Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

in vitro:

- Gene mutation in bacteria (Bacterial reverse mutation assay/Ames test; OECD 471): S. typhimurium TA 97, TA 98, TA 100, TA 1535, TA 1537, TA 1538 and E. coli WP2 uvrA pKM101: negative with and without metabolic activation; CAS# 124-07-2, C8; CAS# 142-62-1, C6; CAS# 112-05-0, C9

- Chromosome aberration (OECD 473): negative with and without metabolic activation; CAS# 112-05-0, C9; CAS# 112-85-6, C22; CAS# 61790-12-3, fatty acids, tall oil

- Gene mutation in mammalian cells (TK locus; OECD 476): negative with and without metabolic activation; CAS# 334-48-5, C10

No properties for genetic toxicity were observed for members of the fatty acids category.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Only short abstract available
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
limited data; only 4 strains tested (strain with A/T base pair as primary mutation site missing); only up to 2500µg/plate tested
Principles of method if other than guideline:
Ames test according to Mutation Research 31, 347 - 364 (1975)
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon
Species / strain / cell type:
S. typhimurium, other: TA 98, TA 100, TA 1535, TA 1537 and TA 1538
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254
Test concentrations with justification for top dose:
4, 20, 100, 500 and 2500 µg/plate
Vehicle / solvent:
- Vehicle/solvent used: Tween 80 / H2O bidest.
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: without metabolic activation: 4-nitro-o-phenylenediamine and sodium azide; with metabolic activation: 2-amino-anthracene
Species / strain:
S. typhimurium, other: TA 98, TA 100, TA 1535, TA 1537 and TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Conclusions:
The test substance did not induce reverse mutations in the absence or presence of S9 mix in the tester strains Salmonella typhimurium TA98, TA100, TA1535, TA1537 and TA1538.
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
strain with AT base pair as primary reversion site missing
GLP compliance:
not specified
Remarks:
The available publication was conducted similar to OECD guideline 471. The GLP status of the study is unclear. Due to the good reliability and quality of the data and for animal welfare reasons, no further testing under GLP conditions was initiated.
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 97
Metabolic activation:
with and without
Metabolic activation system:
induced male Sprague Dawley rat liver S9 (10% and 30% S9 in S9-mix) and induced male Syrian hamster liver S9 (10% and 30% S9 in S9-mix)
Test concentrations with justification for top dose:
10, 33, 100, 333, 1000 µg/plate with (10% S9) and without metabolic activation
33, 100, 333, 1000, 3333 µg/plate with (30% S9) and without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-nitrofluorene or alternatively 4-nitro-o-phenylenediamine (-S9, TA 98); sodium azide (-S9, TA 100 and TA 1535); 9-aminoacridine (-S9, TA 97 and TA 1537); 2-aminoanthracene (+S9, all strains)
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 hours


Evaluation criteria:
If the test chemical was mutagenic to any particular strain of bacterium, the number of histidine-independent colonies arising on those plates will be significantly greater than the corresponding control plates for that strain.
The number of mutant colonies appearing on the positive control plates must be significantly increased over the spontaneous control number for the test to be considered valid. Failure of the positive control chemical to induce mutation is reason to discard the experiment.
If no increase in mutant colonies is seen after testing several strains under several different culture conditions, the test chemical is considered to be nonmutagenic in the Ames test.
Statistics:
Mean values and standard deviation were calculated.
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:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 97
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
slight toxicity without metabolic activation at 1000 and 3333 µg/plate
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: yes, at 1000 and 3333 µg/plate

Table 1. Test results for octanoic acid

 

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

± standard deviation

Base-pair substitution type

Frameshift type

TA 100

TA 1535

TA 97

TA 98

TA 1537

0

95 ± 8.5

28 ± 2.2

124 ± 3.8

21 ± 2.4

9 ± 1.2

33

122 ± 20

21 ± 1.5

127 ± 5.8

22 ± 5

5 ± 0.9

100

114 ± 2.1

22 ± 1.2

137 ± 3

13 ± 1.7

7 ± 1

333

105 ± 3.5

18 ± 2

141 ± 5.8

19 ± 1.3

9 ± 1.9

1000

97p ± 4.4

16p ± 3.1

78p ± 9.5

10p ± 2.5

4p ± 0.7

3333

58p ± 3.9

12p ± 3.5

0x ± 0

10p ± 2.1

7p ± 0.9

Positive controls

SA

SA

9AA

2NF or 4NPD

9AA

Mean No. of colonies/plate

± SD

563 ± 26.1

428 ± 21.9

793 ± 106.2

698 ± 50.8

305 ± 22.6

0

117 ± 5.5

23 ± 2.3

151 ± 10.3

20 ± 4.1

5 ± 1.5

10

104 ± 4.9

16 ± 0.3

153 ± 4.1

15 ± 1.5

6 ± 2.1

33

104 ± 5

19 ± 5.1

167 ± 3.2

15 ± 2.3

5 ± 1.3

100

96 ± 3.8

19 ± 0.9

159 ± 2.3

18 ± 4.2

8 ± 0.3

333

89 ± 2.1

12 ± 1.5

150 ± 8

15 ± 2.8

8 ± 0.7

1000

88p ± 6.7

13p ± 2.7

15x ± 8.7

16p ± 0.9

5p ± 0.3

Positive controls

SA

SA

9AA

2NF or 4NPD

9AA

Mean No. of colonies/plate

± SD

342 ± 0.6

370 ± 13.9

821 ± 13.2

624 ± 29.9

416 ± 43.7

+ (10% HLI)

0

103 ± 7.2

7 ± 0.3

171 ± 4.4

28 ± 5.8

11 ± 3.3

+ (10% HLI)

10

101 ± 6.7

11 ± 1.2

215 ± 9.7

28 ± 6.7

11 ± 2.3

+ (10% HLI)

33

101 ± 2.3

9 ± 1.3

215 ± 4.6

28 ± 4.4

11 ± 1.5

+ (10% HLI)

100

101 ± 11.5

7 ± 0.6

206 ± 16.2

27c ± 2

7 ± 1.2

+ (10% HLI)

333

102 ± 11.3

6 ± 1.3

137 ± 6.4

18 ± 3.1

8 ± 2.3

+ (10% HLI)

1000

83p ± 4.5

11p ± 3.9

38p ± 15.2

19p ± 3.4

10p ± 2.3

+ (10% HLI)

Positive controls

2AA

2AA

2AA

2AA

2AA

Mean No. of colonies/plate

± SD

659 ± 23.3

303 ± 9.5

474 ± 20.5

499 ± 39.3

78 ± 6.7

+ (30% HLI)

0

144 ± 8.2

12 ± 1.2

178 ± 10.3

33 ± 3.1

7 ± 0.3

+ (30% HLI)

33

120 ± 6.2

13 ± 1.3

165c ± 10

32 ± 6.5

11 ± 1.9

+ (30% HLI)

100

128 ± 4.1

16 ± 2.3

170 ± 10.5

32 ± 3.7

8 ± 2.2

+ (30% HLI)

