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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted according to OECD guideline 473 and under GLP.

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Reference substance name:
Tall Oil Pitch
IUPAC Name:
Tall Oil Pitch
Constituent 2
Reference substance name:
Tall-oil pitch
EC Number:
232-414-4
EC Name:
Tall-oil pitch
Cas Number:
8016-81-7
Molecular formula:
UVCB substance molecular formula unknown
IUPAC Name:
8016-81-7
Details on test material:
- Name of test material (as cited in study report): CAS 8016-81-7 (Tall oil pitch)

- Substance type: UVCB

- Analytical purity: 100 %

- Typical composition of test material, percentage of components: acids (22%), Resin acids (9%), Neutrals (16%)

- Lot/batch No.: 2009-04-30-00

- Expiration date of the lot/batch: December 2010

- Stability under test conditions: Not indicated by the Sponsor

- Storage condition of test material: At room temperature

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/beta-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
With metabolic activation:
Experiment I: 10.1, 17.7, 30.9, 54.1, 94.7, 165.8, 290.1, 507.7, 888.5, 1554.9, 2721.1, 4762.0 µg/ml
Experiment II: 3.3, 5.8, 10.1, 17.7, 30.9, 54.1 µg/ml

Without metabolic activation:
Experiment I: 1.9, 3.3, 5.8, 10.1, 17.7, 30.9, 54.1, 888.5, 1554.9, 2721.1, 4762.0 µg/ml
Experiment II: 1.9, 3.3, 5.8, 10.1, 17.7, 30.9, 54.1, 94.7, 165.8, 290.1, 507.7, 888.5, 1554.9, 2721.1, 4762.0 µg/ml
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
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


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


NUMBER OF REPLICATIONS: about 1.5


NUMBER OF CELLS EVALUATED: 100 per culture, except for the positive control in Experiment II in the presence of S9mix, where only 50 metaphases were scored.


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. 100 well spread metaphases per culture were scored for cytogenetic damage on coded slides, except for the positive control in Experiment II in the presence of S9mix, where only 50 metaphases were scored.
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.
A test item is positive for cytogenicity if:
the number of induced structural chromosome aberrations is not in the range of the historical control data and
either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.

A test item can be classified as aneugenic if:
- the number of induced numerical aberrations is not in the range of the historical control data
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:
290.1 µg/ml
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
The test item CAS 8016-81-7 (Tall oil pitch), 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. 100 metaphases per culture were scored for structural chromosomal aberrations, except for the positive control in Experiment II, in the presence of S9 mix, where only 50 metaphases were scored. 1000 cells were counted per culture for determination of the mitotic index.
The highest treatment concentration in this study, 4762.0 µg/mL was chosen with respect to the OECD Guideline for in vitro mammalian cytogenetic tests considering the solubility properties of the test item in an appropriate solvent (THF).
In Experiment I, visible precipitation of the test item in the culture medium was observed at 10.1 µg/mL and above in the absence of S9 mix and at 17.7 µg/mL and above in the presence of S9 mix. In Experiment II, precipitation occurred at 17.7 µg/mL and above in the absence and presence of S9 mix. No relevant influence in the osmolarity or pH value was observed (Exp. I: solvent control: 366 mOsm, pH 7.3 versus 327 mOsm and pH 7.3 at 4762.0 µg/mL; Exp. II: solvent control: 367 mOsm, pH 7.5 versus 321 mOsm and pH 7.4 at 4762.0 µg/mL).
In Experiment I in the absence and presence of S9 mix and in Experiment II in the presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. In Experiment II in the absence of S9 mix cytotoxicity was observed at the highest evaluated concentration (48.9 % of control).
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. The aberration rates of the cells after treatment with the test item (0.0 – 2.0 % aberrant cells, excluding gaps) were close to the range of the solvent control values (0.5 - 1.0 % aberrant cells, excluding gaps) and within the range of the laboratory´s historical solvent control data.
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 (770.0 or 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.
In conclusion, it can be stated that under the experimental conditions reported, the test item CAS 8016-81-7 (Tall oil pitch) did not induce structural chromosomal aberrations in human lymphocytes in vitro, when tested up to cytotoxic and/or precipitating concentrations.
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 CAS 8016-81-7 (Tall oil pitch)

Exp.

Preparation

Test item

Mitotic indices

Aberrant cells

 

 

interval

concentration

in %

in %

 

 

 

in µg/mL

of control

incl. gaps*

excl. gaps*

carrying exchanges

 

 

Exposure period 4 hrs without S9 mix

 

I

22 hrs

Solvent control1

100.0

1.0

1.0

0.0

 

 

 

Positive control2

69.5

13.0

12.5S

3.0

 

 

 

3.3

103.7

1.0

0.5

0.0

 

 

 

5.8

96.5

1.0

1.0

0.0

 

 

 

10.1P

95.1

0.5

0.5

0.0

 

 

Exposure period 22 hrs without S9 mix

 

II

22 hrs

Solvent control1

100.0

1.0

1.0

0.0

 

 

 

Positive control3

51.1

13.0

12.5S

1.5

 

 

 

10.1

78.5

1.5

1.5

0.0

 

 

 

17.7P

97.3

1.5

1.5

0.0

 

 

 

165.8P

61.0

1.0

1.0

0.0

 

 

 

290.1P

48.9

1.0

1.0

0.0

 

 

Exposure period 4 hrs with S9 mix

I

22 hrs

Solvent control1

100.0

1.0

1.0

0.0

 

 

 

Positive control4

38.6

11.0

11.0S

2.5

 

 

 

10.1

100.3

2.0

2.0

0.0

 

 

 

17.7P

98.7

1.0

1.0

0.0

 

 

 

30.9P

108.8

1.0

1.0

0.5

 

II

22 hrs

Solvent control1

100.0

1.0

0.5

0.0

 

 

 

Positive control#5

55.6

36.0

35.0S

3.0

 

 

 

5.8

80.8

1.0

0.5

0.0

 

 

 

10.1

90.8

0.5

0.5

0.5

 

 

 

17.7P

88.2

0.0

0.0

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   THF 0.5 % (v/v)

2     EMS825.0 µg/mL

3     EMS770.0 µg/mL

4   CPA    7.5 µg/mL

5   CPA  15.0 µg/mL

Applicant's summary and conclusion

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

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, CAS 8016-81-7 (Tall oil pitch) is considered to be non-clastogenic in this chromosome aberration test, when tested up to cytotoxic and/or precipitating concentrations.
Executive summary:

The test item CAS 8016-81-7 (Tall oil pitch), dissolved in THF, was assessed for its potential to induce structural chromosomal aberrations in human lymphocytesin vitroin 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 100 metaphases were scored for structural chromosomal aberrations, except for the positive control in Experiment II, in the presence of S9 mix, where only 50 metaphases were scored.

The highest applied concentration in the pre-test on toxicity (4762.0 µg/mL of the test item) was chosen with regard to the solubility properties of the test item in an appropriate solvent (THF) and 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 Experiment I in the absence and presence of S9 mix and in Experiment II in the presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. In Experiment II in the absence of S9 mix cytotoxicity was observed at the highest evaluated concentration.

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.

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