Tetracosanol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation (Bacterial reverse mutation assay / Ames test): the related substance docosan-1-ol was negative with and without activation in S. typhimurium strains TA 98, TA100, TA1535, TA1537 and TA 1538 (OECD TG 471) (Iglesias, 2002b).

Gene mutation (Bacterial reverse mutation assay / Ames test): the related substance Fatty alcohol blend (containing 40.77% C8 and 55.3% C10) was negative with and without activation in S. typhimurium strains TA 98, TA100, TA1535, TA1537 and TA 1538 (similar to OECD TG 471) (Inveresk, 1992).

Cytogenicity in mammalian cells: the related substance Docosan-1-ol was negative with and without activation in Chinese hamster ovary cells (similar to OECD TG 473) (Iglesias, 2002b).
Mutagenicity in mammalian cells: the related substance Docosan-1-ol was negative with and without activation in Chinese hamster lung V79 cells (similar to OECD TG 476) (Iglesias, 2002b).

Mutagenicity in mammalian cells: the related substance Fatty alcohol blend (containing 40.77% C8 and 55.3% C10): negative with and without activation in L5178Y mouse lymphoma cells (similar to OECD TG 476) (Inveresk, 1992).

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:

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, TA 1537, TA 98 and TA 100

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 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. Read across to the registered substance is considered scientifically justified.

Reference 2

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 3

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.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

The restrictions were that the range of strains does not comply with current guidelines. Read-across to the registered substance is considered scientifically justified

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

range of strains does not comply with current guidelines

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine operon

Species / strain / cell type:

S. typhimurium, other: TA 98; TA 100;TA 1535; TA 1537; TA 1538

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver S9

Test concentrations with justification for top dose:

Toxicity test: 33, 100, 333, 1000, 3333, 10000 µg/plate; Main experiment: 1.5, 5, 15, 50, 150, 500 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO was used in preliminary toxicity test, and high levels of toxicity demonstrated. At the concentrations used for the mutation assay, acetone was used as solvent as the levels of test substance could not be detected accurately in analysis when DMSO was the solvent.

- Justification for choice of solvent/vehicle: none given in report

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA 1535 and TA 100 without metabolic activation: 2-aminoanthracene

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA 1537 without metabolic activation

Untreated negative controls:

other:

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA 1538 and TA 98 without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

all strains with metabolic activation

Details on test system and experimental conditions:

ACTIVATION; Aroclor induced rat liver S9; NADP and glucose-6-phosphate as co-factors; 0.5 ml 10% S9 in 2.7 ml agar and test material and test strains.
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION

- Preincubation period: none

- Exposure duration: 2 days

- Expression time (cells in growth medium): 2 days


SELECTION AGENT (mutation assays): histidine-poor agar

NUMBER OF REPLICATIONS: triplicate plates, two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: other: condition of bacterial lawn

Evaluation criteria:

A positive response was recorded if there was a reproducible, dose dependent increase in the number of revertants to at least twice control values for TA 1535, TA 98, TA 1537 and TA 1538, and 1.5 times for strain TA 100.

Statistics:

Mean and standard deviation.

Key result

Species / strain:

S. typhimurium, other: TA 98; TA 100;TA 1535; TA 1537; TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Table 1 Experiment 1: Reversions per plate (mean of 3 plates)

Concentration µg/plate	TA 1535		TA 1537		TA 1538		TA 98		TA 100
	- MA	+ MA	- MA	+ MA	- MA	+ MA	- MA	+ MA	- MA	+ MA
0*	18	23	10	14	23	19	26	39	124	132
Positive control	245	250	1334	150	286	569	186	522	747	598
1.5	18	15	14	16	18	14	24	31	121	145
5	17	17	18	17	19	21	32	35	147	136
15	15	21	16	14	18	20	25	36	130	115
50	14	15	8	16	18	13	22	33	121	110
150	14	19	8	16	13	20	23	27	119	118
500	10	13	2	6	3	7	7	20	58	89

* solvent control acetone

Table 2 Experiment 2: Reversions per plate (mean of 3 plates)

Concentration µg/plate	TA 1535		TA 1537		TA 1538		TA 98		TA 100
	- MA	+ MA	- MA	+ MA	- MA	+ MA	- MA	+ MA	- MA	+ MA
0*	14	16	11	11	20	26	30	31	129	123
Positive control	262	178	1331	166	235	252	224	247	557	523
1.5	13	15	8	10	19	24	28	28	125	112
5	20	12	14	14	19	21	28	34	136	121
15	19	17	14	14	20	25	26	35	137	111
50	21	17	14	7	18	17	21	25	129	105
150	18	16	11	8	13	18	24	27	112	109
500	-	12	-	5	-	6	-	23	-	59

* solvent control acetone

Conclusions:

Fatty alcohol blend was tested in a bacterial reverse mutation assay according to a protocol that is similar to OECD 471 and under GLP in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538. No increase in the number of revertants per plate was observed with or without activation in either the initial assay or the independent repeat assay. Solvent and positive controls gave expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.

