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, 100, 1535, 1537, 1538 (similar to OECD TG 471) (Henkel, 1990).

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: information not required because in vivo information for a closely related substance is available
Mutagenicity in mammalian cells: the related substance 2-ethylhexan-1-ol was negative with and without activation in L5178Y mouse lymphoma cells (similar to OECD TG 476) (Kirby, 1983).

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
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.

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:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
not specified
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:
10 concentrations at log intervals between 0.013 and 1.0 µl/ml
Vehicle / solvent:
not indicated
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
not specified
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 10-12days

SELECTION AGENT (mutation assays): TFT

NUMBER OF REPLICATIONS: single treatement per dose, 10 doses

NUMBER OF CELLS EVALUATED: 3 E+06 calls

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
A result was judged to be positive when a two-fold increase in the number of revertants relative to background was observed.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
> 1.0 µl/ml
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Conclusions:
2-Ethyl hexan-1-ol was tested for mutagenicity to mammalian cells in a valid study using a method similar to OECD TG 476 up to toxic concentrations. No increase in the number of revertants was observed. It was concluded that 2-Ethyl hexan-1-ol is negative for mutagenicity to mammalian cells under the conditions of the test.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04-Apr-1990 to 27-Apr-1990
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
The restrictions were that the range of strains differs from the current guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
(no TA102 or E. coli WP2 uvrA; 2-AA only positive control with S9)
GLP compliance:
yes
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:
Rat liver S9 Aroclor 1254 induced
Test concentrations with justification for top dose:
1st test: 8, 40, 200, 1000 and 5000 µg/plate
2nd test: 6.25, 25, 100, 400 and 1600 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: the test substance was suspended in sorbitan derivative (Tween 80)/water
- Justification for choice of solvent/vehicle: none stated
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 S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
TA1537 without S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylendiamine in TA98 and TA1538 without S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene in all strains with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: none
- Exposure duration: 48 hours

NUMBER OF REPLICATES
- 3
Evaluation criteria:
Combination of: a) plate background of non-reverted bacteria not showing growth reduction relative to respective negative controls; b) spontaneous mutation rates within historical limits; c) at one or more doses tested the substance causes a 2-fold (TA 100) or 3-fold (other strains) increase in mutation rate above control levels.
Statistics:
None
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:
cytotoxicity
Remarks:
5000 µg/plate (except in TA100, no cytotoxicity)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
None

COMPARISON WITH HISTORICAL CONTROL DATA:
The spontaneous mutation frequencies for untreated and solvent treated cultures were within the historical range for the laboratory for all strains in the absence and in the presence of S9.

No increase in the number of revertants relative to the solvent and negative controls was observed in any strain at any concentration in first and second experiments. The results of the first test are presented in tables 1 and 2 below. The results of the second test, conducted over a smaller range of concentrations, were very similar to the first. Table 1 Experiment 1 Plate incorporation: Number of revertants per plate (mean of 3 plates)

 Strain        TA 98        TA 100        TA 1535
 Concentration  µg/plate  + MA  -MA  Toxicity  + MA  -MA  Toxicity  +MA  -MA  Toxicity
 Negative control  44.3  28.7  no  59.3  78.7  no  5.7 5.7   no
 Solvent control  38.7  35.3  no  58.0  79.3  no  4.0  6.7  no
 5000  7.7  0  yes  9.3  20.3  yes  i pp  i pp  yes
 1000  20.3  5.0  yes  55.0  54.7  no  4.0  4.3  no
 200  35.0  38.0  no  68.3  76.0  no  7.3  4.3  no
 40  39.3  31.7  no  64.0  77.0  no  6.7  4.3  no
 8  34.3  40.0  no  61.7  73.3  no  6.3  4.3  no
 Positive control  791.7  519.0  no  765.0  223.3  no  74.0  213.3  no

i pp: inhibition of background lawn and pin-point colonies

Table 2 Experiment 1 Plate incorporation: Number of revertants per plate (mean of 3 plates)

 Strain        TA 1537        TA 1538
 Concentration  µg/plate  + MA  -MA  Toxicity  + MA  -MA  Toxicity
 Negative control  4.0  3.7  no  15.0  10.0  no
 Solvent control  3.7  3.7  no  12.3  8.0  no
 5000  i pp  4.3  yes  11.3  i pp  no
 1000  3.3  3.3  no  10.3  5.7  yes
 200  4.7  2.3  no  13.0  7.0  no
 40  5.0  5.3  no  13.3  8.0  no
 8  4.0  4.3  no  11.3  8.7  no
 Positive control  269.0  354.0  no  1134.3  884.0  no
Conclusions:
In a reliable study, the C6 alcohol Lorol C6 98% did not increase the reverse mutation rate in histidine dependent bacterial strains of Salmonella typhimurium at concentrations of up to 5000 µg/plate in the presence or absence of metabolic activation (cytotoxicity observed at 5000 µg/plate). The study was performed in compliance with GLP. Expected results were obtained with negative (buffer) solvent and positive controls. The repeat experiment gave the same results as the initial test.
It is concluded that the test substance is negative for mutagenicity in bacteria under the conditions of the study.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Chromosome aberration study in rats: the related substance dodecan-1-ol was negative in mice after oral administration (gavage) (Henkel, 1992).

