

2

24/24h

Vehicle control

0.3

0.0

100.0

 

 

12.5 μg/mL

Not determined

Not determined

106.4

 

 

25.0 μg/mL

Not determined

Not determined

80.9

 

 

50.0 μg/mL

0.3

-6.9

100.9

 

 

100.0 μg/mL

0.3

-4.5

96.5

 

 

200.0 μg/mL

0.3

1.2

109.2

 

 

Positive control(EMS 500µg/ml)

2.7*

4.7

121.5

*Frequency statistically significant higher than corresponding control values

Summary result table: experiments with S9 mix

Experiment

Exposure /Preparation

interval

Test group

Genotoxicity

Cytotoxicity

Relative number of binucleated cells with micronuclei per 2 000 cells scored per test group

Proliferation

Index cytostasis

(CBPI) in %

 

Cell count

1

4/24h

Vehicle control

0.6

0.0

100.0

 

 

6.3 μg/mL

Not determined

Not determined

80.9

 

 

12.5 μg/mL

Not determined

Not determined

100.3

 

 

25.0 μg/mL

0.4

-1.4

102.8

 

 

50.0 μg/mL

0.8

-11.2

79.4

 

 

100.0 μg/mL

0.6

-5.4

82.9

 

 

200.0 μg/mL

Not determined

Not determined

81.2

 

 

Positive control(CPP 0.5 µg/ml)

3.0*

21.3

82.1

2

4/44h

Vehicle control

0.6

0.0

100.0

 

 

12.5 μg/mL

Not determined

Not determined

75.9

 

 

25.0 μg/mL

Not determined

Not determined

76.0

 

 

50.0 μg/mL

0.7

4.5

77.2

 

 

100.0 μg/mL

0.4

3.8

90.9

 

 

200.0 μg/mL

0.5

2.6

74.9

 

 

Positive control(CPP 0.5 µg/ml)

4.9*

-11.5

81.2

*Frequency statistically significant higher than corresponding control values

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

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