333

108 ± 11.8

15 ± 0.3

158 ± 6

22 ± 2.5

10 ± 2.1

+ (30% HLI)

1000

83p ± 7.7

9p ± 1.2

77p ± 6.1

14p ± 0.9

8p ± 0

+ (30% HLI)

3333

44p ± 7.8

5p ± 1.9

2p ± 0.9

16p ± 4.5

3p ± 1.7

+ (30% HLI)

Positive controls

2AA

2AA

2AA

2AA

2AA

Mean No. of colonies/plate

± SD

544 ± 33.9

459 ± 37.3

414 ± 25.2

376 ± 14.1

47 ± 3.2

+ (10% RLI)

0

115 ± 10.7

8 ± 2.2

155 ± 7.4

24 ± 2.5

12 ± 1.2

+ (10% RLI)

10

112 ± 9.7

9 ± 0.3

172 ± 5.1

25 ± 3

5 ± 0.3

+ (10% RLI)

33

116 ± 3.3

9 ± 0.6

184 ± 7.2

22 ± 1.7

7 ± 0.6

+ (10% RLI)

100

102 ± 7.6

8 ± 0.7

168 ± 13.7

23 ± 3.2

5 ± 1.2

+ (10% RLI)

333

101 ± 8.5

9 ± 1.2

116 ± 20

20 ± 4.1

7 ± 1.2

+ (10% RLI)

1000

84p ± 7.5

6p ± 0.7

6p ± 0.3

18p ± 1.3

6p ± 1

+ (10% RLI)

Positive controls

2AA

2AA

2AA

2AA

2AA

Mean No. of colonies/plate

± SD

504 ± 29.6

289 ± 27.7

480 ± 8.7

316 ± 45.7

52 ± 1.5

+ (30% RLI)

0

112 ± 7.3

15 ± 2.9

192 ± 7.8

27 ± 3.3

10 ± 1.5

+ (30% RLI)

33

110 ± 3.5

19 ± 1.9

159 ± 6.9

17 ± 3.2

13 ± 2.6

+ (30% RLI)

100

109 ± 7.5

18 ± 4.3

178 ± 5.8

22 ± 3.2

12 ± 2.3

+ (30% RLI)

333

136 ± 12.9

14 ± 3.1

163 ± 15.4

20 ± 1.7

10 ± 2

+ (30% RLI)

1000

111p ± 3

7p ± 1.2

28p ± 4.1

9p ± 0.9

5p ± 1.2

+ (30% RLI)

3333

54p ± 6.8

6p ± 1.2

1p ± 0.7

10p ± 1.5

2p ± 0

+ (30% RLI)

Positive controls

2AA

2AA

2AA

2AA

2AA

Mean No. of colonies/plate

± SD

606 ± 19.7

187 ± 12.5

351 ± 7.9

108 ± 8.7

53 ± 0.6

SA = sodium azide

9AA = 9-aminoacridine

4NPD = 4-nitro-o-phenylenediamine

2NF = 2-nitro-fluorene

2AA = 2-aminoanthracene

SD = standard deviation

HLI = induced male Syrian hamster liver S9

RLI = induced male Sprague Dawley rat liver S9

p = precipitate

x = slight toxicity and precipitate

c = contamination

Conclusions:
Under the experimental conditions described, the test substance was not mutagenic in the Ames test using the Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA97 with and without metabolic activation.
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
only two strains of S. typhimurium were used
GLP compliance:
not specified
Remarks:
The available publication was conducted similar to OECD guideline 471. The GLP status of the study is unclear. Due to the good reliability and quality of the data and for animal welfare reasons, no further testing under GLP conditions was initiated.
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon (for S. typhimurium strains)
trp operon (for E. coli strains)
Species / strain / cell type:
other: S. typhimurium TA98 and TA100
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Species / strain / cell type:
E. coli, other: strain not further specified
Metabolic activation:
with and without
Metabolic activation system:
induced male Sprague Dawley rat liver S9 (10% S9 in S9-mix)
Test concentrations with justification for top dose:
500, 1000, 2500, 3500, 5000 µg/plate with and without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-nitrofluorene or alternatively 4-nitro-o-phenylenediamine (-S9, TA98); sodium azide (-S9, TA100); methyl methanesulfonate (-S9, E. coli pKM101); 2-aminoanthracene (+S9, all strains)
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: two experiments for TA98 and TA100

Evaluation criteria:
If the test chemical was mutagenic to any particular strain of bacterium, the number of histidine-independent colonies arising on those plates will be significantly greater than the corresponding control plates for that strain.
The number of mutant colonies appearing on the positive control plates must be significantly increased over the spontaneous control number for the test to be considered valid. Failure of the positive control chemical to induce mutation is reason to discard the experiment.
If no increase in mutant colonies is seen after testing several strains under several different culture conditions, the test chemical is considered to be nonmutagenic in the Ames test.
Statistics:
Mean values and standard deviation were calculated.
Species / strain:
other: S. typhimurium TA98 and TA100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
decrease in revertants starting at 3500 µg/plate
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
decrease in revertants starting at 3500 µg/plate
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli, other: strain not further specified
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
decrease in revertants starting at 3500 µg/plate
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid

Table 1: Test results of Experiment 1 for TA98, TA100 and E. coli strains

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

± Standard deviation

Base-pair substitution type

Frameshift type

TA100

E. coli WP2 uvrA pKM101

E. coli

TA98

0

135 ± 8

244 ± 19.6

237 ± 4.6

23 ± 2.5

500

124 ± 6.1

221 ± 6.4

216 ± 5

23 ± 1.5

1000

107 ± 7.9

208 ± 3

242 ± 22.3

22 ± 2.6

2500

125 ± 11.8

232 ± 7.5

228 ± 10.1

24 ± 4.3

3500

87 ± 5.9

99 ± 4.9

131 ± 29.2

17 ± 2.6

5000

41 ± 10.3

42 ± 9.4

75 ± 15

10 ± 1.2

Positive control

SA

MMS

MMS

2NF or 4NPD

Mean No. of colonies/plate

± SD

504 ± 55.4

635 ± 7.2

558 ± 31.2

532 ± 43

+

0

116 ± 9

211 ± 9.8

259 ± 1.7

29 ± 3.2

+

500

163 ± 3.4

198 ± 10.4

235 ± 22.3

29 ± 2.6

+

1000

161 ± 17.9

207 ± 27

282 ± 10.7

24 ± 2.1

+

2500

113 ± 14.7

144 ± 13.9

235 ± 20.6

36 ± 2.5

+

3500

114 ± 6.1

97 ± 23

273 ± 16.1

20 ± 3

+

5000

36 ± 20.4

133 ± 32.1

172 ± 8.8

9 ± 0.6

+

Positive control

2AA

2AA

2AA

2AA

Mean No. of colonies/plate

± SD

593 ± 28.5

554 ± 4.5

595 ± 18.7

355 ± 22.9

SA = sodium azide

MMS = methyl methanesulfonate

4NPD = 4-nitro-o-phenylenediamine

2NF = 2-nitro-fluorene

2AA = 2-aminoanthracene

SD = standard deviation

 

