Registration Dossier

Administrative data

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 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: negative with and without activation in Chinese hamster ovary cells (similar to OECD TG 473)

(Iglesias, 2002b).

Mutagenicity in mammalian cells: 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

Referenceopen allclose all

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:
reference to same study
Qualifier:
equivalent or similar to
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.

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:
reference to same study
Qualifier:
equivalent or similar to
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.

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:
reference to same study
Qualifier:
equivalent or similar to
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:
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:
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.

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:
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: 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

Referenceopen allclose all

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:
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 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.

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:
reference to same study
Qualifier:
equivalent or similar to
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 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

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 and 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

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