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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:
25 October 2017 until 16 January 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

Method

Species / strain
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
- Type and identity of media: Dulbeccos's modified Eagle's medium/Ham's F12 medium
- Properly maintained: yes
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9
Test concentrations with justification for top dose:
With metabolic activation:
Experiment I: 4.4, 7.7, 13.5, 23.7, 41.5, 72.6, 127, 222, 667, 2000 µg/mL

Without metabolic activation:
Experiment I: 1.4, 2.5, 4.4, 7.7, 13.5, 23.7, 41.5, 72.6, 127, 222, 667, 2000 µg/mL

Experiment IIA: 1.4, 2.4, 4.2, 7.3, 12.8, 22.4, 39.2, 68.6, 120 µg/mL
Experiment IIB: 16.2, 32.3, 64.6, 74.3, 85.5, 98.3, 113, 130 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility and relatively low cytotoxicity in accordance to the OECD Guideline 473
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
Details on test system and experimental conditions:
Three independent experiments were performed. In Experiment I the exposure period was 4 hours with and without metabolic activation. In Experiment IIA and IIB the exposure period was 4 hours with S9 mix and 22 hours without S9 mix. The chromosomes were prepared 22 hours after start of treatment with the test item. Evaluation of two cultures per dose group.

METHOD OF APPLICATION: in culture medium

DURATION
- Exposure duration: 4 hours (+/- S9 mix) and 22 hours (- S9 mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 22 hours


SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa


NUMBER OF REPLICATIONS: about 1.5


NUMBER OF CELLS EVALUATED: 150 per culture, except for the positive control in Experiment IIB, where only 50 metaphases were evaluated


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
Evaluation criteria:
Evaluation of the cultures was performed (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik") using NIKON microscopes with 100x oil immersion objectives. Breaks, fragments, deletions, exchanges, and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. 150 well spread metaphases per culture were scored for cytogenetic damage on coded slides.
Only metaphases with characteristic chromosome numbers of 46 ± 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined.
In addition, the number of polyploid cells in 500 metaphases per culture was determined (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype). Additionally the number of endomitotic cells scored at the evaluation of polyploid cells was noticed and reported (% endomitotic metaphases).
Statistics:
The statistical significance is confirmed by the Fisher’s exact test (modified) (p < 0.05) using a validated test script of “R”, a language and environment for statistical computing and graphics.

Results and discussion

Test results
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True 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 and IIB, the exposure period was 22 hours without S9 mix. The chromosomes were prepared 22 hours after start of treatment with the test item.
In each experimental group two parallel cultures were analyzed. 150 metaphases per culture were evaluated for structural chromosomal aberrations, except for the positive control in Experiment IIB, 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, 2000 µg/mL was chosen with respect to the OECD Guideline for in vitro mammalian cytogenetic tests.
In Experiment I, precipitation of the test item in the culture medium was observed at 13.5 µg/mL and above in the absence of S9 mix and at 41.5 µg/mL and above in the presence of S9 mix. In addition, precipitation occurred in Experiment IIA, in the absence of S9 mix, at 120 µg/mL and in Experiment IIB in the absence of S9 mix, at 64.6 µg/mL and above.
No relevant influence on osmolarity or pH was observed.
In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentrations, which showed precipitation.
Either with or without metabolic activation 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.
No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.
Either EMS (770 or 660 µg/mL) or CPA (5.0 µg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

Any other information on results incl. tables

Summary of results of the chromosomal aberration study

Exp.

Preparation
interval

Test item
concentration
(µg/mL)

Mitotic indices
(%
of control)

Aberrant cells(%)
incl. gaps*

Aberrant cells(%)
excl. gaps*

Aberrant cells(%)
carrying exchanges

Exposure period 4 hrs without S9 mix

I

22 hrs

Solvent control1

100.0

2.3

2.0

0.0

 

 

Positive control2

66.5

9.3

9.3S

2.0

 

 

4.4

99.2

1.7

1.7

0.0

 

 

7.7

103.1

1.0

0.7

0.0

 

 

13.5P

97.1

1.7

1.7

0.0

Exposure period 22 hrs without S9 mix

II

22 hrs

Solvent control1

100.0

1.7

1.7

0.0

 

 

Positive control3#

39.6

40.0

40.0S

14.0

 

 

16.2

105.7

1.0

1.0

0.0

 

 

32.3

104.0

0.7

0.7

0.0

 

 

64.6P

61.6

1.0

1.0

0.0

Exposure period 4 hrs with S9 mix

I

22 hrs

Solvent control1

100.0

1.3

1.0

0.0

 

 

Positive control4

32.0

11.0

11.0S

0.7

 

 

13.5

97.0

1.7

1.7

0.0

 

 

23.7

89.3

0.7

0.7

0.0

 

 

41.5P

98.1

2.3

2.3

0.0

*      Including cells carrying exchanges

#       Evaluation of 50 metaphases per culture

P      Precipitation occurred at the end of treatment

S       Aberration frequency statistically significant higher than corresponding control values

1       DMSO      0.5 % (v/v)
2         EMS          770 µg/mL
3
         EMS          660 µg/mL
4
       CPA           5.0 µg/mL

Applicant's summary and conclusion

Conclusions:
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 is considered to be non-clastogenic in this chromosome aberration test, when tested up to precipitating concentrations.
Executive summary:

The test item, dissolved in DMSO, was assessed for its potential to induce structural chromosomal aberrations in human lymphocytesin vitro in three independent experiments. The following study design was performed:

 

Without S9 mix

With S9 mix

 

Exp. I

Exp. IIA & IIB

Exp. I

Exposure period

4 hrs

22 hrs

4 hrs

Recovery

18 hrs

-

18 hrs

Preparation interval

22 hrs

22 hrs

22 hrs

In each experimental group two parallel cultures were analyzed. Per culture 150 metaphases were evaluated for structural chromosomal aberrations, except for the positive control in Experiment IIB, where only 50 metaphases were evaluated.

The highest applied concentration in this study (2000 µg/mL of the test item) was chosen with respect to the current OECD Guideline 473.

Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item precipitation in accordance with OECD Guideline 473.

In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentrations, which showed precipitation.

Either with or without metabolic activation 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.

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.