Executive summary:

Fatty alcohol blend was tested in a bacterial reverse mutation assay according to a protocol that is similar to OECD 471 and under GLP in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538. No increase in the number of revertants per plate was observed with or without activation in either the initial assay or the independent repeat assay. Solvent and positive controls gave expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.

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.

Reference 5

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver S9

Test concentrations with justification for top dose:

Toxicity assay: 0.4, 4.3, 43.2, 432, 4320 µg/ml; Mutagenicity assay: 9.4, 18.8, 37.5, 75, 150, 300 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO was used in toxicity assay, acetone in mutagenicity assay
- Justification for choice of solvent/vehicle: due to impurity peaks in the chromatograms, solvent was changed to acetone.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

ACTIVATION: 1.0 ml S9 mix containing 10% S9 and cofactors NADP and glucose-6-phosphate added to give final volume of 10 ml

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: none

- Exposure duration: 4 hours

- Expression time (cells in growth medium): 2 days

- Selection time (if incubation with a selection agent): 11-14 days


SELECTION AGENT (mutation assays): trifluorothymidine

NUMBER OF REPLICATIONS: duplicate cultures, independent repeat experiment

DETERMINATION OF CYTOTOXICITY
- Method: other: cloning efficiency

Evaluation criteria:

A substance was considered positive if there was an increase of at least 1.7 fold in at least one of the highest doses was significant and associated with an increase in mutant numbers and/or an upward trend in the remaining doses, in two experiments under the same activation conditions.

Statistics:

Statistical evaluation was performed if marginal responses were recorded.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

43.2 µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Table 1 Experiment 1 Mutant frequency (average of 3 plates per culture)

Concentration µg/ml	Relative total growth %		Mean mutant count (MC)		Mutant fraction x 10¿¿		Increase over control
	-MA	+MA	-MA	+MA	-MA	+MA	-MA	+MA
Solvent control	81	92	19	24	27	36	-	-
	112	103	32	25	35	36
	102	101	25	31	30	35
	104	103	26	27	31	32
Positive control	75	34	182	151	264	299	8.6	7.6
	63	38	150	135	268	235
9.4	129	85	17	30	21	36	1.0	0.9
	100	88	29	18	39	26
18.8	111	92	27	21	31	25	1.2	0.7
	109	71	39	19	44	24
37.5	72	92	18	24	25	34	0.9	1.0
	79	71	27	27	33	35
75	-	-	NP	NP	-	-	-	-
	-	-	NP	NP	-	-
150	-	-	NP	NP	-	-	-	-
	-	-	NP	NP	-	-
300	-	-	NP	NP	-	-	-	-
	-	-	NP	NP	-

NP = Not plated, too toxic for assessment

Table 2 Experiment 2 Mutant frequency (average of 3 plates per culture)

Concentration µg/ml	Relative total growth %		Mean mutant count (MC)		Mutant fraction x 10¿¿		Increase over control
	-MA	+MA	-MA	+MA	-MA	+MA	-MA	+MA
Solvent control	94	99	25	35	28	43	-	-
	113	110	30	41	26	43
	92	99	29	31	28	37
	-	93	-	30	-	37
Positive control	84	20	152	107	182	315	7.5	7.7
	83	21	172	122	221	298
10	103	-	31	NP	33	-	1.3	-
	98	-	32	NP	39	-
20	128	-	30	NP	29	-	1.1	-
	86	-	28	NP	29	-
30	93	90	21	27	26	30	1.1	0.9
	91	79	29	40	32	39
40	57	90	34	38	48	42	1.3	0.9
	84	94	20	29	22	33
50	32	91	20	31	26	31	1.0	0.8
	29	93	21	32	30	36
60	-	71	NP	36	-	36	-	0.8
	-	56	NP	24	-	28
70	-	37	NP	22	-	25	-	0.6
	-	23	NP	25	-	26
80	-	-	NP	NP	-	-	-	-
	-	-	NP	NP	-	-

NP = Not plated: 3 highest dose levels, too toxic for assessment

Conclusions:

Fatty alcohol blend has been tested according to a protocol that is similar to OECD 476 and under GLP. No increase in the mutant frequency was observed with or without metabolic activation in either the initial or repeat experiment up to cytotoxic concentrations. Solvent and positive controls gave expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.

