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EC number: 242-060-2 | CAS number: 18172-67-3
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- Ecotoxicological Summary
- Aquatic toxicity
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- Short-term toxicity to fish
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Genetic toxicity: in vitro
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 for cross-reference:
- reference to same study
- Reason / purpose for cross-reference:
- reference to other study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 010
- Report date:
- 2010
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- 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. QA statement)
- Type of assay:
- in vitro mammalian chromosome aberration test
Test material
- Reference substance name:
- Turpentine Oil from Pulping Process
- IUPAC Name:
- Turpentine Oil from Pulping Process
- Reference substance name:
- Turpentine, oil
- EC Number:
- 232-350-7
- EC Name:
- Turpentine, oil
- Cas Number:
- 8006-64-2
- IUPAC Name:
- 8006-64-2
Constituent 1
Constituent 2
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.
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