Docosan-1-ol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation (Bacterial reverse mutation assay/ Ames test): negative with and without activation in E.Coli WP2 uvrA strain (OECD TG 471) (Charles River, 2022)

Gene mutation (Bacterial reverse mutation assay / Ames test): negative with and without activation in S. typhimurium strains TA 98, TA100, TA1535, TA1537 and TA 1538 (OECD TG 471) (Iglesias, 2002b).

In addition to the data on the registered substance, in vitro bacterial reverse mutation assays have been commissioned with the analogue substances Icosan-1-ol (CAS 629-96-9, EC 211-119-4) and Tetracosan-1-ol (CAS 506-51-4; EC 208-043-9) and will be conducted according to OECD Test Guideline 471 and in compliance with GLP.

These studies are part of a Category testing strategy and will cover some of the data currently not presented in the above Iglesias publication (historical controls and cytotoxicity data). Please refer to the Category testing strategy and Amendment to Category testing strategy attached to Section 13 for a detailed overview on the planned in vitro genetic toxicity tests in the Category.

Cytogenicity in mammalian cells: negative with and without activation in Chinese hamster ovary cells (similar to OECD TG 473) (Iglesias, 2002b).

In addition to the data on the registered substance, and to cover some of the data not included (or partially presented) in the above Iglesias publication (one concentration evaluated in two of the three test conditions, historical control data, cytotoxicity data, maximum tested concentrationof 10 mM, 2 mg/mL or 2 μl/mL, or that induced 55+5% of cytotoxicity compared to the negative control, or the precipitation of the tested substance), data on the analogue substance (Z)-Octadec-9-enol (CAS 143-28-2; EC 205-597-3) is used as supporting read-across information.

Cytogenicity in mammalian cells: negative with and without activation in human peripheral blood lymphocytes (OECD TG 487) with the analogue substance (Z)-Octadec-9-enol (CAS 143-28-2; EC 205-597-3) (RIFM, 2017 – Full report soon to be available; permission to refer awaited)

Mutagenicity in mammalian cells: negative with and without activation in Chinese hamster lung V79 cells (similar to OECD TG 476) (Iglesias, 2002b).

In addition to the data on the registered substance, an in vitro mammalian cell gene mutation test using the thymidine kinase gene is commissioned with the analogue substance (Z)-Octadec-9-enol (CAS 143-28-2; EC 205-597-3) and will be conducted according to OECD Test Guideline 490 and in compliance with GLP. This study is part of a Category testing strategy and will cover some of the data currently not included in the above Iglesias publication (maximum tested concentration of 10 mM, 2 mg/mL or 2 μl/mL, or that induced 55+5% of cytotoxicity compared to the negative control, or the precipitation of the tested substance; historical control data).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2022-03-22 to 2022-04-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

2020

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Tryptophan locus (E.coli)

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : Trinova Biochem GmbH, Giessen, Germany
- method of preparation of S9 mix : prepared from male Sprague Dawley rats that had been injected intraperitoneally with Arochlor 1254 (500 mg/kg bw).
- concentration or volume of S9 mix and S9 in the final culture medium : 0.5 mL S9 mix in final culture medium (5% v/v in first experiment and 10% v/v in second experiment)
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): not specified

Test concentrations with justification for top dose:

1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate (first experiment)
8.5, 15, 27, 48, 154, 492 μg/plate (second experiment)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: tetrahydrofuran (THF)

- Justification for choice of solvent/vehicle: A solubility test was performed based on visual assessment. The test material formed a clear colourless solution in THF.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

THF

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

other: 2-aminoanthracene (2AA), 15 µg, solvent DMSO

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) : triplicate
- Number of independent experiments : Two experiments

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 10^9 cells/mL
- Test substance added in medium; in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: not applicable
- Exposure duration/duration of treatment: 48 hours
- Harvest time after the end of treatment (sampling/recovery times): not applicable

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition, increase in the size of microcolonies, reduction of the revertant colonies

Rationale for test conditions:

Test Guidelines conditions

Evaluation criteria:

a) The vehicle control and positive control plates (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at the laboratory
b) The selected dose-range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.
A test material is considered negative (not mutagenic) in the test if:
a)The total number of revertants in the tester strain TA100 or WP2 uvrA is not greater than two times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three times the concurrent vehicle control.
b)The negative response should be reproducible in at least one follow-up experiment.
A test material is considered positive (mutagenic) in the test if:
a)The total number of revertants in the tester strain TA100 or WP2 uvrA is greater than two times the concurrent vehicle control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three times the concurrent vehicle control.
b)In case a follow-up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow-up experiment.