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:
weight of evidence
Study period:
11-Feb-1992 to 27-Apr-1992
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:
no
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
other: albino mice, CFW 1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Winkelmann
- Age at study initiation: 7-8 weeks
- Weight at study initiation: males 21-27 g, females 21-26 g
- Assigned to test groups randomly: yes, under following basis: allocated to treatment groups according to randomization table generated by computer programme or manually
- Fasting period before study: yes, overnight until 3-4 hours after dosing
- Housing: males, 1/cage, macrolon cages type I; females, <=3/cage, macrolon cages type II; filled with clean softwood bedding
- Diet (e.g. ad libitum): standard animal diet, Altromin No. 1314, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: >=6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 25 +- 3 (occasionally 20-25)
- Humidity (%): 40 - 50 (occasionally 45-70)
- Air changes (per hr): no data, except "air-conditioned room"
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES (main study): From: 25-Feb-1992 To: 28-Feb-1992
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: arachis oil
- Justification for choice of solvent/vehicle: test material easily soluble at required concentration
- Concentration of test material in vehicle: not stated, but provided a dose level of 5000 mg/kg bw, so 500 mg/ml
- Amount of vehicle (if gavage or dermal): 10 ml/kg bw (main study), 20 ml/kg bw (range finding study)
- Lot/batch no. (if required): no data
- Purity: no data
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: 500 mg/ml in arachis oil (main study)
Duration of treatment / exposure:
single administration
Frequency of treatment:
single administration
Post exposure period:
evaluated at 24, 48, 72 hours after administration
Dose / conc.:
5 000 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): not stated
- Route of administration: oral
- Dose: 20 mg/kg bw
Tissues and cell types examined:
bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: maximum tolerated dose, based on range-finding study (effects seen at 5000 mg/kg were piloerection only)

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): single administration, animals sacrificed 24, 48 and 72 hours after administration

DETAILS OF SLIDE PREPARATION: bone marrow collected from femurs, using foetal calf serum applied via a syringe, into a siliconised centrifuge tube; after centrifugation at 1000 rpm and removal of all but one drop of supernatant, cells of sediment carefully mixed; drop of cell suspension placed on clean, degreased microscope slide and immediately spread; 3 slides/animal; slides air dried at least overnight; stained with Giemsa; air dried and dipped in xylol for 3 minutes

METHOD OF ANALYSIS: 1 slide/animal chosen and given a random code; microscopic evaluation of slides from 5 males and 5 females per treatment group at 1000x magnification; number of micronucleated cells counted in 1000 polychromatic erythrocytes (PCEs)/animal; ratio of
polychromatic to normochromatic erythrocytes determined by counting and differentiating the first 1000 erythrocytes

OTHER: means and standard deviations calculated
Evaluation criteria:
Statistically significant (p<0.05) increase in PCE compared to controls at any sampling time in either sex
Acceptability of test: positive controls induced statistically significant increase in frequency of micronucleated PCEs; solvent control incidence of micronuclei should reasonably fall within historical control range for the testing facility.
Statistics:
Method used: Kastenbaum & Bowman
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
piloerection for 8 hours after administration; no mortality
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 5000 mg/kg bw
- Solubility: used at 250 mg/ml
- Clinical signs of toxicity in test animals: piloerection
- Evidence of cytotoxicity in tissue analyzed: not examined
- Rationale for exposure: based on limit test in rats in which acute oral LD50 was >5000 mg/kg bw
- Harvest times: animals observed for 3 days

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): no statistically significant increase in micronucleus frequency in any treatment group
- Ratio of PCE/NCE (for Micronucleus assay): treated groups similar to controls
- Appropriateness of dose levels and route: maximum tolerated dose of 5000 mg/kg bw used; guideline recommends maximum dose of 2000 mg/kg bw; oral route selected "taking into account the possible route of human exposure during manufacture, handling and use"
- Statistical evaluation: no statistically significant increases in micronuclei in treated groups of either sex; positive control produced a statistically significant increase in micronuclei
- Control incidence of micronuclei: not reported but presumably therefore within historical control range

Lorol 12 did not increase the frequency of micronucleated erythrocytes or the PCE:NCE ratio in mice at any time interval after treatment (24, 48 or 72 hours) at dose levels up to 5000 mg/kg bw when compared to vehicle controls.

