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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
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1993-10-11 to 1994-01-26
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Deviations from the OECD 473 (2014): number of analysed cells was too low. Highest possible dose was not tested (without metabolic activation only). Short-term treatment without metabolic activation was missing. Tables not labelled appropriately. Historical control data is missing. Acceptability/evaluation criteria are missing.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report date:
1994

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
1983-05-26
Deviations:
yes
Remarks:
Highest possible dose was not tested (without metabolic activation only). Tables not labelled appropriately. Evaluation criteria missing.
GLP compliance:
yes (incl. QA statement)
Remarks:
signed 1987-12-07
Type of assay:
other: in vitro mammalian chromosome aberration test

Test material

Constituent 1
Chemical structure
Reference substance name:
2-[({[(1R,2S,5R)-5-methyl-2-(propan-2-yl)cyclohexyl]oxy}carbonyl)oxy]ethan-1-ol
EC Number:
701-039-2
Cas Number:
156324-78-6
Molecular formula:
C13H24O4
IUPAC Name:
2-[({[(1R,2S,5R)-5-methyl-2-(propan-2-yl)cyclohexyl]oxy}carbonyl)oxy]ethan-1-ol

Method

Target gene:
not applicable
Species / strain
Species / strain / cell type:
lymphocytes: human peripheral blood
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: healthy human donor
- Sex and number of blood donors: 1 male
- Whether whole blood or separated lymphocytes were used: whole blood (0.4 mL/culture tube)

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: each culture tube contained 5 mL of Ham's F10 complete medium (500 mL Ham's F10; 100 mL fetal calf serum (heat inactivated); 5 mL penicillin/streptomycin (10 000 IU/m1); 5 mL heparin (5000 IU/m1)), to which 0.1 mL of phytohaem-agglutinin was added. The tubes were sealed and incubated at 37 °C.
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (containing 25 % S9): 4 mM NADP; 25 mM glucose-6-phosphate; 8 mM MgCl2; 33 mM KCl; 100 mM phosphate buffer pH 7.4
Test concentrations with justification for top dose:
Experiment 1/Experiment 2: 10, 30, and 100 µg/mL (without metabolic activation)
Experiment 1/Experiment 2: 30, 100, and 300 µg/mL (with metabolic activation)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
NOTE: two independent experiments were conducted separated by at least 14 days.

TOXICITY TEST:
- test was carried out with the same procedure as described below for the mutagenicity test.
- at least 2000 lymphocytes/ culture were examined
- mitotic index was calculated as the percentage of lymphocytes examined which were in mitosis.

AVERAGE GENERATION TIME.
- method was carried out with the same procedure as described below for the mutagenicity test with the exception that 5-bromodeoxyuridine (BrdU, 30 µM) was added to the cultures, which were treated in the dark for 52 hours until the end of the incubation period.
- Staining technique: after the preparation of microscopic slides, sister chromatids were differentially stained using a modified fluorescence-plus-Giemsa technique (Perry and Wolff, 1974). Briefly, slides aged for 4 to 5 days were stained in 33258 Hoechst dissolved in NaCl/KCl solution, rinsed and mounted in M/15 Sörensen's phosphate buffer (pH 6.8) to irradiate them with a 254nm UV mineralogic lamp. After the exposure, the slides were incubated in 2xSSC solution at 59°C, rinsed in distilled water and stained with 6 % phosphate-buffered Giemsa solution and then mounted as usual.
- estimation of the average generation time: a replicative index based on 200 cells was calculated from the proportions of metaphase cells that completed one, two, or between one and two, or two and three cycles in BrdU. The average generation time (h) was calculated as the number of hours (52) in BrdU divided by the replicative index.
- Classification of metaphase cells:
M1. even, dark staining of both chromatids
M1+: 1 partial S phase in BrdU followed by 1 complete S phase gives regions with sister chromatid differentiation (SCD), and regions with both chromatids still dark
M2: 2 complete S phases in BrdU gives full SCD
M2+: 1 partial followed by 2 complete S phases gives regions with SCD and regions with even, light staining on both chromatids.

MUTAGENICITY TEST (Experiment 1 and Experiment 2):
- test item exposure without metabolic activation: cells were exposed to the test item concentrations for 24 hours.
- test item exposure with metabolic activation: cells were exposed to the test item concentrations and 0.5 mL of S9-mix for 3 hours (37 °C). After 3 hours the cells were centrifuged and washed to remove the test compound and S9-mix. After a further centrifugation the cell pellet was resuspended in complete medium and reincubated at 37°C to give a total of 24 hours incubation.