Table 2: Test results of Experiment 2 for TA98 and TA100

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

± Standard deviation

Base-pair substitution type

Frameshift type

TA100

TA98

0

135 ± 4.9

25 ± 1.2

500

127 ± 9.2

22 ± 1.8

1000

121 ± 7.2

21 ± 1.5

2500

139 ± 3.5

23 ± 3.5

3500

107 ± 7.9

17 ± 3.8

5000

36 ± 9.3

8 ± 2.3

Positive control

SA

2NF or 4NPD

Mean No. of colonies/plate

± SD

545 ± 76.1

636 ± 18

+

0

112 ± 0.9

23 ± 3.3

+

1000

113 ± 6.7

21 ± 0.9

+

2500

120 ± 12.9

27 ± 4

+

5000

112 ± 7.7

32 ± 0.9

+

7500

64 ± 0.6

15 ± 2.4

+

10000

18 ± 3.5

8 ± 2

+

Positive control

2AA

2AA

Mean No. of colonies/plate

± SD

475 ± 16.7

631 ± 31

SA = sodium azide

MMS = methyl methanesulfonate

4NPD = 4-nitro-o-phenylenediamine

2NF = 2-nitro-fluorene

2AA = 2-aminoanthracene

SD = standard deviation

Conclusions:
Under the experimental conditions described, the test substance was not mutagenic in the Ames test using the Salmonella typhimurium strains TA98 and TA100 and the E. coli strain WP2 uvrA pKM101 and one further E. coli strain with and without metabolic activation.
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
only three strains of bacteria were used
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon (for S. typhimurium strains)
trp operon (for E. coli strain)
Species / strain / cell type:
other: S. typhimurium TA98 and TA100
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of induced male Sprague Dawley rats (10% S9 in S9 mix)
Test concentrations with justification for top dose:
Experiment 1 and 2: 1000, 2500, 5000, 7500 and 10000 µg/plate with and without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-nitrofluorene or alternatively 4-nitro-o-phenylenediamine (-S9, TA98); sodium azide (-S9, TA100); methyl methanesulfonate (-S9, E. coli pKM101); 2-aminoanthracene (+S9, all strains)
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 hours



Evaluation criteria:
If the test chemical was mutagenic to any particular strain of bacterium, the number of histidine-independent colonies arising on those plates will be significantly greater than the corresponding control plates for that strain.
The number of mutant colonies appearing on the positive control plates must be significantly increased over the spontaneous control number for the test to be considered valid. Failure of the positive control chemical to induce mutation is reason to discard the experiment.
If no increase in mutant colonies is seen after testing several strains under several different culture conditions, the test chemical is considered to be nonmutagenic in the Ames test.
Statistics:
Mean values and standard deviation were calculated.
Species / strain:
other: S. typhimurium TA98 and TA100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid

Table1: Test results of Experiment 1

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(± Standard deviation)

Base-pair substitution type

Frameshift type

TA100

E. coli WP2 uvrA pKM101

TA98

0

56 ± 6.4

233 ± 11.1

17 ± 2.5

1000

51 ± 3.5

242 ± 15.6

12 ± 0.6

2500

51 ± 1.2

224 ± 7.1

5 ± 1

5000

57 ± 1.2

209 ± 3.5

8 ± 2

7500

47 ± 4.9

225 ± 9.4

9 ± 1.3

10000

51 ± 2.9

242 ± 16.5

6 ± 0.7

Positive control

SA

MMS

2NF or 4NPD

Mean No. of colonies/plate

(± SD)

203 ± 20.8

687 ± 34.3

218 ± 22.7

+

0

53 ± 3

257 ± 5.4

18 ± 2.5

+

1000

53 ± 5.9

261 ± 15.6

14 ± 1.5

+

2500

47 ± 4.1

251 ± 1.2

12 ± 1.2

+

5000

41 ± 2.6

244 ± 8.7

20 ± 3.8

+

7500

43 ± 2.9

270 ± 11

14 ± 0.9

+

10000

60 ± 11.3

281 ± 18.2

13 ± 0.3

+

Positive control

2AA

2AA

2AA

Mean No. of colonies/plate

(± SD)

719 ± 48.6

728 ± 91.8

992 ± 26.7

SA = sodium azide

MMS = methyl methanesulfonate

4NPD = 4-nitro-o-phenylenediamine

2NF = 2-nitro-fluorene

2AA = 2-aminoanthracene

SD = standard deviation

 

Table 2: Test results of Experiment 2

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

± Standard deviation

Base-pair substitution type

Frameshift type

TA100

E. coli WP2 uvrA pKM101

TA98

0

50 ± 3.8

205 ± 20.2

18 ± 4.3

1000

51 ± 5.2

177 ± 11.9

15 ± 1.2

2500

45 ± 3.1

179 ± 7.5

7 ± 1.5

5000

59 ± 3.5

137 ± 16

19 ± 2.3

7500

52 ± 0.3

179 ± 5.2

16 ± 1.5

10000

51 ± 8.1

194 ± 21.8

18 ± 2.1

Positive control

SA

MMS

2NF or 4NPD

Mean No. of colonies/plate

± SD

211 ± 10.7

444 ± 46.8

516 ± 13.8

+

0

53 ± 7.8

216 ± 5.7

28 ± 23.7

+

1000

45 ± 1.2

231 ± 12.1

19 ± 0.9

+

2500

50 ± 5

242 ± 6.4

14 ± 0.7

+

5000

43 ± 2.9

210 ± 17.5

22 ± 0.7

+

7500

57 ± 1.2

208 ± 11.3

24 ± 2

+

10000

61 ± 3.2

247 ± 19

19 ± 4.4

+

Positive control

2AA

2AA

2AA

Mean No. of colonies/plate

± SD

745 ± 61.4

561 ± 51.7

790 ± 13.3

SA = sodium azide

MMS = methyl methanesulfonate

4NPD = 4-nitro-o-phenylenediamine

2NF = 2-nitro-fluorene

2AA = 2-aminoanthracene

SD = standard deviation

Conclusions:
Under the experimental conditions described, the test substance was not mutagenic in the Ames test using the Salmonella typhimurium strains TA98 and TA100 and the E. coli strain WP2 uvrA pKM101 with and without metabolic activation.
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
28 Jun - 23 Aug 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Type of assay:
mammalian cell gene mutation assay
Target gene:
TK locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 complete medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital (80 mg/kg bw) and beta-naphtoflavone (100 mg/kg bw)
Test concentrations with justification for top dose:
Pre-Test:
Experiment 1:
- with and without metabolic activation: 0.5, 2, 4, 6, 8, 10 mM
Experiment 2:
- without metabolic activation. 0.005, 0.05, 0.2, 0.7, 1.3, 2.0 mM

