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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-01-14 to 2015-07-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
2014
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Species / strain
Species / strain / cell type:
lymphocytes: human
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/β-naphthoflavone induced rat liver S9 was used as the metabolic activation system
Test concentrations with justification for top dose:
10.67, 18.67, 32.67, 57.17, 100.04, 175.07, 306.38, 536.16, 938.29, 1642.0 µg/mL (Exp I and IIA without S9 Mix)
100.04, 175.07, 201.11, 268.15, 306.38, 402.22, 536.29, 670.36 µg/mL (Exp. IIB without S9 Mix)
10.67, 18.67, 32.67, 57.17, 100.04, 175.07, 306.38, 536.16, 938.29, 1642.0 µg/mL (Exp I with S9 Mix)
100.04, 175.07, 306.38, 536.16, 938.29, 1642.0 µg/mL (Exp IIA with S9 Mix)
Vehicle / solvent:
DMSO
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: Without S9-Mix: 4 h, 22 h
With S9 Mix: 4 h
- Fixation time: The cultures were harvested by centrifugation 22 h after beginning of treatment

SPINDLE INHIBITOR: Colcemid (final concentration: 0.2 μg/mL)
STAIN: Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: At least 150 metaphases per culture were evaluated

DETERMINATION OF CYTOTOXICITY
- Method: Percentages of mitotic suppression in comparison with the controls by counting 1000 cells per culture in duplicate

Evaluation criteria:
The chromosomal aberration assay will be considered acceptable if it meets the following criteria:
a) The rate of chromosomal aberrations in the solvent controls falls within the historical laboratory control data range.
b) The rate of chromosomal aberrations in the positive controls is statistically significant increased.

A test item can be classified as non-clastogenic if:
− the number of induced structural chromosomal aberrations in all evaluated dose groups is in the range of the historical laboratory control data and
− no statistically significant increase of the rate of structural chromosomal aberrations is observed in comparison to the respective solvent control.

A test item can be classified as clastogenic if:
− the number of induced structural chromosomal aberrations is not in the range of the historical laboratory control data and
− either a concentration-related or a statistically significant increase in the number of cells carrying structural chromosomal aberrations is observed.
Statistics:
The current historical data range together with the statistical significance, confirmed by the Fisher’s exact test (modified) (p < 0.05) using the validated R Script LM.Rnw, should be considered for classification of the test item.

Results and discussion

Test results
Key result
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 examined
Positive controls validity:
valid
Additional information on results:
The test item, dissolved in DMSO, was assessed for its potential to induce chromosomal aberrations in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix.
Three independent experiments were performed. In Experiment I the exposure period was 4 hours with and without S9 mix. In Experiment IIA the exposure period was 4 hours with S9 mix and 22 hours without S9 mix. In Experiment IIB (confirmatory experiment) the exposure period was 22 hours without S9 mix. The chromosomes were prepared 22 hours (in all experiments) after start of treatment with the test item.
In each experimental group two parallel cultures were analysed. At least 150 metaphases per culture were evaluated for structural chromosomal aberrations, except for the positive control in Experiment II, in the absence of S9 mix, where only 50 metaphases were evaluated due to strong clastogenic effects. 1000 cells were counted per culture for determination of the mitotic index.
The highest treatment concentration in this study, 1642.0 µg/mL (approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the OECD Guideline for in vitro mammalian cytogenetic tests.
In Experiment I and IIA, phase separation of the test item in the culture medium was observed at 536.16 µg/mL and above in the absence and presence of S9 mix at the end of treatment.
No relevant influence on osmolarity or pH was observed.
In Experiment I in the absence and presence of S9 mix, in Experiment IIB in the absence of S9 mix and in Experiment IIA in the presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. In Experiment IIA in the absence of S9 mix no cytotoxicity was observed up to the highest evaluated concentration. Toxic effects, indicated by reduced mitotic indices were observed at higher concentrations.
In Experiments I and IIB, in the absence of S9 mix and Experiments I and IIA in the presence of S9 mix, no statistically significant or biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item (0.3 – 3.0% aberrant cells, excluding gaps) exceeded the range of the solvent control values (1.0 – 2.3% aberrant cells, excluding gaps), but were within the range of the laboratory historical solvent control data. In Experiment IIA in the absence of S9 mix a minor but statistically significant increase was observed, following continuous treatment. Therefore a confirmatory experiment with closer concentrations was performed (reported as Exp. IIB). Since no increase was observed in this confirmatory experiment, the observed statistical significant values are judged as biological irrelevant.
Either EMS (660.0 or 770.0 µg/mL) or CPA (7.5 or 15.0 µg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Experiment