NUMBER OF REPLICATIONS: duplicate

CELL HARVESTING:
- 2 hours before the end of the incubation period, the cell division was arrested by the addition of 10 μg/ml Colcemid solution and incubated for further 2 hours.
- 24 hours after beginning of the treatment the cells were harvested by centrifugation.
- cells were resuspended in hypotonic potassium chloride solution and incubated at 37°C.
- then the cells were collected by centrifugation, resuspended, and fixed in 3:1, methanol : glacial acetic acid.
- cells were then washed at least twice in fresh fixative.

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
- cells were centrifuged and resuspended in fresh fixative.
- two or three drops of this suspension were transferred to a microscopic slide and left to air-dry.
- at least two slides/culture were prepared.
- slides were stained in Giemsa solution (10 % in 0.01 M phosphate buffer, pH 6.8) washed in buffer and distilled water and left to air-dry.
- permanent slides were made, after cleaning in xylene, using Eukitt mountant.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE:
- slides were observed under a microscope with a 100x oil immersion objective lens at 1250 fold magnification.
- at least 100 well spread metaphases containing 46 chromosomes from each culture were analysed (total: at least 200 metaphases/test point).
- aberrations were classified using the nomenclature of Savage (1979)*.

*Reference:
- Savage, J. R. K. (1979) Annotation: Classification and relationships of induced chromosomal structural changes, J. Med. Genet., 13, 103- 122.
Rationale for test conditions:
According to the preliminary toxicity test the dose levels were chosen for the main study.
Evaluation criteria:
not specified
Statistics:
Statistical significance was determined according to the methods of Kastenbaum and Bowman (1970)*.
*Reference:
Kastenbaum, M. A., and K. O. Bowman (1970) Tables for determining the statistical significance of mutation frequencies, Mutation Res., 9, 527-549.

Results and discussion

Test results
Key result
Species / strain:
lymphocytes: human peripheral blood
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
please refer to the field "Additional information on results" below
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
AVERAGE GENERATION TIME Of HUMAN PERIPHERAL BLOOD LYMPHOCYTES:
The average generation time was only slightly affected by the test substance both in the presence and absence of S9-mix. Thus a single sampling time at 1.5 times the normal cell-cyle lenth from the beginning of the treatment (24 hours) was used in all independent experiments.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: mitotic index
The highest test item concentration tested resulted in an average of 32 % cytotoxicity in the absence of S9-mix and 49 % in the presence of S9-mix.

MUTAGENICITY TEST (Experiment 1 and Experiment 2):
The test item did not induce an increase in the number of chromosome aberrations in cultured human blood lymphocytes in the absence and presence of a metabolizing system as compared to the controls.

CONTROLS:
- spontaneous value of aberrations observed were within the range previously established in the laboratory.

Please also refer for further information on results to the field "Attached background material" below.




Applicant's summary and conclusion

Conclusions:
The substance was non-clastogenic under the conditions of the study.
According to Regulation (EC) No 1272/2008 and subsequent adaptations, the substance should not be considered to have a mutagenic potential.
Executive summary:

An in vitro mammalian chromosomal aberration test was performed with the test item dissolved in DMSO using human peripheral blood lymphocytes in the absence and presence of metabolic activation. The test procedure was equivalent or similar to the method described in the OECD guideline 473 (1983). Two independent experiments investigating induction of chromosomal aberration were performed.

Cells were treated in the absence of metabolic activation at test item concentrations of 10, 30, and 100 µg/mL for 24 hours (experiment 1 and experiment 2). With metabolic activation, cells were treated with the test item at concentrations of 30, 100, and 300 µg/mL for three hours (experiment 1 and experiment 2). After 3 hours. the test item and S9 -mix were removed and the cells were resuspended in complete medium and reincubated to give a total of 24 hours incubation. Each treatment was established in duplicate. Negative and positive controls were also run concurrently. The effect of the test item on the cell cycle time of human peripheral blood lymphocytes both in the presence and absence of metabolic activation was investigated.

The highest concentration tested resulted in an average of 32 % cytotoxicity in the absence of S9-mix and 49 % in the presence of S9-mix. Furthermore, the test compound affected only slightly the cell-cycle time of human peripheral blood lymphocytes both in the presence and absence of S9-mix. Therefore, a sampling time of 1.5 times the normal cell-cycle length from the beginning of the treatment (24 hours) was used in all independent experiments. The test item did not induce an increase in the number of chromosome aberrations in cultured human blood lymphocytes in the absence and presence of a metabolizing system.

Thus, the substance was non-clastogenic under the conditions of this study.

According to Regulation (EC) No 1272/2008 and subsequentadaptations, the substance should not be considered to have a mutagenic potential.