Alcohols, C16-18, ethoxylated

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro gene mutation in bacteria (OECD 471, Ames): negative with and without metabolic activation

Conclusion based on data obtained with alcohols, C16-18 (even numbered), ethoxylated, < 2.5 EO (CAS No. 68439-49-6, EC No. 939-518-5) and all available data on gene mutation in bacteria in the Alcohol Ethoxylates (AE) category in a Weight-of-Evidence approach.

 

In vitro cytogenicity / chromosome aberration in mammalian cells (OECD 473): negative with and without metabolic activation

Conclusion based on data obtained with alcohols, C16-18 (even numbered), ethoxylated, < 2.5 EO (CAS No. 68439-49-6, EC No. 939-518-5).

 

In vitro gene mutation in mammalian cells (OECD 476, HPRT): negative with and without metabolic activation

Conclusion based on data obtained with alcohols, C16-18 (even numbered), ethoxylated, < 2.5 EO (CAS No. 68439-49-6, EC No. 939-518-5).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

29 Aug - 17 Nov 1997

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted in 1983

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted in 2020

Deviations:

yes

Remarks:

test conducted in 4 valid strains only; no historical control data provided; 2-aminoanthracene used as sole positive control for tests with S9 mix

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministerium für Umwelt, Raumordnung und Landwirtschaft des Landes Nordrhein-Westfalen, Düsseldorf, Germany

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

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 treated with phenobarbital and beta-napthoflavone..
- source of S9 : CCR (Cytotest Cell Research GmbH & Co. KG), Roßdorf, Germany
- method of preparation of S9 mix Obtained from livers of male Wistar rats dosed on 3 consecutive days with 80 mg/kg bw phenobarbital (i.p.) plus 80 mg/kg bw beta-naphthoflavone (oral), animals were killed 24 h after last administration. Livers were removed from the animals, homogenised and the supernatant of the 9000 x g centrifugation step (the 'S9 fraction') was frozen immediately. S9 mix was prepared freshly before use. One part of S9 fraction was mixed with 9 parts of a cofactor solution resulting in the following mixture: 10% S9 fraction, 33 mM KCI, 5 mM glucose-6-phosphate, 4 mM NADP, 100 mM Na2HP04/NaH2P04 (pH 7.4), 8 mM MgCl2.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Protein content, sterility and activity of the preparation in the S. typhimurium gene mutation assay were certified by the vendor.

Test concentrations with justification for top dose:

Plate incorporation: 0, 50, 160, 500, 1600 and 5000 µg/plate
Preincubation (Test 1): 0, 50, 150, 300, 900 and 1500 µg/plate
Preincubation (Test 2): 0, 10, 25, 50, 100 and 150 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone
- Test concentration: 25 µl/plate (plate incorporation and preincubation test)

Untreated negative controls:

yes

Remarks:

untreated cells

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-aminoanthracene (2.5 µg/plate in DMSO, + S9, all strains)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments : 3 (plate incorporation test, preincubation (Test 1) and preincubation (Test 2))

METHOD OF TREATMENT/ EXPOSURE: in agar (plate incorporation, Experiment 1) and pre-incubation (Experiment 2)
Plate incorporation test
In a sterile tube,
- 0.025 mL of the appropriately diluted test material (or 0.025 mL of the solvent, or 0.1 mL [0.05 mL for TA 1535] of the strain specific positive control substance)
- 0.5 mL phosphate buffer (or 0.5 mL S9 mix in the experiment with metabolic activation)
- 2 mL of molten trace histidine supplemented top agar at approx. 45 °C
- 0.1 mL of the bacterial overnight culture were mixed.
Mixing was done in triplicate, for each bacterial strain and for each concentration of the test material. The mixture was then poured onto the surface of minimal agar plates. These plates were incubated at 37 °C for 72 h and then the number of revertant colonies was counted.