Main Test:
Experiment 1:
- with metabolic activation: 0.70, 0.82, 0.94, 1.06, 1.18, 1.30, 1.42, 1.54 mM
- without metabolic activation. 0.22, 0.46, 0.58, 0.70, 0.82, 0.94, 1.06, 1.18 mM
Experiment 2:
- with metabolic activation: 1.0, 1.12, 1.24, 1.36, 1.48, 1.60, 1.72, 1.84 mM
- without metabolic activation. 0.0005, 0.001, 0.002, 0.005, 0.01, 0.06, 0.18, 0.3 mM
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: RPMI cell culture medium
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
since medium was used as solvent, no further solvent control was necessary
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: -S9: methylmethanesulfonate (10 µg/mL, dissolved in 0.9% NaCl); ethylemethanesulphanate (200 and 500 µg/mL, dissolved in medium); +S9: benzo(a)pyrene (3.5 µg/mL, dissolved in DMSO (1% final concentration in medium))
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Experiment 1: 4 h (short-term exposure) with and without metabolic activation
Experiment 2: 4 h (short-term exposure) with metabolic activation and 24 h (long-term exposure) without metabolic activation
- Expression time (cells in growth medium): 3 days (short-term exposure) or 2 days (long-term exposure)
- Selection time (if incubation with a selection agent): 11 - 14 days
- Fixation time (start of exposure up to fixation or harvest of cells): 13 - 18 days

SELECTION AGENT (mutation assays): 5 µg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: triplicates each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth; cloning efficiency; mitotic index

OTHER:
Small and large colonies were differentiated, as small colonies are capable to indicate chromosomal mutations
Evaluation criteria:
There are several criteria for a positive result:
- clear and dose-related increase in the mutant frequency
- biologically relevant response (at least a 2-fold increase of mutant frequencies related to the respective negative control values and higher than the historical range of negative controls) for at least one of the dose groups
- combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (slow growth colonies) indicated by a low large/Small colonies ratio (1.5 times the ratio of clastogenic control MMS and/or B[a]P) is an indication for potential clastogenic effects and/or chromosomal aberrations.

The test substance is considered to be negative if there is no biologically relevant increase in the induction of mutant cells above concurrent control levels, at any dose level.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 1.54 mM with metabolic activation and at 1.18 mM without metabolic activation in experiment 1, respectively; at 1.84 mM with metabolic activation and at 0.30 mM without metabolic activataion in experiment 2, respectively.
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: within the physiological range
- Effects of osmolality: within the physiological range
- Precipitation: in the pre-test with metabolic activation from concentrations of 4 mM and higher

RANGE-FINDING/SCREENING STUDIES:
All mutant values were found to be within the range of the historical control data of the test facility BSL Bioservice (about 51 to 170 mutants per 10^6 cells)

COMPARISON WITH HISTORICAL CONTROL DATA:
All mutant values were found to be within the range of the historical control data of the test facility BSL Bioservice (about 51 to 170 mutants per 10^6 cells)
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Colony sizing was performed for the highest concentrations of the test item and for the negative and positive controls. A mutation frequency above 2 in combination with an increased occurrence of small colonies (defined by slow growth and/or morphological alteration of the cell clone), indicated by a low large/small colony ratio (ratio of the clastogenic controls MMS and/or B[a]P with a coefficient of 1.5), is an indication for potential clastogenic effects and/or chromosomal aberrations.

Although in experiment 1 with metabolic activation an increased number of small colonies was noted at doses of 1.30 mM and 1.42 mM (26 and 31 small colonies, respectively, compared to 11 and 9 at control) all dose groups were considered as not clastogenic since no mutagenicity was found at these doses.

All other dose groups in the other experiments were also found not to be clastogenic, respectively.

Table 1: Experiment I - 4 h exposure - With Metabolic Activation

Concentration
[mM]

Cloning efficiency [%]

Relative Total Growth [%]

Mutants per 1E+06 surviving cells

Mutation factor

Colony Sizing

Quotient Large/Small

0

100

100

86.77

1

3.66

0.7

96.63

95.51

95.86

1.10

--

0.82

104.12

92.71

88.56

1.02

--

0.94

109.36

95.60

76.43

0.88

--

1.06

110.11

78.73

78.58

0.91

--

1.18

109.36

60.06

86.55

1.00

--

1.30

116.10

40.14

100.88

1.16

1.77

1.42

112.36

31.61

121.24

1.40

1.55

1.54

96.63

9.84

139.92

1.61

2.78

B[a]P, 3.5 µg/mL

99.63

69.03

623.89

7.19

1.24

B[a]P:Benzo[a]pyrene

  

Table 2: Experiment I - 4 h exposure - Without Metabolic Activation 

Concentration
[mM]

Cloning efficiency [%]

Relative Total Growth [%]

Mutants per 1E+06 surviving cells

Mutation factor

Colony Sizing

Quotient Large/Small

0

100

100

79.39

1

2.75

0.22

100.33

90.09

78.53

0.99

--

0.46

86.38

79.76

124.63

1.57

--

0.58

91.03

79.70

77.88

0.98

--

0.70

98.34

89.53

70.89

0.89

--

0.82

98.34

71.30

69.28

0.87

--

0.94

95.02

64.50

62.74

0.79

1.60

1.06

99.00

47.66

74.59

0.94

3.17

1.18

91.69

11.04

97.49

1.23

1.12

EMS, 500 µg/mL

86.38

62.27

1337.77

16.85

--

MMS, 10 µg/mL

85.71

66.62

841.03

10.59

0.69

EMS:Ethyl methane sulphonate

MMS:Methyl methane sulphonate

 Table 3: Experiment II - 4 h Exposure - With Metabolic Activation

Concentration
[mM]

Cloning efficiency [%]

Relative Total Growth [%]

Mutants per 1E+06 surviving cells

Mutation factor

Colony Sizing

Quotient Large/Small

0

100

100

82.64

1.00

2.07

1

96.97

85.91

82.23

1.00

--

1.12

101.68

89.08

61.54

0.74

--

1.24

98.99

86.20

86.92

1.05

--

1.36

99.66

83.65

75.75

0.92

--

1.48

90.91

64.04

118.05

1.43

--

1.60

96.07

35.51

70.23

0.85

2.38

1.72

91.58

38.25

84.77

1.03

2.13

1.84

98.99

19.98

98.81

1.20

4.73

B[a]P, 3.5 µg/mL

86.20

60.39

829.02

10.03

0.89

B[a]P:Benzo[a]pyrene

 

 

Table 4: Experiment II - 24 h exposure - Without Metabolic Activation

Concentration
[mM]

Cloning efficiency [%]

Relative Total Growth [%]