Statistics:

Not specified

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

precipitation from 52 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: not examined
- Data on osmolality: not examined
- Possibility of evaporation from medium: not examined
- Water solubility: not examined
- Precipitation and time of the determination: not examined
- Definition of acceptable cells for analysis: not examined


RANGE-FINDING/SCREENING STUDIES (if applicable): not examined

STUDY RESULTS
- Concurrent vehicle negative and positive control data : see in tables attached

Ames test:
- Signs of toxicity : None
- Individual plate counts : See in tables attached
- Mean number of revertant colonies per plate and standard deviation : See in tables attached


HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: Range (89-2027: without S9; 109-1642: with S9); Mean (1312: without S9; 424: with S9); SD (369: without S9; 189: with S9); 95% upper limit (2035: without S9; 795: with S9); 95% lower limit (589: without S9; 53: with S9)
- Negative (solvent/vehicle) historical control data: Range (9-61: without S9; 9-68: with S9); Mean (21: without S9; 25: with S9); SD (8: without S9; 10: with S9); 95% upper limit (37: without S9; 44: with S9); 95% lower limit (5: without S9; 6: with S9)

Conclusions:

Docosan-1-ol (CAS 661-19-8, EC 211-546-6) has been tested in a reliable 5th-strain test conducted according to OECD Test Guideline 471 and in compliance with GLP, using Escherichia Coli (E.coli) strain WP2 uvrA via the plate incorporation methodology. No dose-related increase in the number of revertant colonies in the tester strain WP2 uvrA was observed, both in the absence and presence of metabolic activation in the first experiment. These results were confirmed in the follow up experiment. Appropriate positive and solvent controls were added and gave expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

no data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

no cross linking strain was used

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

(no TA102 or E. coli WP2 uvrA; 2-aminoanthracene only positive control with metabolic activation)

Principles of method if other than guideline:

Well-conducted study according to a protocol very similar to OECD guideline 471

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

liver microsomal fractions from male rats prepared by "established methods"

Test concentrations with justification for top dose:

10, 100, 333, 667, and 1000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: solubility

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA100, TA1535 without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-O-phenylenediamine

Remarks:

TA98, TA1537, TA1538 without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: aminoanthracene

Remarks:

all strains with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: none
- Exposure duration: no data

NUMBER OF REPLICATES:
- two independent experiments, both with and without metabolic activation
- each concentration (including controls) tested in triplicate

DETERMINATION OF CYTOTOXICITY
- Method: no data

Evaluation criteria:

To be considered positive in TA100, >=2x increase in revertants over spontaneous rate; in TA98, TA1535, TA1537 and TA1538, >=3x increase; alternatively a concentration-dependent increase irrespective of 2- or 3-fold increase

Statistics:

none

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

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:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: not determined, but number of revertants reduced in TA 98 at 1000 µg/plate

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:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: not determined, but number of revertants not reduced at 1000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: insoluble
- Precipitation: no data
- Other confounding effects: no data

RANGE-FINDING/SCREENING STUDIES: yes, no data presented

COMPARISON WITH HISTORICAL CONTROL DATA: no

ADDITIONAL INFORMATION ON CYTOTOXICITY: none

Remarks on result:

other: No mutagenic potential

Table 1 Revertants per plate (mean of 3 plates)

