Alcohols, C16-18, ethoxylated

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 Jan - 3 Nov 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

adopted in 1983

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Version / remarks:

adopted in 2016

Deviations:

yes

Remarks:

pleaser refer to "Principles of method if other than guideline".

Principles of method if other than guideline:

Deviations to OECD guideline 473 (2016): only 200 metaphases evaluated per condition, cytotoxicity measured by counting the number of cells at the end of the culture period relative to control, instead of RPD or RICC; no information on karyotype stability or mycoplasma contamination check, acceptability and evaluation criteria differ from those specified in guideline

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Hexadecan-1-ol

EC Number:

253-149-0

EC Name:

Hexadecan-1-ol

Cas Number:

36653-82-4

Molecular formula:

C16H34O

IUPAC Name:

hexadecan-1-ol

Constituent 2

Reference substance name:

2-(hexadecyloxy)ethanol

EC Number:

218-373-5

EC Name:

2-(hexadecyloxy)ethanol

Cas Number:

2136-71-2

Molecular formula:

C18H38O2

IUPAC Name:

2-(hexadecyloxy)ethanol

Constituent 3

Reference substance name:

2-[2-(Hexadecyloxy)ethoxy]ethanol

EC Number:

610-892-9

Cas Number:

5274-61-3

Molecular formula:

C20H42O3

IUPAC Name:

2-[2-(Hexadecyloxy)ethoxy]ethanol

Constituent 4

Reference substance name:

2-[2-[2-(Hexadecyloxy)ethoxy]ethoxy]ethanol

Cas Number:

4484-59-7

Molecular formula:

C22H46O

IUPAC Name:

2-[2-[2-(Hexadecyloxy)ethoxy]ethoxy]ethanol

Constituent 5

Reference substance name:

2-[2-[2-(2-hexadecoxyethoxy)ethoxy]ethoxy]ethanol

Cas Number:

5274-63-5

Molecular formula:

C24H50O5

IUPAC Name:

2-[2-[2-(2-hexadecoxyethoxy)ethoxy]ethoxy]ethanol

Constituent 6

Reference substance name:

Octadecan-1-ol

EC Number:

204-017-6

EC Name:

Octadecan-1-ol

Cas Number:

112-92-5

Molecular formula:

CH3(CH2)17OH

IUPAC Name:

octadecan-1-ol

Constituent 7

Reference substance name:

2-(octadecyloxy)ethanol

EC Number:

218-374-0

EC Name:

2-(octadecyloxy)ethanol

Cas Number:

2136-72-3

Molecular formula:

C20H42O2

IUPAC Name:

2-(octadecyloxy)ethanol

Constituent 8

Reference substance name:

2-[2-(Octadecyloxy)ethoxy]ethanol

EC Number:

605-213-8

Cas Number:

16057-43-5

Molecular formula:

C22H46O3

IUPAC Name:

2-[2-(Octadecyloxy)ethoxy]ethanol

Constituent 9

Reference substance name:

2-[2-[2-(Octadecyloxy)ethoxy]ethoxy]ethanol

EC Number:

610-180-8

Cas Number:

4439-32-1

Molecular formula:

C24H50O

IUPAC Name:

2-[2-[2-(Octadecyloxy)ethoxy]ethoxy]ethanol

Constituent 10

Reference substance name:

2-[2-[2-(2-octadecoxyethoxy)ethoxy]ethoxy]ethanol

Cas Number:

59970-10-4

Molecular formula:

C26H54O5

IUPAC Name:

2-[2-[2-(2-octadecoxyethoxy)ethoxy]ethoxy]ethanol

Constituent 11

Reference substance name:

2-[2-[2-[2-(2-octadecoxyethoxy)ethoxy]ethoxy]ethoxy]ethanol

Cas Number:

71093-13-5

Molecular formula:

C28H58O6

IUPAC Name:

2-[2-[2-[2-(2-octadecoxyethoxy)ethoxy]ethoxy]ethoxy]ethanol

Test material form:

liquid

Method

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: The cell line used was obtained from the University of Leiden in 1987.
- Suitability of cells: Cell type selected is listed as one of the recommended cell types in OECD guideline 473.
- Normal cell cycle time: The cells have a generation time of approx. 12 h.
- Methods for maintenance in cell culture: The cells were grown as monolayers. Cells were trypsinised from stock flasks and resuspended in fresh culture medium at densities of 0. I x 10E6 or 0.05 x 10E6 cells/mL. These cells, in 5 mL volumes, were dispensed into tissue culture flasks. The high and low cell densities were for cultures harvested at 24 or 48 h post treatment respectively. The cultures were established approx. 20 h before testing.