Preincubation test
In a sterile tube, a 0.1 mL aliquot of each one of the bacterial overnight cultures was mixed with a 0.5 mL volume of S9 mix (for tests with metabolic activation) or phosphate-buffer (for tests without metabolic activation). Then either 100 µL [50 µL for TA 1535] of the appropriate positive control, 25 µL of the solvent, or 25 µL of the test substance solution were added. The tubes were incubated at 30 °C for 30 min with gentle agitation. At the end of the incubation period, 2 mL of molten top agar was added to each tube, mixed briefly and poured onto minimal agar plates. These plates were incubated at 37 °C for 72 h and then the number of revertant colonies was counted.

Data collection
Bacterial colonies were counted on an Artek Model 880 Colony Counter which was calibrated for each test to check the counting accuracy.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: clearance of the bacterial background lawn, titer of the overnight culture and reduction in the number of spontaneous revertants

Evaluation criteria:

For a test compound to be considered positive, it must (in two independent experiments) cause at least a doubling in the mean revertants per plate of at least one tester strain. This increase must be accompanied by a dose response towards increasing concentrations of the test article. A test article that does not meet these criteria will be considered non-mutagenic in bacteria. Single increases in revertant frequencies, which are not dose-related and not reproducible in two independent tests are considered non-relevant. If however these increases do occur in both tests, this will be taken as an indication of a mutagenic effect.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

precipitates were noted in the first preincubation test; in the plate incorporation test, limit concentrations were used

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

precipitates were noted in the first preincubation test; in the plate incorporation test, limit concentrations were used

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

precipitates were noted in the first preincubation test; in the plate incorporation test, limit concentrations were used

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

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
- Precipitation and time of the determination: In the preincubation test (Test 1), precipitation of the test item in the agar was noted at 150 µg/plate and above for all strains in the presence and absence of metabolic activation. Therefore the preincubation test was repeated at lower concentration levels (Test 2). In the plate incorporation test, no precipitation occurred at any concentration for any strain, neither in the presence nor absence of metabolic activation.

CYTOTOXICITY:
There was no cytotoxicity observed up to and including the highest concentration in all strains and all experiments, neither with nor without metabolic activation.

STUDY RESULTS:
In the plate incorporation test with TA 98 (+S9) at 500 µg/plate the revertant frequency was significantly increased (quotient 2.1). As this significant increase was not dose related and not reproducible it was an effect by chance and not due to a mutagenic effect of the test substance.
In preincubation test 1 the test substance precipitated in the agar even at relatively low concentrations. Therefore, the preincubation test was repeated with lower concentrations (10 - 150 µg/plate) as preincubation test 2.
In preincubation test 1 with TA 1535 (+S9) at 900 µg/plate the revertant frequency was significantly increased (quotient 2.1 ). As this significant increase was not dose related and not reproducible it was an effect by chance and not due to a mutagenic effect of the test substance.
All four bacterial strains exhibited a positive mutagenic response with the positive controls tested both with and without metabolic activation by S9 mix. Negative (solvent) controls were also tested with each strain, and the mean numbers of spontaneous revertants were considered acceptable. All criteria for a valid study were met as described.

HISTORICAL CONTROL DATA: not provided in the study report

Detailed information are provided under 'Attached background material'.

Conclusions:

Under the conditions of the test, the test item was not mutagenic in S. typhimuirum strains TA98, TA100, TA1535 and TA1537 with and without metabolic activation.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

weight of evidence

Justification for type of information:

Please refer to the category justification provided in the category object.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

For a detailed assessment of the potential of Alcohol Ethoxylates (AE) to induce gene mutation in bacteria, please refer to the category justification attached to the category object.

Conclusions:

Applying read-across based on grouping of substances (category approach), no potential to induce gene mutation in bacteria with and without metabolic activation is predicted for the target substance.

Executive summary:

The available data on gene mutation in bacteria in the Alcohol Ethoxylates (AE) category indicate no potential for the target substance to exhibit any mutagenic activity. As explained in the category justification, the differences in molecular structure and composition between the target substance and the members of the AE category are unlikely to lead to differences in the mutagenic potential.

Reference 3

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

adopted in 1983

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

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.

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.

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”.

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

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.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 Jan - 26 Apr 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

adopted in 1984

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

Version / remarks:

adopted in 2016

Deviations:

yes

Remarks:

cytotoxicity evaluated based on cloning efficiency; no information on karyotype stability or mycoplasma contamination check of cells, no positive historical control data provided, acceptability and evaluation criteria differ from those spec. in guideline

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministerium für Umwelt, Raumordnung und Landwirtschaft des Landes Nordrhein-Westfalen, Düsseldorf, Germany

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

HPRT locus

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Chinese Hamster Ovary (CHO-K1), Flow Laboratories, Meckenheim, Germany, stored under liquid nitrogen.

MEDIA USED
- Medium for harbouring spontaneous mutations prior to treatment: HAT: Ham's F12 medium with 10% FCS, 2 mM L-Glutamine, 100 IU/mL Penicillin, 100 µg/ml Streptomycin, 200 mM glycin, 5 µM thymidine, 10 µM hypoxanthine, 3.2 µM aminopterin.
- Complete culture medium: H10: Ham's F12 medium with 10% fetal calf serum (FCS), 2 mM L-Glutamine, 100 IU/mL Penicillin, 100 µg/mL Streptomycin.
- Treatment medium: H0: Ham's F12 medium with 2 mM L-Glutamine, 100 IU/mL Penicillin, 100 ug/mL Streptomycin
- Selection medium: H6TG: Ham's F12 medium with 10% FCS, 2 mM L-Glutamine, 100 IU/mL Penicillin, 100 µg/mL Streptomycin, 10 µg/mL 6-thioguanine. H6TG does not contain hypoxanthine.
- Incubation (CO2 concentration, humidity level, temperature): The CHO cells were cultured at 37 °C, 5% CO2 and approx. 95% rel. humidity.

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 treated with Aroclor 1254.
- Source of S9: CCR (Cytotest Cell Research GmbH & Co. KG), Roßdorf, Germany (lots #060694 and #191294)
Obtained from the liver of male Wistar rats (weight approx. 220-320 g) which received a single i.p. injection of Aroclor 1254 (500 mg/kg body weight) in olive oil 5 days prior to the preparation of the S9 fraction. The livers were removed from the animals, homogenised, and the supernatant of the 9000 x g centrifugation step (the "S9 fraction") was frozen immediately.
- method of preparation of S9 mix: S9 mix was prepared freshly before use. 3 parts of S9 fraction were mixed with 7 parts of the following cofactor solution:- 43 mM KCl (final conc. in S9 mix: 30 mM), - 14 mM MgCl2 (final conc. in S9 mix: 10 mM), - 7 mM Glucose-6-phosphate (final conc. in S9 mix: 5 mM), - 4.7 mM NADP (monosodium salt) (final conc. in S9 mix: 4 mM), - 59 mM Na2HPO4/NaH2PO4, pH 7.4 (final conc. in S9 mix: 50 mM)
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Protein content, sterility and activity of the preparation in the S. typhimurium gene mutation assay were certified by CCR. Additionally, the ethoxyresorufin O-dealkylase activity is determined bimonthly to assure a sufficient activity of the S9 fraction.

Test concentrations with justification for top dose:

Preliminary cytotoxicity test:
With and without S9 mix: 1, 1.8, 3, 6, 10, 18, 30, 60 and 100 µg/mL (4 h exposure)

Main mutation assay:
With and without S9 mix: 1.8, 6, 18, 60 and 100 µg/mL (4 h exposure)

Justification for top concentration: The highest concentration used in the preliminary toxicity test was chosen with regard to the solubility of the test item. The dose range of the main experiments was set according to the data generated in the preliminary cytotoxicity test.

Vehicle / solvent:

- Vehicle/solvent used: 1% ethanol

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

1% ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

ethylmethanesulphonate

Details on test system and experimental conditions:

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

METHOD OF TREATMENT:
- Cell density at seeding: 2 x 10E5 cells/ 25 cm flask
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 h
- Expression time (cells in growth medium between treatment and selection): 9 days
- Selection time: 6 days
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: After the expression period, five cultures with 1 x 10E6 cells/75 cm flask were seeded in selective medium including trifluorothymidine (TFT, H6TG medium). The viability (cloning efficiency) was determined by seeding 200 cells per 60 mm dish in complete culture medium (without TFT).

SELECTION AGENT: 10 μg/mL trifluorothymidine (TFT)

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cloning efficiency 1 (survival) and 2 (viability)

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- Method: Mutant frequency (MF)

Evaluation criteria:

A test compound will be reported as being mutagenic in the HPRT test with CHO cells if it causes a statistically significant, dose related increase in mutant frequency at concentrations of the test substance resulting in geater than 20% cell survival. In addition, a positive response is claimed only, if the mean mutant frequency in treated cultures reaches a value significantly above the maximum spontaneous mutant frequency (of approx. 20/10E6 viable cells). Statistical significance is determined on the basis of a t-test ("Two-sample analysis").

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
Solubility testing with the test item revealed a solubility limit of approx. 100 µg/mL cell culture medium with 1% ethanol. Therefore, 100 µg/mL was selected as top concentration for the main mutation experiment. Up to and including the concentration of 100 µg/mL, no cytotoxicity was observed.

STUDY RESULTS
In the test without metabolic activation, there was a statistically significant increase in mutant frequency at 6 and 18 µg/mL in the first experiment. The finding occured without dose-response relationship and were therefore considered to have no biological meaning. In the test with metabolic activation, a statistically significant increase in mutant frequency at 1.8, 18, 60 and 100 µg/mL in the second experiment only. All values remained within the range of historical negative control data, therefore the findings were considered not to indicate a mutagenic potential of the test item.

CYTOTOXICITY
There was no cytotoxicity up to and including the highest tested concentration of 100 µg/mL in the presence or absence of metabolic activation.

HISTORICAL CONTROL DATA
The solvent and the positive controls showed the expected results and the obtained values remained within the historical control data.
- Positive historical control data: not provided in the study report
- Negative (solvent/vehicle) historical control data: 0-20 mutants/ 10E6 cells

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

Conclusions:

Under the conditions of the test the test item did not induce mutations in the Tk locus in CHO-K1 cells in the presence or absence of metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro gene mutation in bacteria

Data on gene mutation in bacteria in vitro are available for alcohols, C16-18 (even numbered), ethoxylated, < 2.5 EO (CAS No. 68439-49-6, EC No. 939-518-5) as well as several member substances of the Alcohol Ethoxylates (AE) category.

 

Study with alcohols, C16-18 (even numbered), ethoxylated, < 2.5 EO (CAS No. 68439-49-6, EC No. 939-518-5)

Mutagenicity of alcohols, C16-18 (even numbered), ethoxylated, < 2.5 EO (CAS No. 68439-49-6, EC No. 939-518-5) in bacteria was assessed in a study performed according to OECD guideline 471 under GLP conditions (Sasol, 1997). S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 were treated with the test substance using the plate incorporation method as well as the preincubation method, both with and without the addition of a rat liver S9 mix. The dose ranges for the plate incorporation test were 50, 160, 500, 1600 and 5000 µg/plate; for the first preincubation test 50, 150, 300, 900 and 1500 µg/plate and for the second one 10, 25, 50, 100 and 150 µg/plate. All tests were done in triplicates. The vehicle (acetone) and negative (untreated) control plates produced counts of revertant colonies within an acceptable range. All positive controls used in the test induced marked increases in the frequency of revertant colonies, both with and without the metabolising system. A reproducible mutagenic activity of the test substance to any of the tester strains was not observed with and without metabolic activation. Thus, under the conditions of this test the test substance is regarded as not mutagenic in bacteria.

 

Studies in the AE category

Studies investigating in vitro gene mutation in bacteria are available for the following AE substances:

 

CAS No.	EC No.	Substance	Study protocol	Hazard conclusion
27252-75-1	500-058-1	Octan-1-ol, ethoxylated	OECD 471	Negative, with and without metabolic activation
68439-50-9	500-213-3	Alcohols, C12-14, ethoxylated	OECD 471	Negative, with and without metabolic activation
			Similar OECD 471	Negative, with and without metabolic activation
68439-49-6	939-518-5	Alcohols, C16-18 (even numbered), ethoxylated, < 2.5 EO	OECD 471	Negative, with and without metabolic activation
9005-00-9	500-017-8	Octadecan-1-ol, ethoxylated	Similar OECD 471	Negative, with and without metabolic activation

 

Evaluation of gene mutation in bacteria as observed in studies

All available study results indicate a clear lack of mutagenic potential. No indication of an increase in revertant colony counts is observed in any study. Positive and vehicle control experiments yielded the expected results, demonstrating the adequacy of the test systems and metabolic activation systems. Based on all available data on in vitro gene mutation in bacteria in the AE category, it is predicted that the AE substances are not mutagenic in bacteria either in the presence or the absence of metabolic activation.

This evaluation is considered sufficiently conclusive for the hazard assessment and classification and labelling of the AE substances. For a detailed evaluation of the genotoxic potential of the substances in the AE category, please refer to the category justification attached to the category object.

 

In vitro cytogenicity / chromosome aberration in mammalian cells

Data on cytogenicity / chromosome aberration in mammalian cells in vitro are available for alcohols, C16-18 (even numbered), ethoxylated, < 2.5 EO (CAS No. 68439-49-6, EC No. 939-518-5).

The clastogenic potential was assessed in a chromosomal aberration test with alcohols, C16-18 (even numbered), ethoxylated, < 2.5 EO (CAS No. 68439-49-6, EC No. 939-518-5) in mammalian cells according to OECD guideline 473 under GLP conditions (Sasol, 1995b). Chinese hamster ovary cells (CHO) were exposed to 313, 625, 1250, 2500 and 5000 µg/mL in the presence and 1.25, 2.5, 5, 10, 20, 39 and 78 µg/mL in the absence of metabolic activation. Positive and vehicle (1% ethanol) control cultures were included in each assay. No increases in the number of chromosome aberrations in the presence or absence of metabolic activation were seen at any concentration tested. Appropriate reference mutagens used as positive controls showed a significant increase in chromosome aberrations, thus indicating the sensitivity of the assay, and the efficacy of the S9 mix. Hence, the test substance is regarded as not clastogenic. The study demonstrates the lack of a clastogenic potential. It is concluded that AE substances are generally not clastogenic.

This evaluation is used for the hazard assessment and classification and labelling of the AE substances. For a detailed evaluation of the genotoxic potential of the substances in the AE category, please refer to the category justification attached to the category object.

 

In vitro gene mutation in mammalian cells

Data on gene mutation in mammalian cells in vitro are available for alcohols, C16-18 (even numbered), ethoxylated, < 2.5 EO (CAS No. 68439-49-6, EC No. 939-518-5).

The mutagenic potential in mammalian cells was assessed with alcohols, C16-18 (even numbered), ethoxylated, < 2.5 EO (CAS No. 68439-49-6, EC No. 939-518-5) in a HPRT assay according to OECD guideline 476 under GLP conditions (Sasol, 1995c). Following pre-tests with the concentration ranging from 1-100 µg/mL, the latter being the solubility limit of the test substance, Chinese hamster ovary (CHO) cells were exposed for 4 h to concentrations of 1.8, 6, 18, 60 and 100 µg/mL in the absence and presence of a metabolic activation system with (rat liver S9 mix). No dose-related increases in mutant colony numbers were obtained in two independent experiments with the test substance in either the presence or absence of S9 mix. Appropriate reference mutagens used as positive controls produced highly significant increases in mutation frequency indicating the sensitivity of the assay. Therefore, the test substance is regarded as not mutagenic in mammalian cells. The study demonstrates the lack of a mutagenic potential. It is concluded that AE substances are generally not mutagenic in mammalian cells.

This evaluation is used for the hazard assessment and classification and labelling of the AE substances. For a detailed evaluation of the genotoxic potential of the substances in the AE category, please refer to the category justification attached to the category object.

Justification for classification or non-classification

The available data on genetic toxicity obtained with alcohols, C16-18 (even numbered), ethoxylated, < 2.5 EO (CAS No. 68439-49-6, EC No. 939-518-5) and with other members of the Alcohol Ethoxylates (AE) category do not meet the criteria for classification according to the CLP Regulation (EC) No. 1272/2008 and are therefore conclusive but not sufficient for classification.