Concentration µg/plate	TA 98		TA100		TA1535		TA1537		TA1538
	- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9
Negative control	15.3	17.0	82.3	833.7	5.7	8.0	5.0	4.0	14.3	16.7
0*	15.3	15.7	83.3	77.3	8.3	8.3	7.0	5.0	14.7	15.0
10.0	14.0	19.0	70.7	80.3	10.7	9.7	3.0	5.0	14.3	14.0
100.0	9.3	15.3	85.0	82.0	7.7	9.0	4.0	4.3	14.7	15.7
333.3	11.7	15.7	80.0	79.7	8.0	12.3	4.7	5.0	15.0	15.3
666.6	12.0	12.7	74.0	82.3	8.3	6.3	4.3	5.3	14.0	15.3
1000	6.7	8.0	76.0	86.3	4.7	3.3	3.7	5.0	13.7	14.7
Positive control	1573	2337.7	1158	2414	601.7	345	109.7	85.3	1980	495.7

* Solvent control with

Conclusions:

In a valid and reliable study, behenyl alcohol (C22) did not increase the reverse mutation rate in histidine dependent bacterial strains of Salmonella typhimurium in the presence or absence of metabolic activation at concentrations up to and including 1000 µg/plate. It is concluded that the test substance is negative for mutagenicity in bacteria under the conditions of the test.

Executive summary:

In the bacterial mutagenicity study, S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 1538 bacteria were incubated with 10, 100, 333, 667, and 1000 µg/plate of test material, with and without metabolic activation. The test material did not increase the reverse mutation rate in histidine dependent bacterial strains of Salmonella typhimurium in the presence or absence of metabolic activation up to limit concentration. The study concludes that the test substance is negative for mutagenicity in bacteria under the conditions of the test. The study was conducted according to a test protocol that is comparable to the appropriate OECD test guideline, with the exception that no cross linking strain was used.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

not stated

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

without detailed documentation

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Principles of method if other than guideline:

Well-conducted study according to protocol very similar to OECD guideline 473

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not applicable

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: minimum essential medium
- Properly maintained: no data
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

liver microsomal fraction from male rats prepared according to Ames et al., 1977

Test concentrations with justification for top dose:

0.6, 10.0 and 20.0 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: solubility

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

ethylmethanesulphonate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

with metabolic activation

Positive control substance:

cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: not applicable
- Exposure duration: 4 hours
- Expression time (cells in growth medium): not applicable
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 7 and 24 (or 28) hours at 20 µg/ml, 18 hours at 0.6, 10 and 20 µg/ml

SPINDLE INHIBITOR (cytogenetic assays): Colcemid, 0.2 µg/ml

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2 cultures per concentration

NUMBER OF CELLS EVALUATED: 100 per slide, 200 per concentration

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Evaluation criteria:

To be considered positive, either a statistically significant, concentration-related increase in the number of structural chromosome aberrations, or a statistically significant positive response at one of the concentrations

Statistics:

Chi-squared test performed for cells with aberration (excluding gaps)

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: presumably >20 µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: insoluble
- Precipitation: no data
- Other confounding effects: no data

RANGE-FINDING/SCREENING STUDIES: yes, but no data presented

COMPARISON WITH HISTORICAL CONTROL DATA: no data

ADDITIONAL INFORMATION ON CYTOTOXICITY: at 20 µg/ml, mitotic index not reduced, plating efficiency not reduced

Remarks on result:

other: No mutagenic potential

Table 1 Cytogenicity: 7 hour fixation. Aberrations in 200 cells

Activation	Concentration µg/ml	Percent aberrant cells
		incl gaps	excl gaps	exchanges
Without	0*	4.0	1.5	0
	20	2.5	0.5	0
With	0*	4.0	1.5	0
	20	7.0	2.5	0

* Solvent control with ethanol

** Only 100 cells counted for positive controls

 

Table 2 Cytogenicity: 18 hour fixation. Aberrations in 200 cells

Activation	Concentration µg/ml	Percent aberrant cells
		incl gaps	excl gaps	exchanges
Without	Negative control	5.5	1.5	0
	0*	4.0	1.5	0.5
	0.6	4.5	2.0	0
	10	4.0	1.0	0.5
	20	3.0	0.5	0
	Positive control**	12.0	9.0	4.0
With	Negative control	2.5	1.5	0
	0*	2.5	1.5	0.5
	0.6	5.5	3.0	0.5
	10	4.0	2.5	0
	20	4.0	2.5	0.5
	Positive control**	16.0	13.0	5.5