Mutants per 1E+06 surviving cells

Mutation factor

Colony Sizing

Quotient Large/Small

0

100

100

104.66

1

2.61

0.0005

95.24

94.64

76.34

0.73

--

0.001

101.36

103.58

68.22

0.65

--

0.002

94.56

95.29

66.56

0.64

--

0.005

101.36

105.73

52.63

0.50

--

0.01

102.04

98.98

64.06

0.61

--

0.06

102.72

96.75

61.54

0.59

3.30

0.18

96.60

55.56

91.91

0.88

2.24

0.30

91.16

19.77

99.26

0.95

1.94

EMS, 200 µg/mL

70.75

34.84

2516.55

24.05

--

MMS, 10 µg/mL

59.18

27.33

2625.00

25.08

0.81

EMS: Ethyl methane sulphonate

MMS: Methyl methane sulphonate

Conclusions:
In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item decanoic acid is considered to be non-mutagenic in the mouse lymphoma thymidine kinase locus using the cell line L5178Y.
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Comparable to guideline study with acceptable restrictions. The mutation frequency provided in the supporting information (Table 4) was incomprehensible. Based on the given values for mutants and viable cells, the mutant colonies per 1E+06 surviving cells and the mutation factor were re-calculated.
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
no information on dose selection rationale: test concentrations (-S9: up to 0.08 mM; +S9: 0.03 - 0.13 mM) are very low and cover only a small dose range (-S9: 0.07 - 0.08 mM)
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay
Target gene:
TK locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: Fischer's medium for leukemic cells of mice supplemented with 10% horse serum and 0.02% pluronic F-68
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9-mix), prepared from the livers of Sprague Dawley rats treated with Aroclor 1254 (500 mg/kg bw)
Test concentrations with justification for top dose:
0.021, 0.022, 0.023, 0.024 and 0.025 µL/mL without S9
0.01, 0.021, 0.031 and 0.041 µL/mL with S9
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: ethyl methylsulfonate (4.7E-06 M) or methyl methanesulfonate (10 - 20 µg/mL) with metabolic activation; 3-methylcholanthrene (1.86E-05 M) or dimethylbenz[a]anthracene (0.5 - 4 µg/mL) without metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h
- Expression time (cells in growth medium): 2 days after the end of the treatment, cells were plated in soft agar medium containing TFT for 10-12 days.
- Selection time (if incubation with a selection agent): 10-12 days
- Fixation time (start of exposure up to fixation or harvest of cells): 12-14 days

SELECTION AGENT (mutation assays): trifluorothymidine (TFT; 3 µg/mL)
STAIN (for cytogenetic assays):

NUMBER OF REPLICATIONS: duplicate cultures

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

OTHER: small and large colonies were differentiated, as small colonies are capable to indicate chromosomal mutations

Evaluation criteria:
Two evaluation criteria were applied:
1. a 2-fold increase in mutant frequency elicits a positive result
2. a concentration-related increase in mutant frequency and one or more dose levels with 10% or total growth exhibiting mutant frequencies ≥ 100 mutants per 1E+06 clonable cells over the background lawn elicits a positive result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
starting at 0.024 µL/mL without S9 and at 0.041 µL/mL with S9
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid

Table 1: Mouse lymphoma test - 4 h exposure - without metabolic activation 

Concentration
[µL/mL]

Relative Total Growth [%]

Viable cells (average) per 200 seeded cells

Mutants (average)

Mutant colonies per 1E+06 surviving cells1

Mutation factor

0 (DMSO)

100

178

58

65.16

1

0.021

79

163

55

67.48

1.04

82

166

56

67.47

1.04

0.022

63

145

52

71.72

1.10

87

174

61

70.11

1.08

0.023

62

148

51

68.92

1.06

70

146

56

76.71

1.18

0.024

70

168

51

60.71

0.93

37

107

46

85.98

1.32

0.025

37

135

50

74.07

1.14

51

156

58

74.36

1.14

Positive control

23

69

228

660.87

10.14

1 mutants x 10^6 / value of survived cells

 

Table 2: Mouse lymphoma test - 4 h exposure - with metabolic activation

Concentration
[µl/mL]

Relative Total Growth [%]

Viable cells (average) per 200 seeded cells

Mutants (average)

Mutant colonies per 1E+06 surviving cells1

Mutation factor

0 (DMSO)

100

158

59

74.6835

1

0.01

102

163

57

69.94

0.94

113

177

63

71.19

1.05

0.021

84

164

69

84.15

1.13

0.031

58

162

59

72.84

0.98

68

164

61

74.39

1.00

0.041

11

160

82

102.5

1.37

Positive control

26

89

311

698.88

9.36

1 mutants x 10^6 / value of survived cells

 

Conclusions:
Under the experimental conditions reported, no increase in mutation frequency was observed for linolenic acid compared to the control. Therefore, the test substance was considered not to be mutagenic in mammalian cells.
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Comparable to guideline study with acceptable restrictions. The mutation frequency provided in the supporting information (Table 4) was incomprehensible. Based on the given values for mutants and viable cells, the mutant colonies per 1E+06 surviving cells and the mutation factor were re-calculated.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
no information on dose selection rationale: very low concentrations (-S9: 0.02 - 0.08 mM; +S9: 0.03 - 0.2 mM) were tested
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay
Target gene:
TK locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: Fischer's medium for leukemic cells of mice supplemented with 10% horse serum and 0.02% pluronic F-68
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9-mix), prepared from the livers of Sprague Dawley rats treated with Aroclor 1254 (500 mg/kg bw)
Test concentrations with justification for top dose:
0.005, 0.011, 0.018 and 0.024 µL/mL without S9
0.01, 0.02, 0.04, 0.05 and 0.06 µL/mL with S9
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: ethyl methylsulfonate (4.7E-06 M) or methyl methanesulfonate (10 - 20 µg/mL) with metabolic activation; 3-methylcholanthrene (1.86E-05 M) or dimethylbenz[a]anthracene (0.5 - 4 µg/mL) without metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h
- Expression time (cells in growth medium): 2 days after the end of the treatment, cells were plated in soft agar medium containing TFT for 10-12 days.
- Selection time (if incubation with a selection agent): 10-12 days
- Fixation time (start of exposure up to fixation or harvest of cells): 12-14 days

SELECTION AGENT (mutation assays): trifluorothymidine (TFT; 3 µg/mL)

NUMBER OF REPLICATIONS: duplicate cultures

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

OTHER: small and large colonies were differentiated, as small colonies are capable to indicate chromosomal mutations

Evaluation criteria:
Two evaluation criteria were applied:
1. a 2-fold increase in mutant frequency elicits a positive result
2. a concentration-related increase in mutant frequency and one or more dose levels with 10% or total growth exhibiting mutant frequencies ≥ 100 mutants per 1E+06 clonable cells over the background lawn elicits a positive result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 0.06 µL/mL with S9
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid

Table 1: Mouse lymphoma test - 4 h exposure - with metabolic activation 

Concentration
[µL/mL]

Relative Total Growth [%]

Viable cells (average) per 200 seeded cells

Mutants (average)

Mutant colonies per 1E+06 surviving cells1

Mutation factor

0 (DMSO)

100

148

59

79.73

1

0.01

105

147

58

78.91

0.99

111

147

57

77.55

0.99

0.02

99

139

63

90.65

1.14

99

136

68

100

1.25

0.04

96

146

62

84.93

1.07

102

145

65

89.66

1.12

0.05

79

153

70

91.50

1.15

68

165

63

76.36

0.96

0.06

54

151

53

70.20

0.88

29

150

60

80

1.00

Positive control

58

118

193

327.12

4.10

1mutants x 10^6 / value of survived cells

 

