1,2-Propanediol, 3-(cyclohexyloxy)-

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:

experimental study

Adequacy of study:

key study

Study period:

04 Aug 2010 to 27 Aug 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

adopted in 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

3-(cyclohexyloxy)propane-1,2-diol

EC Number:

805-622-3

Cas Number:

10305-41-6

Molecular formula:

C9H18O3

IUPAC Name:

3-(cyclohexyloxy)propane-1,2-diol

Constituent 2

Reference substance name:

3-cyclohexloxypropane-1,2-diol

IUPAC Name:

3-cyclohexloxypropane-1,2-diol

Details on test material:

- Name of test material (as cited in study report): SDX-3012
- Physical state: liquid
- Analytical purity: > 99%
- Lot/batch No.: 019X9
- Expiration date of the lot/batch: Oct.2010
- Storage condition of test material: room temperature (20.6-21.2 °C), nitrogen substitution
- Other: flash point: 159 °C

Method

Target gene:

Not applicable

Species / strain

Species / strain / cell type:

other: Chinese Hamster Lung cells (CHL/IU)

Details on mammalian cell type (if applicable):

- Type and identity of media:
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Phenobarbital (PB) and 5,6-Benzoflavone (BF)

Test concentrations with justification for top dose:

Short time (6 h) treatment: 435, 870 and 1740 µg/mL with and without metabolic activation
Continuous (24 h) treatment:435, 870 and 1740 µg/mL without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

mitomycin C

Remarks:

Continuous treatment: Mitomycin C - 0.05 µg/mL in water, -S9. Short-term treatment: Mitomycin C - 0.05 µg/mL in water, -S9 and Benzo[a]pyrene -20 µg/mL in DMSO, + S9.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 6 h (with metabolic activation), 6h and 24 ( without metabolic activation)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 h


SPINDLE INHIBITOR (cytogenetic assays): colcemid 0.2 µL/mL medium
STAIN (for cytogenetic assays): Giemsa 3% solution (in 0.01 mol/L Sörenson phosphate buffer, pH 6.8) for approximately 20 min.

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: at least 100 metaphase chromosome spreads per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index.

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
- other: Structural chromosome aberrations were classified into chromatid break (ctb), chromatid exchange (cte), chromosome break (csb), chromosome exchange (cse) or other (o). When several gaps or breaks were evident in metaphase, these were recorded as a fragment (frg). Chromosomes with multiple aberrations were recorded as aberrant cells. Two types of aberrations, such as chromatid and chromosome gaps (g) were recorded separately from structural aberrations. An achromatic lesion narrower than the width of one chromatid was recorded as a gap. Gaps (g) were not recorded as structural aberrations and were not included in the calculations of the aberrations rates.

Evaluation criteria:

The frequencies of chromosome aberration cells except gaps were determined in accordance with the criteria of Toshio Sofuni as follows:
-Mean frequencies of chromosome aberration cells below 5% = Negative (-)
-Mean frequencies of chromosome aberration cells above 5% - below 10% = Equivocal positive (±)
-Mean frequencies of chromosome aberration cells abelow 10% = Positive (+)

Statistics:

For the frequency of cells with chromosome aberrations, SAS program (version 9.1.3, SAS Institute Inc., USA) was used. When the result was negative, Fisher’s exact method was used for comparison of the negative control group with the test substance groups or the positive control group (significance level: 0.05)

Results and discussion

Test results

Species / strain:

other: Chinese Hamster Lung cell line (CHL/IU)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

but tested upto limit concentration

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: In order to determine the high dose for the main study, the cell growth inhibition study was conducted. The cell growth inhibition was less than 50% at all dose levels (6.80, 13.6, 27.2, 54.4, 109, 218, 435, 870 and 1740 µg/mL) for the 6 h treatment, with and without metabolic activation and for the 24 h treatment without metabolic activation. The deposition of the test substance was not evident at the time of treatment of the test substance and at the end of the incubation. Therefore, 1740 µg/mL (approximately 10 mM) in the main study was selected as the high dose in the short time treatment with and without metabolic activation and continuous treatment without metabolic activation. The high dose was serially diluted by a ratio of 2 to a produce total of 3 dose levels.

COMPARISON WITH HISTORICAL CONTROL DATA: The chromosomal aberrations for the negative and positive controls were within the range of the historical control data.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 2: Summary of main study

Test substance	Dose (µg/mL)	S9 mix	Trt-Rec Time (h)	Number of cells analysed	Number of cells with structure aberrations						Gap %
					ctb	cte	csb	cse	frg	Total%
Water	0	-	6-18	100	0	0	0	0	0	0 (0.0)	0 (0.0)
				100	0	0	0	0	0
SDX-3012	435	-	6-18	100	0	0	0	0	0	0 (0.0)	0 (0.0)
				100	0	0	0	0	0
	875	-	6-18	100	0	0	0	0	0	0 (0.0)	0 (0.0)
				100	0	0	0	0	0
	1740	-	6-18	100	1	0	0	0	0	1 (0.5)	0 (0.0)
				100	0	0	0	0	0
MMC	0.05	-	6-18	100	7	8	0	0	0	31* (15.5)	1 (0.5)
				100	6	10	0	0	0
Water	0	+	6-18	100	0	0	0	0	0	0 (0.0)	0 (0.0)
				100	0	0	0	0	0
SDX-3012	435	+	6-18	100	0	0	0	0	0	0 (0.0)	2 (1.0)
				100	0	0	0	0	0
	875	+	6-18	100	0	0	0	0	0	0 (0.0)	0 (0.0)
				100	0	0	0	0	0
	1740	+	6-18	100	0	0	0	0	0	0 (0.0)	1 (0.5)
				100	0	0	0	0	0
B[a]P	20	+	6-18	100	6	10	0	0	0	34* (17.0)	1 (0.5)
				100	4	16	0	1	0
Water	0	-	24-0	100	0	0	0	0	0	0 (0.0)	1 (0.5)
				100	0	0	0	0	0
SDX-3012	435	-	24-0	100	1	0	0	0	0	1 (0.5)	2 (1.0)
				100	0	0	0	0	0
	875	-	24-0	100	0	0	0	0	0	0 (0.0)	0 (0.0)
				100	0	0	0	0	0
	1740	-	24-0	100	0	0	0	0	0	0 (0.0)	0 (0.0)
				100	0	0	0	0	0
MMC	0.05	-	24-0	100	6	7	0	0	0	27* (13.5)	3 (1.5)
				100	2	12	0	1	0

Aberration; gap: chromatid and chromosome gap, ctb: chromatid break, cte: chromatid exchange, csb: chromosome break, cse: chromosome exchange, frg: fragmentation.

Trt-Rec time: Treatment-Recovery times

MMC: Mitomycin C, B[a]P: Benzo[a]pyrene

* p<0.05, Significant difference from negative control by Fisher’s exact test

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative