Fatty alcohols, C12-18 (even numbered), manuf. of, destn. residues

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In Vitro • Ames (OECD 471): Negative (+/- metabolic activation) • In vitro mouse lymphoma assay (OECD 476): Negative (+/- metabolic activation) • Chinese Hamster Ovary cell chromosome aberration assay (OECD 473): Negative (+/- metabolic activation)

Link to relevant study records

Reference

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:

2009-10-02 to 2009-12-14

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well-documented guideline study according to GLP. Read-across from analogue substance (Alcohols, C18-22, distn. residues). For details please refer to the read-across report.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

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):

- Type and identity of media: Ham's F-10 containing HEPES buffer and supplemented with minocycline (basic medium, medium for treatment in the presence of S9 mix and for washing cultures before or after treatment), basic medium with 10% (v/v) foetal bovine serum (medium for cell growth and treatment in the absence of S9 mix)
- Periodically checked for Mycoplasma contamination: yes

Additional strain / cell type characteristics:

other: Modal chromosome number: 21

Metabolic activation:

with and without

Metabolic activation system:

S9 mix prepared from S9 fraction obtained from male rats dosed with Arochlor 1254

Test concentrations with justification for top dose:

First test:
1. Experiment: 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, 100 and 200 µg/ml
2. Experiment: 25, 50, 100 and 200 µg/ml

Vehicle / solvent:

- Vehicle/solvent used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: with S9 mix: cyclophosphamid (CP); without S9 mix: methyl methanesulphonate (MMS)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
1. Experiment: -S9/+S9: 6 hours
2. Experiment: -S9: 22 hours; +S9: 6 hours
- Fixation time (start of exposure up to fixation or harvest of cells):
1. Experiment: -S9/+S9: 24 hours
2. Experiment: -S9: 24 and 48 hours; +S9: 24 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid (0.1 µg/ml)
STAIN (for cytogenetic assays): 5% Giemsa

NUMBER OF REPLICATIONS: two cultures per dose level, controls and vehicle control

NUMBER OF CELLS EVALUATED: 200 metaphases per dose level (100 per replicate)

DETERMINATION OF CYTOTOXICITY
- Method: Slides were examined for evidence of metaphase cells and signs of cellular necrosis. From the cell counts, the number if cells recovered per culture, was calculated. This was then compared with the number of cells (mean of 2 cultures) recovered from the vehicle control cultures.

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Other: During the first test, the osmotic pressure of selected concentrations of the test substance was measured; observations of precipitation were made at the end of the treatment period.

Evaluation criteria:

The results for test item and positive control treated cultures are evaluated by comparison with the concurrent vehicle control cultures and with historical negative control data. A negative response was recorded if responses from the test item treated cultures are within the 95% confidence limits for the historical negative control data.
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 the historical negative control data or greater than double the frequency of an elevated vehicle or untreated control culture if appropriate.
A test was positive if the response in at least one acceptable dose level was significant by the criterion described above.

A test item was positive if Test 1 was positive, as described above or if one of the tests was positive and the other test gave indications of activity. These indications may be suspicious levels of aberrant cells (between 95% and 99% confidence limits).
Experiments that met in part the criteria for a positive response, or marginally met all the criteria, were classed as inconclusive.

Statistics:

No statistical analysis was performed.

Species / strain:

Chinese hamster Ovary (CHO)

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:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: the colour of the medium was not changed by the test substance indicating no change of the pH
- Effects of osmolality: no effect on osmotic pressure at selected concentrations tested
- Precipitation: precipitation was noted at dose levels of 100 and 200 µg/ml in both the presence and absence of S9 mix in experiment 1 and in cultures treated with 200 µg/ml in both the presence and absence of S9 in experiment 2

RANGE-FINDING/SCREENING STUDIES: No toxicity was noted in any of the cultures treated with test substance up to 200 µg/mL

COMPARISON WITH HISTORICAL CONTROL DATA: The vehicle control cultures had levels of structural and numerical aberration within the 95% confidence limits of the historical negative control data.

Table 1a. Aberration Data: Experiment 1
Exposure duriation / Harvest Time	S9 mix	Concentration (µg/ml)	Aberration frequency	Aberrant Cell Frequency (%)
			(Lesions/ Cell)	Including Gaps	Excluding Gaps
6 h / 24 h	-	0 (DMSO)	0.00	0	0
		0 (DMSO)	0.00	0	0
		50	0.00	0	0
		50	0.00	0	0
		100	0.00	0	0
		100	0.00	0	0
		200	0.00	0	0
		200	0.00	0	0
		30 µg/ml MMS	0.04	3	3
		40 µg/ml MMS	0.29	16	14
6 h / 24 h	+	0 (DMSO)	0.00	0	0
		0 (DMSO)	0.00	0	0
		50	0.00	0	0
		50	0.02	2	1
		100	0.00	0	0
		100	0.00	0	0
		200	0.00	0	0
		200	0.01	1	0
		40 µg/ml CP	0.18	14	13
		50 µg/ml CP	0.13	11	8
Table 1b. Aberration Data: Experiment 2
Exposure duriation / Harvest Time	S9 mix	Concentration (µg/ml)	Aberration frequency	Aberrant Cell Frequency (%)
			(Lesions/ Cell)	Including Gaps	Excluding Gaps
22 h / 24 h	-	0 (DMSO)	0.00	0	0
		0 (DMSO)	0.01	1	0
		50	0.01	1	0
		50	0.00	0	0
		100	0.00	0	0
		100	0.00	0	0
		200	0.00	0	0
		200	0.01	1	1
		30 µg/ml MMS	0.06	5	3
		40 µg/ml MMS	0.29	11	10
22 h / 48 h	-	0 (DMSO)	0.00	1	0
		0 (DMSO)	0.00	0	0
		50	0.00	0	0
		50	0.00	0	0
		100	0.00	0	0
		100	0.00	0	0
		200	0.00	0	0
		200	0.00	0	0
		30 µg/ml MMS	0.06	3	2
		40 µg/ml MMS	0.25	13	12
6 h / 24 h	+	0 (DMSO)	0.01	1	0
		0 (DMSO)	0.00	0	0
		50	0.00	0	0
		50	0.01	1	0
		100	0.00	0	0
		100	0.00	0	0
		200	0.00	0	0
		200	0.00	0	0
		40 µg/ml CP	0.02	1	1
		50 µg/ml CP	0.10	8	8
Table 2: Polyploid data
Concentration (µg/ml)	No. Of Diploid cells	No. of Polyploid cells		Frequency of Polyploid cells
		Normal	Endoploid
0 (DMSO)	300	0	0	0.00
0 (DMSO)	300	1	0	0.33
50	300	0	0	0.00
50	300	1	0	0.33
100	300	0	0	0.00
100	300	1	0	0.33
200	300	2	0	0.66
200	300	0	0	0.00

Conclusions:

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

It was concluded that Alcohols, C18-22 Distn. Residues were not clastogenic when tested with Chinese hamster ovary cells in vitro, when tested up to a concentration of 200 µg/mL.
This result is used in a read-across approach in the REACH registration of Alcohols, C12 -18, distn. residues.

Executive summary:

All cultures treated with Alcohols, C18-22, distn. residues had levels of structural aberrations within the 95% confidence limits for a negative response. An extra assessment of polyploidy was carried out on the cultures treated in the absence of S9 mix and harvested at 48 h. All the cultures treated with Alcohols, C18-22, distn. residues had levels of polyploidy within the 95% confidence limits for a negative response.

This result is used in a read-across approach in the REACH registration of Alcohols, C12 -18, distn. residues.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

In Vitro:

A suite of in vitro mutagenicity studies were conducted on alcohols, C18-22, distillation residues (CAS No. 1160164-88-4), including the Ames test, in vitro mouse lymphoma assay and the CHO chromosome aberration assay. All these in vitro studies were negative for mutagenicity.

 

A GLP compliant OECD 471 bacterial reverse mutation assay was conducted with alcohols, C18-22, distillation residues (CAS No. 1160164-88-4). Revertant colonies were assessed following treatment with 6 / 20 / 60 / 200 / 600 / 2000 µg/plate of the test item in the absence and presence of S9 metabolic activation. It was concluded that the test item was not mutagenic in strains of Salmonella typhimurium and Escherichia coli, when tested in the absence and presence of metabolic activation up to and beyond its limit of solubility in the test system.

The mammalian cell gene mutation potential of alcohols, C18-22, distillation residues (CAS No. 1160164-88-4) was assessed in mouse lymphoma L5178Y cells. The test item was tested in the presence and absence of metabolic activation at doses of 25, 50, 100 and 200 µg/ml (not cytotoxic but up to precipitating concentrations). Alcohols, C18-22 distillation residues are not mutagenic in mouse lymphoma L5178Y cells, in either the absence or the presence of S9 mix, when tested in dimethylsulphoxide up to and beyond its limit of solubility in the test system.

A GLP compliant OECD 473 Chromosomal Aberrations Assay with Chinese Hamster Ovary Cell Cultures in vitro was conducted with alcohols, C18-22, distillation residues (CAS No. 1160164-88-4). It was concluded that alcohols, C18-22 distillation residues were not clastogenic when tested with Chinese hamster ovary cells in vitro, when tested up to a concentration of 200 µg/mL.

On the basis of the consistently negative study results from the in vitro genotoxicity data available (above), alcohols, C18-22 distillation residues are non-genotoxic. This result is used in a read-across approach in the REACH registration of Alcohols, C12 -18, distn. residues.

Justification for selection of genetic toxicity endpoint
GLP guideline study.
No substances specific data are available for Alcohols, C12-18, distn. residues. Therefore, available data for the analogue substance Alcohols. C18-22, distn. residues are used. Details on the read-across justification are summarized in the attached read-across report.

Justification for classification or non-classification

These findings do not warrant the classification of alcohols, C18-22, distillation residues as genotoxic under the new Regulation (EC) 1272/2008 on classification, labeling and packaging of substances and mixtures (CLP) or under the Directive 67/518/EEC for dangerous substances and Directive 1999/45/EC for preparations.

This finding is used in a read-across approach in the REACH registration of Alcohols, C12 -18, distn. residues.