Table 2: Mouse lymphoma test - 4 h exposure - without metabolic activation 

Concentration
[µL/mL]

Relative Total Growth [%]

Viable cells (average) per 200 seeded cells

Mutants (average)

Mutant colonies per 1E+06 surviving cells1

Mutation factor

0 (DMSO)

100

149

55

73.83

 1

0.005

93

137

53

77.37

1.05

105

151

50

66.23

0.90

0.011

97

148

57

77.03

1.04

97

147

55

74.83

1.01

0.018

84

137

53

77.37

1.05

92

154

55

71.43

0.97

0.024

73

141

46

65.25

0.88

69

131

58

88.55

1.20

Positive control

32

83

266

640.96

8.68

1mutants x 10^6 / value of survived cells

Conclusions:
Under the experimental conditions reported, no increase in mutation frequency was observed for linoleic acid compared to the control. Therefore, the test substance was considered not to be mutagenic in mammalian cells.
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Please refer to the category justification provided in IUCLID section 13.
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Key result
Species / strain:
other: S. typhimurium TA98 and TA100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
other: S. typhimurium TA98 and TA100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
decrease in revertants starting at 3500 µg/plate
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
decrease in revertants starting at 3500 µg/plate
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli, other: strain not further specified
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
decrease in revertants starting from 3500 µg/plate
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Source CAS 142-62-1
Conclusions:
Based on available read-across studies, octanoic acid is considered to be not mutagenic in strains with AT base pairs at the primary reversion site with and without metabolic activation.
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Please refer to the category justification provided in IUCLID section 13.
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 1.54 mM with metabolic activation and at 1.18 mM without metabolic activation in experiment 1, respectively; at 1.84 mM with metabolic activation and at 0.30 mM without metabolic activataion in experiment 2, respectively.
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
starting at 0.024 µL/mL without S9 and at 0.041 µL/mL with S9
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 0.06 µL/mL with S9
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: Source CAS 334-48-5
Conclusions:
Based on available read-across studies, octanoic acid is considered to be non-mutagenic in the mouse lymphoma thymidine kinase locus using the cell line L5178Y.


Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
not applicable
Species / strain / cell type:
mammalian cell line, other: Chinese hamster lung (CHL) cells
Details on mammalian cell type (if applicable):
- Type and identity of media: Eagle-MEM liquid medium
- Properly maintained: yes
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 from rat liver, induced with phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose:
-S9 mix (short-term exposure): 0, 875, 1750, 3500 µg/mL
+S9 mix (short-term exposure): 0, 875, 1750, 3500 µg/mL
-S9 mix (24 h continuous exposure): 0, 350, 700, 1400, 2800 µg/mL
-S9 mix (48-hour continuous exposure): 0, 288, 575, 1150, 2300 µg/mL
Vehicle / solvent:
1.0% carboxymethylcellulose sodium
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: continuous exposure: mitomycin C (0.05 µg/mL for 24 hours and 0.025 µg/mL for 48 hours); short-term exposure: cyclophosphamide (12.5 µg/mL)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 3 days
- Exposure duration: 6 (short-term exposure), 24, 48 h

STAIN (for cytogenetic assays): Giemsa

NUMBER OF CELLS EVALUATED: 200

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:
The frequency of polyploid cells or cells with abnormal structure of each test group were determined according to the criteria of Ishidate.
Species / strain:
mammalian cell line, other: Chinese hamster lung (CHL) cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: insoluble in water, soluble in alcohol, ether, chloroform and acetone
- Precipitation: observed on the slide of continuous exposure high dose group

RANGE-FINDING/SCREENING STUDIES: see 'Any other information on material and method incl. tables'

COMPARISON WITH HISTORICAL CONTROL DATA: this test was valid, since the frequency of chromosomal aberration in positive control was within background data.

Table 1: Results of growth inhibition test

Test item

Concentration

in µg/mL

Survival in %

Exposure period 24 h, without S9 mix

1% CMC▪Na

 

100

Test substance

272

97.7

454

95.2

756

96.5

1260

83.1

2100

62.1

3500

37.8

Exposure period 48 h, without S9 mix

1% CMC▪Na

 

100

Test substance

272

101.1

454

104.6

756

98.9

1260

89.7

2100

75.9

3500

1.3

Exposure period 6 h, without S9 mix

1% CMC▪Na

 

100

Test substance

272

109.6

454

93.7

756

102.5

1260

110.7

2100

89.8

3500

95.1

Exposure period 6 h, with S9 mix

1% CMC▪Na

 

100

Test substance

272

84.3

454

85.9

756

85.3

1260

86.5

2100

68.3

3500

76.3

 

 

Table 2: Results of chromosome aberration test

Test item

Concentration

Aberrant cells in %

Polyploid cells in %

 

in µg/mL

with gaps

without gaps

Exposure period 24 h, without S9 mix

1% CMC▪Na

 

0.5

0.5

0.0

MMC

0.05

56.0

52.5

0.5

Test substance

350

1.0

1.0

0.5

700

1.0

0.5

0.5

1400

0.5

0.0

0.0

2800

Toxic

Exposure period 48 h, without S9 mix

1% CMC▪Na

 

0.0

0.0

0.5

MMC

0.025

58.5

54.5

0.0

Test substance

288

1.0

1.0

0.0

575

0.5

0.0

0.5

1150

2.0

1.5

0.0

2300

Toxic

Exposure period 6 h, without S9 mix

1% CMC▪Na

 

1.5

0.5

0.5

CP

12.5

0.5

0.0

0.5

Test substance

875

0.5

0.5

0.0

1750

3.0

2.5

0.0

3500

1.0

0.5

0.5

Exposure period 6 h, with S9 mix

1% CMC▪Na

 

1.5

1.0

0.0

CP

12.5

63.5

61.5

0.0

Test substance

875

2.5

1.5

1.0

1750

2.0

2.0

0.0

3500

2.0

2.0

0.0

CMC▪Na: carboxymethylcellulose sodium (solvent)

MMC: Mitomycin C; CP: Cyclophosphamide (positive controls)

 

Conclusions:
The test substance did not induce structural chromosomal aberrations in the absence or presence of an exogenous metabolic activation system.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
not applicable
Species / strain / cell type:
lymphocytes: cultured peripheral human
Details on mammalian cell type (if applicable):
- Type and identity of media: F10 complete culture medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: not required. Cell cultures were started within 4 hours after blood collection.
- Periodically checked for karyotype stability: not required. Cell cultures were started within 4 hours after blood collection.
- Periodically "cleansed" against high spontaneous background: not required. Cell cultures were started within 4 hours after blood collection.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
9000g supernatanet (S9) from Aroclor 1254 induced rat liver
Test concentrations with justification for top dose:
Experiment I:
With and without metabolic activation: 100, 333, 420, 480, 520*, 750* µg/mL
Experiment II:
without metabolic activation: 10, 33, 100, 240, 300 µg/mL
with metabolic activation: 333, 420, 480, 520* µg/mL