Executive summary:

Fatty alcohol blend has been tested according to a protocol that is similar to OECD 476 and under GLP. No increase in the mutant frequency was observed with or without metabolic activation in either the initial or repeat experiment up to cytotoxic concentrations. Solvent and positive controls gave expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Mouse micronucleus study: the related substance docosan-1-ol was negative in bone marrow (similar to OECD TG 474) (Iglesias, 2002b).

Micronucleus study in mice: the related substance Fatty alcohol blend (containing 40.77% C8 and 55.3% C10) was negative after oral administration (Inveresk, 1992).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 November 1991 to 11 February 1332

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

only 1000 PCE per animal were scored for micronuclei

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited, Manston Road, Kent

- Age at study initiation: 5-7 weeks

- Weight at study initiation: 27-30 g (males) 18-23 g (females)

- Assigned to test groups randomly: yes

- Fasting period before study: no information

- Housing: individually in polypropylene/stainless steel cages

- Diet: ad libitum

- Water: ad libitum

- Acclimation period: at least 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19

- Humidity (%): 38

- Air changes (per hr): no information

- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil

- Justification for choice of solvent/vehicle: none given; standard vehicle

- Concentration of test material in vehicle: sufficient to give required dose in appropriate volume of vehicle
- Amount of vehicle (if gavage or dermal): 10 mg/ml/day

Duration of treatment / exposure:

Animals were dosed on three consecutive days.

Frequency of treatment:

daily

Post exposure period:

96 hours

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 (positive control, low and mid dose) or 10 (vehicle control, high dose)

Control animals:

yes, concurrent vehicle

Positive control(s):

- Positive control substance: cyclophosphamide

- Justification for choice of positive control(s): none given - standard positive control

- Route of administration: no information

- Doses / concentrations: 40 mg/ kg bw

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: no deaths occurred in preliminary toxicity assay

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Animals dosed at 0, 24 and 48 hours; samples taken at 72 and 96 hours

DETAILS OF SLIDE PREPARATION: Air dried slides were fixed in methanol then stained with 1% May-Grunwald for 5 minutes then counterstained in 15% Giesma for 15 minutes

METHOD OF ANALYSIS: 1000 PCE scored for micronuclei; PCE/NCE ratio was determined for 300 cells, using x 1000 oil immersion objective

Evaluation criteria:

An increase in micronucleus frequency of greater than 0.28%.

Statistics:

No statistical evaluation described.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Table 1 Results of in vivo micronucleus study

Treatment mg/kg.bw /day	Time of dosing (h)	Time of sampling (h)	Sex	No. of surviving dosed mice	Erythrocytes
					Polychromatic cells (PCE)			Mean PCE/NCE
					PCE Analysed	No. of MN-PCE	% MN-PCE
Negative control	0+24+48	72	M/F	10	10000	14	0.14	0.93
		96	M/F	10	10000	10	0.10	1.02
Positive control	0+24+48	72	M/F	10	10000	150*	1.50	0.46
500	0+24+48	72	M/F	10	10000	9	0.09	1.00
1000	0+24+48	72	M/F	10	10000	9	0.09	0.94
2000	0+24+48	72	M/F	10	10000	8	0.08	0.86
		96	M/F	10	10000	24	0.24	0.90

PCE = Polychromatic erythrocytes                                                           

MN-PCE = Micronucleated PCE

MN-PCE = Micronucleated NCE

* = Positive response in PCE

Conclusions:

Fatty alcohol blend has been tested according to OECD 474 and under GLP. Male and female mice were dosed with 500, 1000 and 2000 mg/kg bw. No increase in the number of micronucleated PCE was observed (1000 PCE scored per animal). It is concluded that the test substance is negative for the induction of micronuclei under the conditions of the test. No toxicity to bone marrow or general toxicity was observed.

Executive summary:

Fatty alcohol blend has been tested according to OECD 474 and under GLP. Male and female mice were dosed with 500, 1000 and 2000 mg/kg bw. No increase in the number of micronucleated PCE was observed (1000 PCE scored per animal). It is concluded that the test substance is negative for the induction of micronuclei under the conditions of the test. No toxicity to bone marrow or general toxicity was observed.

Reference 2

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

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

There is no information on the genetic toxicity of tetracosan-1-ol, so key studies were chosen from studies on closely related linear or branched alcohols of similar chain length, including a study on an unsaturated alcohol. The choice of key study was based on reliability and similarity of chain length. The data available from standard in vitro and in vivo genetic toxicity assays for all 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.