MEDIA USED
- Type and composition: The basic medium (Ham's F-10) containing HEPES buffer, was supplemented with the antibiotic minocycline. For cell growth and treatment in the absence of S9 mix, foetal bovine serum (10% v/v) was added. The medium used for treatment in the presence of S9 mix and for washing cultures before or after treatment, was serum free.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: Cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats.
- method of preparation of S9 mix: S9 enzymes were prepared from the livers of adult, male Fischer rats. S9 was stored in sterile plastic tubes immersed in liquid nitrogen. To prepare S9 mix, Ham's F-10 was added to pre-weighed cofactors: nicotinamide adenine dinucleotide phosphate (NADP) disodium salt and glucose-6-phosphate (G-6-P) disodium salt, giving final concentrations in the S9 mix of NADP di-Na salt: 4 mM (3.150 mg/mL) and glucose-6-phosphate di-Na salt: 25 mM (7.605 mg/mL). This solution was immediately filter-sterilised by passage through a 0.2 pm disposable filter assembly and mixed 9:1 (v/v) with the S9.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Enzymic activity of each batch of S9 was characterised by testing selected pre-mutagens in an Ames test with S. typhimurium TA 1538. S9 batches used demonstrated, within each test, a satisfactory clastogenic response in cells treated with cyclophosphamide (CPI-I).

Test concentrations with justification for top dose:

Test 1:
6 h exposure with metabolic activation, 24 h harvest: 313, 625, 1250*, 2500* and 5000* µg/mL
24 h exposure without metabolic activation, 24 h harvest: 1.25, 2.5, 5*, 10*, 20*, 39 and 78 µg/mL

Test 2:
6 h exposure with metabolic activation, 24 and 48 h harvest: 313, 625, 1250*, 2500* and 5000* µg/mL
24 h exposure without metabolic activation, 24 and 48 h harvest: 1.25, 2.5*, 5*, 10*, 20, 39 and 78 µg/mL
*: concentrations used for evaluation of chromosome aberrations

Justification for top dose: Dose levels were selected based on a preliminary experiment, in which the highest dose in the presence of S9 mix was 5000 µg/mL and the highest dose in the absence of S9 mix was 156 µg/mL-

Vehicle / solvent:

- Vehicle/solvent: 1% ethanol
- Test formulation preparation: The test material was dissolved in ethanol and placed in a sonicating water bath for 30 min.

Controls

Untreated negative controls:

yes

Remarks:

untreated cells

Negative solvent / vehicle controls:

yes

Remarks:

1% ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 6 and 24 h
- Harvest time: 24 and 48 h after beginning of treatment.

SPINDLE INHIBITOR:
0.1 µg/mL colcemid was added approx. 2 h prior to harvest.

SLIDE PREPRATION AND METAPHASE ANALYSIS:
- Methods of slide preparation and staining technique used including the stain used: Mitotic cells were harvested by gently tapping flasks to release these cells from the monolayer. Cells were sedimented by centrifugation (approx. 210 g) and treated with hypotonic solution (l% trisodium citrate) for 15 min at room temperature. The cells were then fixed using 4 mL of freshly prepared fixative (methanol:glacial acetic acid, 3:1). Two further changes of fixative were made. Slides were prepared by dropping the cell suspension on to clean, grease-free slides. For both experiments, 3 slides per culture were prepared.
- Number of cells spread and analysed per concentration: 200 (100 cells per culture)
- Criteria for scoring chromosome aberrations: Metaphases were assessed by light microscopy. The following structural aberrations were recorded: chromosome and chromatid breaks, gaps and fragments, complex structures such as exchanges, dicentric and rings, double minutes, uncondensed chromosomes, translocations, pulverised chromosomes, multiple aberrations. In addition, aneuploidy, endoreduplication and polyploidy were assessed.
- Determination of polyploidy: yes
- Determination of endoreplication: yes

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: From the cell counts, the number of cells recovered per culture, was calculated. This was compared with the number of cells (mean of 2 cultures) recovered from the vehicle control cultures. A dose level was considered to be toxic if die cell count was reduced to less dian 60% of the vehicle control cultures or if consistent evidence of changes to cell morphology was observed.