Preparation interval

Test item concentration in µg/mL

Mitototic indices in % of control

incl. Gaps

aberrant cells in % excl. Gaps

with exchanges

Exposure period 4 h without S9-Mix

I

22 h

solvent control

100.0

2.7

2.3

0.0

Positive control

56.2

8.7

8.3

1.7

306.38

98.2

2.7

2.7

0.0

536.16

81.2

1.3

1.0

0.0

938.29

118.5

3.0

2.3

0.3

1642.00

71.0

1.7

1.7

0.0

Exposure period 22 h without S9 Mix

IIA

22 h

Solvent control

100.0

1.7

1.0

0.0

Positive control

33.4

51.9

51.9

9.0

175.07

111.8

1.3

1.3

0.0

306.38

114.5

3.2

3.0

0.2

536.16

96.3

3.5

3.5

0.0

Exposure period 22 h without S9 Mix

IIB

22 h

Solvent control

100.0

1.3

1.3

0.0

Positive control

53.7

20.3

20.0

5.7

402.22

81.1

1.0

0.7

0.0

536.29

86.3

3.0

3.0

0.3

670.36

67.7

2.0

1.7

0.0

Exposure period 4 h with S9 Mix

I

22 h

Solvent control

100.0

1.0

1.0

0.0

Positive control

41.6

11.0

10.7

1.7

306.38

89.0

0.3

0.3

0.0

536.16

97.6

2.7

2.3

0.0

938.29

114.8

2.3

2.3

0.0

1642.00

117.7

2.0

1.7

0.3

IIA

22 h

Solvent control

100.0

2.3

2.3

0.0

Positive control

85.2

13.0

13.0

0.7

175.07

80.8

2.0

1.3

0.3

306.38

87.9

2.7

1.7

0.0

536.16

86.3

3.0

2.7

0.0

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosomal aberrations in human lymphocytes in vitro.
Therefore, the test item is considered to be non-clastogenic in this chromosome aberration test, when tested up to insoluble concentrations (phase separation) and in Experiment IIA up to the highest evaluable concentration.
Executive summary:

The test item, dissolved in DMSO, was assessed for its potential to induce structural chromosomal aberrations in human lymphocytes in vitro in three independent experiments. 150 metaphases were evaluated for structural chromosomal aberrations, except for the positive control in Experiment II, in the absence of S9 mix, where only 50 metaphases were evaluated.

The highest applied concentration in this study (1642.0 µg/mL of the test item, approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the current OECD Guideline 473.

Dose selection of the cytogenetic experiment was performed considering the toxicity data and the solubility of the test item in accordance with the OECD Guideline 473. In Experiment I in the absence and presence of S9 mix, in Experiment IIB in the absence of S9 mix and in Experiment IIA in the presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. In Experiment IIA in the absence of S9 mix no cytotoxicity was observed up to the highest evaluated concentration. Toxic effects, indicated by reduced mitotic indices were observed at higher concentrations.

In Experiments I and IIB, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. Only in Experiment IIA a minor but statistically significant increase was observed in the absence of S9 mix following continuous treatment, therefore a confirmatory experiment with closer concentrations was performed (reported as Exp. IIB).

No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures. Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with structural chromosome aberrations. In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosomal aberrations in human lymphocytes in vitro.

Therefore, is considered to be non-clastogenic in this chromosome aberration test, when tested up to insoluble concentrations (phase separation) and in Experiment IIA up to the highest evaluable concentration.