* = precipitation
Vehicle / solvent:
- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: low solubility of pelargonic acid
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: mitomycin C and cyclophosphamide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hours
- Exposure duration:
First experiment: with S9: 3 hours. Without S9: 3, 24, and 48 hours.
Second experiment: with S9: 3 hours. Without S9: 24 and 48 hours.
- Expression time (cells in growth medium): 48 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 51- 96 hours


SPINDLE INHIBITOR (cytogenetic assays): colchicine


NUMBER OF REPLICATIONS: 2


NUMBER OF CELLS EVALUATED: 200/dose level/experiment


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index


OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
Evaluation criteria:
A result was considered positive if a statistically significant (p<0.05) increase of the number of cells with chromosome aberrations was seen.
Statistics:
Chi-squrae test
Species / strain:
lymphocytes: cultured peripheral human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: at 480 µg/mL the pH was lowered to 7.34, compared to pH=7.48 of the solvent control
- Effects of osmolality: no
- Precipitation: yes; at 520 and 750 µg/mL in the first and second experiment

RANGE-FINDING/SCREENING STUDIES: yes

COMPARISON WITH HISTORICAL CONTROL DATA: yes

ADDITIONAL INFORMATION ON CYTOTOXICITY: yes
Conclusions:
Pelargonic acid did not induce chromosomal aberrations and was not clastogenic in human lymphocytes with and without metabolic activation
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 Jun - 16 Aug 2001
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP - Guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
no additional experiment with continuous treatment (-S9 mix) and no confirmatory experiments (the test substance is considered negative, since clastogenic response only occurs at overtly toxic concentrations); no cytoxicity data for positive controls
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
only one harvest time; only one idependent experiment
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
not applicable
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: Ham's F-10 containing HEPES buffer supplemented with the antibiotic minocycline and 10% (v/v) foetal bovine serum (FBS only for cell growth and treatement in the absence of S9 mix)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9-mix), prepared from the liver of male Fisher rats treated with Aroclor 1254
Test concentrations with justification for top dose:
5, 10 and 20 µg/mL with metabolic activation
39, 78 and 156 µg/mL without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: cyclophosphamide (CPH), 30 and 40 µg/mL, +S9 mix; methyl methanesulphonate (MMS), 30 and 40 µg/mL, -S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 6 h with and without S9 mix
- Fixation time (start of exposure up to fixation or harvest of cells): 24 h

SPINDLE INHIBITOR (cytogenetic assays): Colcemid (0.1 µg/mL)
STAIN (for cytogenetic assays): Giemsa


NUMBER OF REPLICATIONS: 1 (treatments with test item or controls were performed on duplicate cell cultures)

NUMBER OF CELLS EVALUATED: 100 per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
A dose level was considered to be toxic if the cell count was reduced to less than 50% of the mean vehicle control values or if consistent evidence of changes to cell morphology was observed.

Evaluation criteria:
The results for test item and positive control treated cultures are evaluated by comparison with the concurrent vehicle control cultures and with historical negative control data.
A negative response was recorded, if responses from the test item treated cultures were within the 95% confidence limits for the historical negative control data.
The response at a single dose was classified as significant if the percent of aberrant cells was consistently greater than the 99% confidence limits for the historical negative control data or greated than double the frequency of an elevated vehicle or untreated control culture if appropriate.
A test was positive if the response in at least one acceptable dose level was significant by the criterion described above.
A test item was positive if both test 1 and 2 were positive, as described above or if the second test was positive after the first test gave indications of activity. These indications may be suspicious levels of aberrant cells (between 95% and 99% confidence limits).
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
other: positive responses were noted at overtly toxic concentrations: +S9: at 20 µg/mL and -S9: at 156 µg/mL
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
+S9: at 20 µg/mL; -S9: at 156 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The test substance did not change the colour of the culture medium, therefore no pH measurement was made.
- Effects of osmolality: There was a slight decrease in osmotic pressure as the concentrations increased, but this decrease was not noted in the concentrations assessed for aberrations.
- Precipitation: Observation of precipitation were made before cultures were washed out at the end of the treatment period. Precipitation was noted in cultures treated with 156-5000 µg/mL in the presence and in cultures treated with 313-5000 µg/mL in the absence of S9 mix.

RANGE-FINDING/SCREENING STUDIES: 9 dose levels ranging from 20 µg/mL up to 5000 µg/mL were used for cytotoxicity testing with and without S9 mix. In the absence of S9 mix, toxicity was observed in the cultures treated with 156-5000 µg/mL with reduced cell counts noted. In the cultures treated with 313-5000 µg/mL there were no metaphases cells present for assessment. The experiment in the presence of S9 mix was repeated due to toxicity. Test concentrations of 2.5, 5, 10, 20, 30 and 40 µg/mL were used with S9 mix in the repeat test. In the presence of S9 mix, toxicity was was observed in the cultures treated with 20-40 µg/mL. Reduced cell counts were noted in these cultures (less than 50% of the vehicle control cultures) and no metaphase cells were present for assessment in the cultures treated with 30 and 40 µg/mL.

COMPARISON WITH HISTORICAL CONTROL DATA:
The results obtained for the negative controls were compared with historical control data. The negative controls were within the range of historical negative control data.

Table 1. Test results.

 

Test item

Concentration

Mitotic Index

Aberrant cells in %

 

in µg/mL

in %

with gaps

without gaps

Exposure period 6h, fixation time 24h, without S9 mix

DMSO

1.0%

0.99

0

0

1.01

0

0

MMS

30

-

16

14

40

-

25

25

Test substance

20

0.65

-

-

0.86

-

-

39

0.86

0

0

0.83

0

0

78

0.86

0

0

0.71

0

0

156

0.14

8

7

0.15

13

12

313

0

-

-

0

-

-

625

0

-

-

0

-

-

1250

0

-

-

0

-

-

2500

0

-

-

0

-

-

5000

0

-

-

0

-

-

Exposure period 6h, fixation time 24h, with S9 mix

DMSO

1.0%

0.91

1

0

1.09

0

0

CP

30

-

5

4

40

-

20

16

Test substance

2.5

1.23

-

-

1.11

-

-

5

0.75

1

0

0.72

0

0

10

0.75

3

1

0.8

0

0

20

0.08

9

8

0.17

12

10

30

0

-

-

0

-

-

40

0

-

-

0

-

-

MMS: Methyl methanesulphonate; CP: Cyclophosphamide (positive controls)

  

Only at overtly cytotoxic concentrations (mitotic index ≤ 17% of negative control) of 156 µg/mL without S9 mix and 20 µg/mL with S9 mix increases in the number of aberrations were noted. At these concentrations the number of metaphase cells was sparse and in the cell culture treated with 156 µg/mL without S9 mix slightly rounded cells were observed, indicating changes of cell morphology. It is known, that high cytotoxicity causes artefacts in form of aberrations in in vitro chromosomal aberration assays. Hence, the increase in aberrant cells at the overtly toxic concentrations is considered as artefact. Thus, the test substance is considered to be not clastogenic.

