Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Recent GLP study conducted according to OECD guideline 473 without any deviation
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent

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:
Phenobarbital and beta-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Experiment I: 0.032 - 5.0 µL/mL (with and without S9); Experiment II: 0.006 to 0.93 µL/mL (without S9), 0.30 to 5.0 µL/mL (with S9)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: THF
- 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
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:
Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and without metabolic activation. In Experiment II 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 (about 1.5 cell cycles)

SPINDLE INHIBITOR (cytogenetic assays): Colcemid

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicate cultures, experiment repeated

NUMBER OF CELLS EVALUATED: At least 100 per culture

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. At least 100 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.
Statistics:
Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05).

Results and discussion

Test results
Species / strain:
lymphocytes:
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 0.17 µL/mL , 22 h exposure
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
The test item Turpentine oil CAS 8006-64-2, dissolved in THF, 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.
Two independent experiments were performed. In Experiment I, the exposure period was 4 hours with and without S9 mix. In Experiment II, the exposure period was 4 hours with S9 mix and 22 hours without S9 mix. The chromosomes were prepared 22 hours (Exp. I & II) after start of treatment with the test item.
In each experimental group two parallel cultures were analysed. At least 100 metaphases per culture were scored for structural chromosomal aberrations. 1000 cells were counted per culture for determination of the mitotic index.
The highest treatment concentration in this study, 5.0 µL/mL was chosen with respect to the OECD Guideline for in vitro mammalian cytogenetic tests.
In this study, no precipitation of the test item in the culture medium was observed. A decrease in the osmolarity and no relevant increase in the pH value was observed (Exp. I: solvent control: 496 mOsm, pH 7.6 versus 316 mOsm and pH 7.6 at 5.0 µL/mL; Exp. II: solvent control: 387 mOsm, pH 7.5 versus 360 mOsm and pH 7.5 at 0.93 µg/mL). Phase separation was observed in Experiment I at 1.63 µL/mL and above in the absence and presence of S9 mix and in Experiment II at 0.93 µL/mL in the absence and at 0.93 µL/mL and above in the presence of S9 mix.
In Experiment I in the absence of S9 mix, concentrations showing clear cytotoxicity were not scorable for cytogenetic damage. In Experiment II in the absence of S9 mix, cytotoxicity was observed at the two highest evaluated concentrations (54.3, 49.1 % of control). In Experiment I in the presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. However, in Experiment II in the presence of S9 mix the highest applied concentration was not evaluable due to low metaphase numbers.
In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. In Experiment I in the absence and presence of S9 mix and in Experiment II in the presence of S9 mix the aberration rates of the cells after treatment with the test item (0.0 - 3.0 % aberrant cells, excluding gaps) were in the range of the solvent control values (0.5 – 3.0 % aberrant cells, excluding gaps) and within the range of the laboratory´s historical solvent control data. In Experiment II after continuous treatment with the test item the lowest evaluated concentration 0.032 µg/mL slightly exceeded the laboratory’s historical control range. Since no statistical significance and no dose-dependency were observed the finding is not biologically relevant.
No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.
In both experiments, either EMS (825.0 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: strain/cell type: human lymphocytes
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Summary of results of the chromosomal aberration study with Turpentine oil CAS 8006-64-2

Preparation

interval

 

Test item

concentration in µL/mL

Mitotic indices

in % of control

Aberrant cells

in %

incl. gaps*

excl. gaps*

carrying exchanges

Exposure period 4 hrs without S9 mix. Experiment 1

22 hrs

 

 

 

 

Solvent control1

100.0

1.0

1.0

0.0

Positive control2

51.6

11.0

10.5S

3.5

0.099

95.5

1.5

1.0

0.0

0.17

94.1

1.5

1.5

1.0

0.30

70.3

1.5

1.5

0.0

Exposure period 22 hrs without S9 mix. Experiment 2

22 hrs

 

 

 

 

 

Solvent control1

100.0

2.0

1.5

0.0

Positive control2

40.3

27.5

25.0S

2.5

0.032#

84.8

3.8

3.0

0.0

0.057

86.8

4.0

2.0

0.0

0.099

54.3

2.0

1.5

0.0

0.17

49.1

1.0

0.5

0.0

Exposure period 4 hrs with S9 mix Experiments 1 and 2

22 hrs, expt 1

 

 

 

 

Solvent control1

100.0

0.5

0.5

0.0

Positive control3

63.9

12.5

12.0S

3.0

1.63

99.7

0.5

0.0

0.0

2.86

102.6

0.5

0.5

0.0

5.00

81.0

1.0

1.0

0.0

22 hrs, expt 2

 

 

 

 

Solvent control1

100.0

3.5

3.0

0.5

Positive control4

51.0

8.5

8.5S

0.5

0.93

86.6

1.5

1.5

0.0

1.63

91.9

0.5

0.5

0.0

2.86

91.0

1.5

0.5

0.0

*  Including cells carrying exchanges

#   Evaluation of 200 metaphases per culture

S  Aberration frequency statistically significant higher than corresponding control values

1   THF 0.5 % (v/v)

2     EMS825.0 µg/mL

3   CPA  15.0 µg/mL

4   CPA    7.5 µg/mL

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

Turpentine oil CAS 8006-64-2 has been tested in a valid study according to OECD TG 473 under GLP. Under the experimental conditions reported, the test item did not induce structural chromosomal aberrations in human lymphocytes in vitro in the initial experiment, nor in the repeat experiment with longer exposure. The vehicle and positive controls gave expected results. Therefore, Turpentine oil CAS 8006-64-2 is considered to be non-clastogenic in this chromosome aberration test, when tested up to cytotoxic or the highest evaluable concentrations.
Executive summary:

Test item Turpentine oil CAS 8006-64-2, dissolved in THF, was assessed for its potential to induce structural chromosomal aberrations in human lymphocytes in vitro in two independent experiments. The following study design was performed:

 

Without S9 mix

With S9 mix

 

Exp. I

Exp. II

Exp. I & II

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 analysed. Per culture at least 100 metaphases were scored for structural chromosomal aberrations.

The highest applied concentration in the pre-test on toxicity (5.0 µL/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 in accordance with OECD Guideline 473.

In Experiment I in the absence of S9 mix, concentrations showing clear cytotoxicity were not scorable for cytogenetic damage. In Experiment II in the absence of S9 mix, cytotoxicity was observed at the two highest evaluated concentrations. In Experiment I in the presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. In Experiment II in the presence of S9 mix the highest applied concentration was not evaluable due to low metaphase numbers.

In both independent experiments, 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 (p < 0.05) in cells with structural chromosome aberrations.