Evaluation criteria:

Structural and numerical chromosomal aberrations were evaluated for lesions per cell, percentage of aberrant cells including cells with gaps only, percentage of aberrant cells excluding cells with gaps only, percentage of aneuploid cells and percentage of polyploid cells (normal and endoreduplicated) from additional assessment of ploidy.
A negative response was recorded if responses from the test material treated cultures are within the 95% confidence limits for historical negative controls. The response at a single dose was classified as significant if the percent of aberrant cells is consistently greater than the 99% confidence limits for a negative historical negative controls or greater than double the frequency of an elevated vehicle control or untreated culture if appropriate.
An experiment was positive if the response in at least the one acceptable dose level is significant by the criterion described above and is associated with an increase in aberrant cells in at least one other dose level. A test material was positive if both experiments were positive, as described above or if the second test was positive after the first test gave indications of activity. These indications may be suspicious levels of aberrant cells (benveen 95% and 99% confidence limits) at extreme or sub toxic dose levels.
An inconclusive response was recorded for experiments that met, in part or marginally, the criteria for a positive response.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the absence of S9 mix, cultures treated with 20-156 µg/mL (Test 1) and 10-78 µg/mL (Test 2) were judged to be toxic.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: The test substance did not change the colour of the culture medium, therefore no pH measurements were made.
- Data on osmolality: During the first test, the osmolality of selected concentrations of the test material was measured. The test substance did not affect the osmolality of the culture medium, therefore no further measurements were made.
- Precipitation and time of the determination: Observations of precipitation were made before cultures were washed out at the end of the treatment period. Cloudy solutions were noted in cultures treated with 78-156 µg/mL and lumps of precipitate were observed in cultures treated with 313-5000 µg/mL.

STUDY RESULTS:
STRUCTURAL CHROMOSOMAL ABERRATIONS RESULTS
In general, cultures treated with the test substance had levels of structural aberrations within the confidence limits of a negative response. There were 2 exceptions. In Test 2, with S9 mix, there were 2 cultures with suspicious levels of aberrant cells excluding gaps. As these responses were not reproduced in duplicate cultures or dose related, they were considered sporadic.
All vehicle and untreated control cultures had levels of structural aberrations within the 95% confidence limit of the historical negative control data. The positive control substances, CPH in the presence and MMS in the absence of S9 mix, induced structural aberrations. These results demonstrated the sensitivity of the test system.

NUMERICAL ABERRATIONS
An additional assessment of polyploidy was carried out in cultures harvested at 48 h. In the absence of S9 mix, both cultures treated with 10 µg/mL had positive responses and the cultures treated with 5 µg/mL had one positive and one suspicious response. There was an unusually high level of endoreduplicated cells in one of the cultures treated with 10 µg/mL. In the presence of S9 mix, one of the untreated control cultures had a slightly elevated incidence of polyploidy. A doubling over the elevated result was required to judge a culture as positive. There was no induction of polyploidy in the presence of S9 mix.

CYTOTOXICITY:
The test substance was found to be non-toxic to CHO cells in vitro when tested in the presence of S9 mix. In the absence of S9 mix, cultures treated with 20-156 µg/mL (Test 1) and 10-78 µg/mL (Test 2) were judged to be toxic. Toxicity was evident from reduced cell counts (< of vehicle control) and changes in the cell morphology. The toxicity in the absence of S9 mix may have been due to the longer exposure time.

HISTORICAL CONTROL DATA:
The solvent and the positive controls showed the expected results and the obtained values remained within the historical control data. For details on the historical control data please refer to the tables under “Attached background material”.

Any other information on results incl. tables

Detailed additional information is provided under 'Attached background material'.

Applicant's summary and conclusion

Conclusions:

Under the conditions of the test, the test item did not induce chromosomal aberrations in Chinese Hamster Ovary (CHO) cells in the presence or absence of metabolic activation.