* Solvent control with ethanol

** Only 100 cells counted for positive controls

Table 3 Cytogenicity: 18 hour fixation. Aberrations in 200 cells

Activation	Concentration µg/ml	Percent aberrant cells
		incl gaps	excl gaps	exchanges
Without	0*	6.0	2.5	0.5
	20	3.5	2.0	0
With	0*	1.0	0.5	0
	20	4.0	2.5	0.5

* Solvent control with ethanol

** Only 100 cells counted for positive controls

Conclusions:

In a reliable study, according to a protocol that is similar to OECD 473, behenyl alcohol (C22) did not increase the incidence of chromosome aberrations in Chinese hamster V79 cells in the presence or absence of metabolising fraction at concentrations up to 20 µg/ml. There was no evidence of cytotoxicity at this dose level.

Executive summary:

In an in vitro chromosome aberration study, Chinese hamster lung fibroblasts (V79) were incubated with 0.6, 10.0 and 20.0 µg/ml of test material dissolved in ethanol for 4 hours, with and without metabolic activation.

The test substance did not increase the incidence of chromosome aberrations in Chinese hamster V79 cells in the presence or absence of metabolic activation when tested up to limit concentration. There was no evidence of cytotoxicity at this dose level. The study was comparable to guideline without detailed documentation (publication). It is considered that read across to the registered substance is valid and scientifically justifiable.

Reference 4

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

yes

Remarks:

Full report was not available

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes: Human peripheral blood lymphocytes

Metabolic activation:

with and without

Metabolic activation system:

in the presence and absence of metabolic activation (S9)

Test concentrations with justification for top dose:

concentrations up to 1000 μg/mL

Vehicle / solvent:

DMSO

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Details on test system and experimental conditions:

Exposure for 3 or 24 hours

Rationale for test conditions:

Not specified

Evaluation criteria:

Not specified

Statistics:

Not specified

Key result

Species / strain:

lymphocytes: Human peripheral blood lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Additional information on results:

A statistically significant increase in the frequency of micronucleated binucleated cells was observed in the 3-hour and 24-hour treatments without S9. However, the observed MNBN frequencies in both test conditions were within the vehicle historical control ranges and were not indicative of clastogenic effects. (Z)-Octadec-9-enol did not induce micronucleated binucleated cells in the 3-hour treatment with S9.

Conclusions:

(Z)-octadec-9-enol has been tested in a valid study according to OECD Test Guideline 487 and in compliance with GLP in human peripheral blood lymphocytes. A statistically significant increase in the frequency of micronucleated binucleated cells was observed in the 3-hour and 24-hour treatments without S9. However, the observed MNBN frequencies in both test conditions were within the vehicle historical control ranges and were not indicative of clastogenic effects. (Z)-Octadec-9-enol did not induce micronucleated binucleated cells in the 3-hour treatment with S9. It is concluded that the test substance is not clastogenic under the conditions of this test.

Reference 5

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

no data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Well-conducted study according to a protocol very similar to OECD guideline 476

GLP compliance:

not specified

Type of assay:

mammalian cell gene mutation assay

Target gene:

HGPRT

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: no data
- Properly maintained: no data
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data

Metabolic activation:

with and without

Metabolic activation system:

no data, but for Ames test, liver microsomal fractions from male rats prepared by "established methods"

Test concentrations with justification for top dose:

2.0, 7.5, 15.0, and 20.0 ug/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol, final concentration in culture medium <=1% v/v
- Justification for choice of solvent/vehicle: solubility

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

ethylmethanesulphonate

Remarks:

1.0 ug/ml

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

with metabolic activation

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

15.4 ug/ml

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: not applicable
- Exposure duration: 4 hours
- Expression time (cells in growth medium): no data
- Selection time (if incubation with a selection agent): no data
- Fixation time (start of exposure up to fixation or harvest of cells): no data

SELECTION AGENT (mutation assays): thioguanine

NUMBER OF REPLICATIONS:
- 2 independent experiments, both with and without metabolic activation