Conclusion:                                                                                                                                                        

The category C6-24 LCAAs do not have a genotoxic potential.

 Genetic toxicity of LCAAs

	CAS	CHEMICAL NAME	Bacterial mutagenicity	Bacterial mutagenicity	Mammalian cytogenicity	Mammalian cytogenicity	Mamalian mutagenicity	Mamalian mutagenicity	In vivostudies	In vivostudies
			Result (Rel.)	Reference	Result (Rel.)	Reference	Result (Rel.)	Reference	Result (Rel.)	Study Type*(Ref)
C6	111-27-3	Hexan-1-ol	Neg; (1)	Henkel, 1990
C7, 8 and 9		Alcohols, C7-9	Neg. (1)	Shell, 1996
C8	111-87-5	Octan-1-ol	Neg; (2)	Henkel, 1982a; HLS, 1996k
C8-10	none	Fatty alcohol blend (40.7% C8 and 55.3% C10) Supporting Substance	Neg(2)	Dillon, D.M., McCartney, M.A. (1992)			Neg (1)	Cattanach, P., Riach, C. (1992)	Neg (1)	Micronucleus (Holstrom, M., Innes, D. (1992))
C10	112-30-1	Decan-1-ol	Neg (4) 2 strains only	(HLS, 1996l)
C12	112-53-8	Dodecan-1-ol	Neg. (1)l	(Thompson, 1996a)Shimizu, 1985					Neg. (2)	Micronucleus; (Banduhn, 1992)
C12 and 13	75782-87-5	Alcohols, C12-13	Neg (2, >80% lin)	Dean, 1980
C12 and 13	740817-83-8	Alcohols, C12-13-branched and linear	Neg (1 50% lin),	Sasol, 1998f	Neg (1 (50% lin)	Sasol, 1998 g
C12	67762-25-8	C12-18 Alcohols, Type B Supporting	Neg (2)Ames	Henkel 1982c
C 12-15	90604-40-3	Alcohols, C12-15-branched and linear	Neg (1)	Ballantyne, 1996
C14	112-72-1	Tetradecan-1-ol	Neg (1)	Thompson, 1996b
C16	36653-82-4	Hexadecan-1-ol	Neg (1)	Thompson, 1996c
C16	36653-82-4	Hexadecan-1-ol	Neg. (2)	Henkel, 1981d
C16	68002-94-8	C16-18 and C18 Unsaturated Supporting	Neg. Ames (2)	Banduhn, 1989)
C18	112-92-5	Octadecan-1-ol	Neg (1)	Thompson, 1996d					Neg (2) MN	Hachiya, 1982
C18	112-92-5	Octadecan-1-ol	Neg(2)	Henkel, 1981f
C18	97552-91-5	C18-22 Alcohol Supporting	Neg. Ames (2)	Banduhn 1995
C22	661-19-8	Docosan-1-ol	Neg (2),	Iglesias, 2002a, Thompson, 1997	Neg (2),	Iglesias, 2002a	Neg (2)	Iglesias, 2002a	Neg (2)	Micronucleus Iglesias, 2002aª
C24-32		D-002*** Supporting substance							Neg (4)	MN; Dom. Leth.Rodeiro 1998a

* MN: Mouse bone marrow micronucleus test; Dom. Leth. Mouse Dominant Lethal test; UDS: Unscheduled DNA Synthesis assay

** Tested in S. typhimurium TA 98 and TA100, only.

***Mixture of very long chain fatty alcohols from hydrolysed beeswax

References:                                                                                                                                                       

Ashby, J., Tennant, R.W., 1991. Definitive relationships among chemical structure, carcinogenicity, and mutagenicity for 301 chemicals tested by the US NTP. Mutation Research 257, 229–306.            

WHO, 1999. Technical Report Series 884 Evaluation of certain food additives and contaminants. 49th Report of the Joint FAO/WHO Expert Committee on Food Additives (JECFA), Geneva.

Justification for classification or non-classification

Tetracosan-1-ol is a member of the category aliphatic alcohols. The category members contain no structural elements which may be of concern for potential mutagenic activity. In vitro tests over the carbon range (C6-22) of the long chain alcohols category members (primary aliphatic alcohols) and supporting substances (C5-C24-34) are negative. Evidence from in vivo studies on other category members supports the conclusion that these alcohols are not genotoxic in vivo.

Based on the available data, tetracosan-1-ol does not require classification for genetic toxicity according to Regulation (EC) No 1272/2008.