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:
From April 07 to August 04, 2008
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP study conducted according to OECD test Guideline No. 473 without any deviation. The study was fully reliable (Klimisch score = 1), however the reliability score was lowered to 2 which is the maximum score for read-across. The supporting substance is considered adequate for read-across purpose (see IUCLID section 13 for additional justification).
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:
2008
Report date:
2008

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
Principles of method if other than guideline:
not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
Inspection date: 2006-09-02
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Reference substance name:
2-pentylcyclopentan-1-ol
EC Number:
283-187-3
EC Name:
2-pentylcyclopentan-1-ol
Cas Number:
84560-00-9
IUPAC Name:
2-pentylcyclopentanol
Test material form:
not specified
Details on test material:
- Name of test material (as cited in study report): Cyclopentol HC
- Physical state: no data
- Storage condition of test material: +2 - +8°C

Method

Target gene:
not applicable
Species / strain
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
Blood samples were obtained from a healthy donor not receiving medication. For this study, blood was collected from a female donor (32 years old) for the 1st experiment and from 34 year-old female and 26 year-old male donor for experiment II. Blood samples were drawn by venous puncture and collected in heparinised tubes.
- Type and identity of media: Blood cultures were set up in bulk within 24 hrs after collection in 75 cm² cell culture flasks. The culture medium was DMEM:F12 (Dulbecco's modified eagle medium/ Ham's F12 medium; mixture 1:1; Life Technologies GmbH, 76339 Eggenstein, Germany) containing 10 % FCS (fetal calf serum). The antibiotic solution contains 10,000 U/mL penicillin and 10,000 µg/mL streptomycin. Additionally, the medium was supplemented with Phytohemagglutinin (final concentration 3 µg/mL), the anticoagulant heparin (25,000 U.S.P.-U/mL), and HEPES (final concentration 10 mM).
- All incubations were done at 37° C in a humidified atmosphere with 5.5 % CO2 (94.5 % air).
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/beta-naphthoflavone induced rat liver S-9
Test concentrations with justification for top dose:
Experiment I: 10.5, 18.4, 32.2, 56.4*, 98.7*, 172.7*, 302.3, 529.0, 925.7, 1620.0 µg/L (+/- S9 mix) - up to 10 mM.
Experiment II: 3.2, 5.7, 9.9, 17.4, 30.5*, 53.3*, 93.3*, 163.3, 285.7, 500 µg/L (- S9 mix).
Experiment II: 10.5, 18.4, 32.2, 56.4, 98.7*, 17207*, 302.3*, 529, 925.7, 1620 µg/L (+ S9 mix) - up to 10 mM.
* = evaluated.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO (final concentration in the culture medium was 0.5 % v/v)
- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
culture medium
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
825 µg/mL (6.64 mM), Exp I. 660 µg/mL (5.32 mM), Exp II. Dissolved in nutrient medium.
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation
Untreated negative controls:
yes
Remarks:
culture medium
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
30 µg/mL (0.106 mM), Exp I. 15 µg/mL (0.053 mM), Exp II. Dissolved in saline (0.9 % NaCl w/v)
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 50-80 hours.
- Exposure duration: Exp I: 4 hours. Exp II: 4 hours with S9-mix, 22 hours without S9-mix.
- Expression time (cells in growth medium): 18 hours (except Exp II without S9-mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 22 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid (0.2 µg/mL) was added to the cultures 3 hours before harvesting.
STAIN (for cytogenetic assays): Giemsa or according to the fluorescent plus Giemsa technique, respectively.

NUMBER OF REPLICATIONS: duplicates

NUMBER OF CELLS EVALUATED: At least 1000 cells were counted per culture for determination of the mitotic index. For structural chromosome aberrations at least 100 cells per culture were scored. The number of polyploid cells was scored in 250 metaphase cells.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

OTHER: Exp II with S9-mix was repeated 3 times due to missing cytotoxicity or technical reasons.
Evaluation criteria:
Acceptability of the assay:
The chromosomal aberration assay is considered acceptable if it meets the following criteria:
a) The number of aberrations found in the solvent controls falls within the range of historical laboratory control data range: 0.0 - 4.0 % aberrant cells, exclusive gaps.
b) The positive control substances should produce significant increases in the number of cells with structural chromosome aberrations, which are within the range of the laboratory’s historical control data:
EMS (330-880 µg/mL) : 4 - 47 %
CPA (15-45 µg/mL): 7.5 - 40 %

A test item is classified as non-mutagenic if:
-the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of our historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps).
- no significant increase of the number of structural chromosome aberrations is observed.
A test item is classified as mutagenic if:
- the number of induced structural chromosome aberrations is not in the range of our historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps).
- and either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.

Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include the polyploids and endoreduplications. The following criteria is valid:
A test item can be classified as aneugenic if:
- the number of induced numerical aberrations is not in the range of our historical control data (0.0 – 0.8 % polyploid cells).
Statistics:
Statistical significance was confirmed by means of the Fisher’s exact test (p < 0.05). However, both biological and statistical significance should be considered together. If the criteria for the test item mentioned above are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.

Results and discussion

Test results
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 applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no relevant increase observed. In Exp 1 pH solvent = 7.6 versus pH 1620 µg/mL = 7.3.
- Effects of osmolality: no relevant increased observed. In Exp 1 solvent = 365 mOsm versus 1620 µg/mL = 365 mOsm.
- Precipitation: In Experiment I, no visible precipitation of the test item in the culture medium was observed. However, phase separation occurred at 925.7 Lg/mL and above in the absence of S9 mix and at 529.0 Lg/mL and above in the presence of S9 mix at the end of treatment. In addition, phase separation occurred in Experiment II, in the presence of S9 mix, at 302.3 Lg/mL and above.

RANGE-FINDING/SCREENING STUDIES: since the cultures fulfilled the requirements for cytogenetic evaluation, the preliminary test was designated Experiment 1.

COMPARISON WITH HISTORICAL CONTROL DATA: the aberration rates of the cells after treatment with the test item (0.5-3.0 %, exl. gaps) were slightly above the range of solvent control values (0.5-1.0%, excl. gaps) but were within the range of laboratory's historical control data: 0.0-4.0%, excl. gaps.
In both experiments, either EMS or CPA showed distinct increases in cells with structural chromosome aberrations.

ADDITIONAL INFORMATION ON CYTOTOXICITY: In Experiment I, in the absence and the presence of S9 mix, and in Experiment II in the presence of S9 mix, concentrations showing clear cytotoxicity were not scorable for cytogenetic damage. In Experiment II, in the absence of S9 mix, cytotoxicity indicated by clearly reduced mitotic indices could be observed at the highest evaluated concentration (47.1 % of control).
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

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 both experiments, no biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (0.0 – 0.2 %) as compared to the rates of the solvent controls (0.0 – 0.2 %).

The proliferation index of the lymphocytes in solvent control cultures in Experiment I with and without S9 mix (4 hrs treatment; 1.25 and 1.52, respectively), in Experiment II with and without S9 mix (1.10 and 1.88, respectively) was checked by analysing the proportion of mitotic cells in the 1st, 2nd and 3rd metaphase (M1, M1+, M2 and M3) indicating that the lymphocytes divided about 1.5 times within the early preparation interval. This is also proven by the occurrence of sufficient numbers of mitotic cells and by a clear clastogenicity observed after treatment with the positive control substances.

Applicant's summary and conclusion

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

Under the test conditions, the test material was considered to be non-clastogenic to human lymphocytes in vitro.
Executive summary:

In an in vitro chromosome aberration test performed according to OECD guideline No 473 and in compliance with GLP, human lymphocyte were exposed to the test material diluted in DMSO. Three treatment conditions were used, i.e.

- 4 hours exposure with the addition of an induced rat liver homogenate metabolising system in standard co-factors (S9-mix) with cell harvest after 18 hours,

- 4 hours exposure with cell harvest after 18 hours in the absence of activation,

- 22 hours continuous exposures in the absence of activation.

The dose range for evaluation was selected from a series of 10 dose levels on the basis of toxicity.

In Experiment I, in the absence and the presence of S9 mix, and in Experiment II in the presence of S9 mix, concentrations showing clear cytotoxicity were not scorable for cytogenetic damage. In Experiment II, in the absence of S9 mix, the mitotic indices were clearly reduced below 50 % of control 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 increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures

All the positive control treatments gave significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.

Under the test conditions, the test material was considered to be non-clastogenic to human lymphocytes in vitro.

This study is considered as acceptable and satisfies the requirement for in vitro mammalian chromosome aberration assay. The supporting substance is considered adequate for read-across purpose (see IUCLID section 13 for additional justification).