Mean values per group in the micronucleus test with 1-Dodecanol (Lorol C12-99)

a) Number of micronucleated cells per 1000 polychromatic erythrocytes (PCE)

b) Ratio of polychromatic to normochromatic erythrocytes (PCE/NCE)

Treatment group; (sampling time)

Species and sex

Dose mg/kg

Micronucleated cells 1000 PCE

Ratio of PCE/NCE

Mean

Range

Mean

Range

Negative control (24 hours) arachis oil

male mice

10 ml/kg

3.60

0 - 9

1.11

0.80 - 1.31

female mice

10 ml/kg

2.00

0 - 4

1.34

1.02 - 1.07

Positve control (24 hours) cyclophosphamide

male mice

20

13.40

10 - 16

1.21

0.90 - 1.72

female mice

20

10.80

7 - 14

0.95

0.67 - 1.28

1-Dodecanol (Lorol C12-99)

 

 

 

 

 

 

 

Limit dose (24 hours)

male mice

5000

2.60

0 - 5

1.08

0.94 - 1.26

female mice

5000

2.40

2 - 3

1.01

0.90 - 1.18

Limit dose (48 hours)

male mice

5000

3.00

1 - 4

0.89

0.48 - 1.16

female mice

5000

2.00

0 - 5

1.18

0.90 - 1.68

Limit dose (72 hours)

male mice

5000

2.60

2 - 4

1.65

0.91 - 2.14

female mice

5000

1.60

0 - 4

1.33

1.08 - 1.55

 

Conclusions:
Dodecan-1-ol has been tested a reliable study, conducted according to OECD guideline 474, no genotoxicity was seen in mice after a single oral dose of 5000 mg/kg bw. The study was performed in compliance with GLP.
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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

The only information directly relating to hexan-1-ol is a bacterial mutagenicity study, which is a reliable study and therefore key. For endpoints where there is no information on hexan-1-ol, key studies were chosen from studies on closely related linear or branched alcohols of similar chain length. 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.

Hexan-1 -ol has been tested for mutagenicity to bacteria, in a study which was conducted according to the OECD TG 471, compliant with GLP. No evidence of a test-substance related increase in the number of revertants was observed with or without metabolic activation in Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 1538 in the initial or repeat experiments up to limit concentrations. Appropriate positive, solvent and negative controls were included and gave the expected results. It is concluded that the test substance is inegative for mutagenicity to bacteria under the conditions of the test (Henkel, 1990).

Fatty alcohol blend (containing 40.77% C8 and 55.3% C10) has been tested for mutagenicity to bacteria, in a study which was conducted according to OECD TG 471, compliant with GLP. No evidence of a test-substance related increase in the number of revertants was observed with or without metabolic activation inSalmonella typhimuriumstrainsTA 1535, TA 1537, TA 98, TA 100 and TA 1538 in the initial or repeat experiments up to cytotoxic concentration. Appropriate positive and solvent controls were included and gave the expected results. It is concluded that the test substance is negative for mutagenicity to bacterial under the conditions of the test (Inveresk, 1992).

Fatty alcohol blend (containing 40.77% C8 and 55.3% C10) has been tested for mutagenicity in Mouse lymphoma L5178Y cells according to OECD TG 476, and under GLP. No test-substance induced increase in the number of mutations was observed when tested up to cytotoxic concentrations. Appropriate solvent and positive controls were included and gave expected results. It is concluded that the test substance is negative for mutagenicity to mammalian cells under the conditions of the study (Inveresk, 1992).

Dodecan-1-olhas been tested for the induction of micronuclei in mice according to OECD TG 474 and in compliance with GLP. No evidence for a test substance induced increase in the incidence of micronucleated normochromatic erythrocytes in mice bone marrow. Appropriate solvent and positive controls were included and gave expected results. It is concluded that the test substance does not cause damage to chromosomes under the conditions of the test (Benduhn, 1992).

Fatty alcohol blend has been tested according to OECD 474 and under GLP. No evidence for a test substance induced increase in the incidence of micronucleated PCE was observed (1000 PCE scored per animal). Appropriate solvent and positive controls were included and gave expected results. 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.

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.

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

104-76-7

2-ethylhexan-1-ol

Supporting Substance

Neg; (2)

Kirby, 1983

 

 

Neg; (2)

Kirby, 1983

Neg; (2)

MN;Dom. Leth

(Putman, 1983; WHO, 1993)

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 beeeswax

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

Hexan-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 including a study for hexan-1-ol [Ames]. Evidence from in vivo studies on other category members supports the conclusion that these alcohols are not genotoxic in vivo. Negative data of reliability 1 or 2 in support of this conclusion are available for 2-ethyl hexanol (supporting) [negative chromosome aberration assay] and 1-dodecanol [negative micronucleus assay].