NUMBER OF CELLS EVALUATED: no data

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

To be considered positive, statistically significant concentration-related increase in mutant frequency, or a reproducible and statistically significant positive response for at least one concentration

Statistics:

no data

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: presumably >20 µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: insoluble
- Precipitation: no data
- Other confounding effects: no data

RANGE-FINDING/SCREENING STUDIES: yes, but no data presented

COMPARISON WITH HISTORICAL CONTROL DATA: no data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- With metabolic activation: mean relative cell survival over the test concentrations ranged from 89.1% (20 ug/ml) to 93.8% (15 ug/ml).
- Without metabolic activation: mean relative cell survival ranged from 96% (15 ug/ml) to 120.2 % (20 ug/ml).

Remarks on result:

other: No mutagenic potential

Table 1 Results of mutagenicity in V79 cells (mean of 2 cultures)

Concentration µg/ml	Mean relative cell survival (%)		Mean mutants per culture		Mutant colonies per 10 E06 cells
	-MA	+MA	-MA	+MA	-MA	+MA
Negative	105.1	98.2	2.7	4.8	8.65	47.4
0*	100	100	4.5	2.1	14.7	8.75
2	101.3	92.55	4.1	6.3	12.5	21.65
7.5	102.4	93.6	5.4	4.9	16.1	15.75
15	96.0	93.8	3.4	1.7	12.3	6.35
20	120.2	89.1	4.3	4.4	17.9	16.95
Positive control	67.3	104.6	156.7	39.1	1143.7	163.4

Conclusions:

In a reliable study, behenyl alcohol (C22) did not increase the gene mutation rate in Chinese hamster V79 cells in the presence or absence of metabolic activation at concentrations up to 20 ug/ml. It is concluded that the test substance is negative for mutagenicity in mammalian cells under the conditions of this test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Mouse micronucleus study: negative in bone marrow (similar to OECD TG 474) (Iglesias, 2002b).

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

not stated

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Only 1000 erythrocytes were scored per animal, full experimental details were not reported, toxicity details were lacking. It was not compliant with GLP.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

(only 1000 PCEs per animal scored for micronuclei)

Principles of method if other than guideline:

Well-conducted study according to protocol very similar to OECD guideline 474

GLP compliance:

not specified

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BRL Tierfarm Fullinsdorf, Switzerland
- Age at study initiation: >=10 weeks
- Weight at study initiation: no data
- Assigned to test groups randomly: no data
- Fasting period before study: 18 hours, but continued to receive water ad libitum
- Housing: Markrolon Type 1 cages with wire mesh tops and granulated soft wood bedding
- Diet (e.g. ad libitum): standard pellet diet, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21
- Humidity (%): not regulated
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: no data

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: polyethylene glycol
- Justification for choice of solvent/vehicle: no data
- Concentration of test material in vehicle: no data [calculated: 5, 15 and 50 mg/ml]
- Amount of vehicle (if gavage or dermal): 10 ml/kg bw
- Lot/batch no. (if required): no data
- Purity: no data

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: few details; test material suspended in vehicle

Duration of treatment / exposure:

single administration

Frequency of treatment:

single administration

Post exposure period:

none

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

150 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Justification for choice of positive control(s): no data
- Route of administration: presumably oral gavage
- Doses / concentrations: 40 mg/kg bw

Tissues and cell types examined:

bone marrow cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: based on previous study - 500 mg/kg bw estimated to be the "maximum attainable dose"
TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): 24, 48 and 72 hours after dosing

DETAILS OF SLIDE PREPARATION: femurs removed, marrow flushed out with foetal calf serum, cell suspension centrifuged and supernatant discarded, small drop of cell pellet spread on slide, air dried, stained with May-Grunwald, mounted; 1 slide/sample

METHOD OF ANALYSIS: 1000 polychromatic erythrocytes (PCEs) scored for micronuclei; polychromatic:normochromatic (PCE:NCE) ratio scored

OTHER: only 5/sex per dose level evaluated

Evaluation criteria:

To be considered positive, either a statistically significant dose-related increase in the number of micronucleated PCEs or a reproducible, statistically significant positive response for at least one dose level

Statistics:

Mann-Whitney test

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

not specified

Remarks:

presumably toxic at >500 mg/kg bw

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: no data
- Solubility: no data
- Clinical signs of toxicity in test animals: no data
- Evidence of cytotoxicity in tissue analyzed: no data
- Rationale for exposure: no data
- Harvest times: no data
- High dose with and without activation: no data
- Other: presumably toxic above 500 mg/kg bw since this maximum dose was chosen for the main study on the basis of the results of the range-finding study

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): 0.03-0.09% for vehicle controls, 0.04-0.10% for test material treated, 0.71% for positive control
- Ratio of PCE/NCE (for Micronucleus assay): 1.05-1.27 for vehicle controls, 0.98-1.55 for test material treated, 0.93 for positive control
- Appropriateness of dose levels and route: appropriate (top dose was apparently the maximum tolerated dose, oral route relevant to humans)
- Statistical evaluation: no statistically significant increases in the frequency of micronuclei in mice treated with the test material; statistical significance not presented for positive control

Toxicity unclear, but possibly one male and one female mouse [per group?] died either spontaneously or due to gavage error.

Table 1 Results of micronucleus assay 24 hour sampling time

Treatment	Suspending agent	Low dose	Mid dose	High dose
Concentration mg/kg bw	0	40	50	150
Harvest time	24	24	24	24
Micronucleated PCE (%)	0.03	0.71	0.07	0.08
Ratio PCE/NCE	1.27	0.93	0.98	1.07

Table 2 Results of micronucleus assay 48 hour sampling time

Treatment	Suspending agent	Test substance	Test substance	Test substance
Concentration mg/kg bw	0	50	150	500
Harvest time	48	48	48	48
Micronucleated PCE (%)	0.09	0.1	0.04	0.05
Ratio PCE/NCE	1.05	1.06	1.01	1.23

Table 3 Results of micronucleus assay 72 hour sampling time

Treatment	Suspending agent	Low dose	Mid dose	High dose
Concentration mg/kg bw	0	50	150	500
Harvest time	72	72	72	72
Micronucleated PCE (%)	0.09	0.09	0.05	0.07
Ratio PCE/NCE	1.41	1.33	1.55	1.46

Conclusions:

In a reliable study, behenyl alcohol (C22) did not increase the incidence of micronuclei in mouse bone marrow cells after a single oral gavage dose of up to 500 mg/kg bw.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Information is available for all the REACH genetic toxicity endpoints. The data available from standard in vitro and in vivo genetic toxicity assays for docosan-1-ol (CAS 661-19-8; EC 211-546-6) and related substances show no evidence of mutagenic potential.

Discussion of trends in the Category of C6-24 linear and essentially-linear aliphatic alcohols:

The in vitro and in vivo data available for members of the category and supporting substances indicate that the C6-24 alcohols are not genotoxic. In addition, the category of LCAAs under consideration does not contain any structural elements that are of concern for potential mutagenic activity (Ashby and Tenant, 1991). Furthermore, primary LCAAs (linear and branched) in the range C1 to C5 do not have a mutagenic potential (Bevan, 2001; OECD SIDS butan-1-ol, 2001). Moreover, in a review by WHO-JECFA a series of 22 saturated aliphatic branched-chain primary LCAAs and the corresponding aldehydes and acids in the range C4 to C8 showed no activity in a battery of in vitro and in vivo mutagenicity tests (WHO, 1999). On this basis it is concluded that the category of LCAAs does not have a mutagenic potential and that read across within the category can be justified. Where data gaps exist, the gap is filled by read-across from reliable evidence within the C6-24 Alcohols Category, where possible using interpolation between at least two reliable studies using higher and lower carbon number test substances.

It is concluded that the category C6-24 LCAAs do not have a genotoxic potential.

Justification for classification or non-classification

Based on the available data, docosan-1-ol (CAS 661-19-8; EC 211-546-6) does not require classification for genetic toxicity according to Regulation (EC) No 1272/2008.