Conclusions:
Chromosome aberrations were induced only at overtly toxic concentrations. No chromosomal aberrations were observed at non-toxic concentrations with and without metabolic activation. Therefore, the test substance is not considered to be clastogenic.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Please refer to the category justification provided in IUCLID section 13.
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Key result
Species / strain:
mammalian cell line, other: Chinese hamster lung (CHL) cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Key result
Species / strain:
lymphocytes: cultured peripheral human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
other: positive responses were noted at overt toxic concentrations: +S9: at 20
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
+S9: at 20 µg/mL; -S9: at 156 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: Source CAS 112-85-6
Conclusions:
Based on available read-across studies, octanoic acid is not expected to induce structural chromosomal aberrations in the absence or presence of an endogenous metabolic activation system.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No in vivo study is available.

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Human health effects in regard to genetic toxicity are predicted from adequate and reliable data for source substances by read-across to the target substance within the group applying the group concept in accordance with Annex XI, Item 1.5, of Regulation (EC) No 1907/2006.

No genotoxic potential for fatty acids is expected since fatty acids are found in all living organisms where they are fulfilling fundamental physiological functions. The negative genetic toxicity for fatty acids is demonstrated by gene mutation tests in bacteria (Ames test) with members of the fatty acid category including short-, mid- and long-chain fatty acids as well as mixtures of fatty acids, in cytogenicity tests with C9 fatty acid (nonanoic acid), C18:1 fatty acid (oleic acid), C22 fatty acid (docosanoic acid) and fatty acids, tall oil and in gene mutation assays in mammalian cells with C10 fatty acid (decanoic acid) and with C18 unsaturated fatty acids (linoleic and linolenic acid). Further, one exposure related observation study in humans is included which analyses the oxidative DNA damage after dietary supplementation with C18:2 fatty acid (linoleic acid) in peripheral blood lymphocytes. Due to the large number of available Ames tests of members of the fatty acids category only those which have been performed with the target substance octanoic acid as well as with the source substances hexanoic acid and nonanoic acid were summarised and presented.

Gene mutation in bacteria

There are two studies available analysing the genetic toxicity of octanoic acid in bacteria similar to OECD guideline 471.

In a bacterial gene mutation assay octanoic acid was tested in the S. typhimurium strains TA 97, TA 98, TA 100, TA1535 and TA1537 with and without metabolic activation (NTP, 1985). Using the preincubation method 10, 33, 100, 333 and 1000 µg octanoic acid/plate with 10% metabolic activation (S9) did not reveal any genotoxicity. Also concentrations of 33, 100, 333, 1000 and 3333 µg/plate with 30% metabolic activation (S9) did not reveal any genotoxicity. Also when conducted without metabolic activation, concentrations of up to 3333 µg/plate did not reveal any increase in the mean number of revertants compared to the control. For strains TA 98, TA 100, TA1535 and TA1537, no cytotoxicity was observed but the test was conducted up to precipitating concentrations. For strain TA 97 slight cytotoxicity was noted without metabolic activation at 1000 and 3333 µg/plate.

In another study, S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 were incubated with octanoic acid in concentrations of 4, 20, 100, 500 and 2500 µg/plate with and without metabolic activation (1981). Octanoic acid did not induce gene mutation in bacteria. This study was insufficient for assessment due to limited documentation. 

Since the available Ames tests with octanoic acid did not use a strain with an AT base pair at the primary reversion site, further studies with the source substances hexanoic acid (CAS# 142-62-1) and nonanoic acid (CAS# 112-05-0) were considered in the assessment of genetic toxicity.

In an Ames test, hexanoic acid was tested in the S. typhimurium strains TA 98 and TA 100 and in E. coli WP2 uvr A pKM 101, which reflects a bacteria strain with an AT base pair at the primary reversion site (NTP, 2005). Using the preincubation method 1000, 2500, 5000, 7500 and 10000 µg hexanoic acid/plate with and without metabolic activation did not reveal any genotoxicity. No cytotoxicity was observed up to 10000 µg/plate.

The genetic toxicity of nonanoic acid was tested in an Ames test similar to OECD guideline 471 in the S. typhimurium strains TA 98 and TA 100 and in E. coli WP2 uvr A pKM 101, which reflects a bacteria strain with an AT base pair at the primary reversion site (NTP, 2009). A second not further specified E.coli strain was also tested. Using the preincubation method 500, 1000, 2500, 3500 and 5000 µg nonanoic acid/plate with and without metabolic activation did not reveal any genotoxicity in two independent experiments. Cytotoxicity was observed at 3500 µg/plate.

Thus, based on these results octanoic acid is considered not mutagenic in bacteria.

Cytogenicity in mammalian cells

Nonanoic acid (CAS# 112-05-1) was tested for its ability to induce chromosomal aberrations in cultured human peripheral lymphocytes in a study conducted according to OECD guideline 473 and under GLP conditions (2001). Following a range finding pre-test, two independent experiments were conducted, both with and without metabolic activation (S9 mix; contained 9000 g supernatant from Aroclor 1254-induced male Wistar rat liver). The test concentrations in the definitive test ranged from 100 to 750 µg/mL. Precipitation was seen at 520 and 750 µg/mL. The pH at the highest non-precipitating dose level of 480 µg/mL was lowered to 7.34, compared to pH=7.48 of the solvent control. A mitotic index below 50% of the control indicated cytotoxicity at 750 µg/mL in the first experiment and at 240 µg/mL and above in the second.

A statistically significantly increased number of cells with chromosome aberrations was only seen at the toxic concentration of 750 µg/mL (mitotic index 38%, with and without metabolic activation), which was considered to be not biologically relevant. The positive controls showed the expected increase in the rate of chromosome aberrations, thus indicating the sensitivity of the assay.

Therefore, it was concluded that nonanoic acid did not induce chromosomal aberrations and was not clastogenic in human lymphocytes with and without metabolic activation.

 

A further in vitro mammalian chromosome aberration test was conducted with docosanoic acid (CAS# 112-85-6) in accordance with GLP and OECD guideline 473 and Japanese Guidelines for Screening Mutagenicity Testing of Chemicals (2002). Properly maintained Chinese hamster lung (CHL) cells were treated with docosanoic acid dissolved in 1% carboxymethylcellulose sodium at concentrations of 875, 1750 and 3500 µg/mL for 6 hours with and without metabolic activation by S9 mix prepared from phenobarbital- and 5,6-benzoflavone-induced rat livers. The highest test concentration of 3500 µg/mL reflect 0.01 M of the test substance as required in the in OECD guideline 473. In addition, the cells were incubated with 350, 700, 1400, 2800 µg/mL without metabolic activation for 24 hours and with 288, 575, 1150 and 2300 µg/mL without metabolic activation for 48 hours, respectively. The highest concentration of the test item used was set to the maximum one showing no apparent cytotoxic effects during continuous treatment. No increase in chromosomal aberrations nor polyploidy were observed up to the maximum concentration under short-term and continuous treatment with and without metabolic activation.The positive controls included during short-term and continuous exposure showed the expected results and thus verified the sensitivity of the assay.

 

Fatty acids, tall oil (CAS# 61790-12-3), which consists predominantly of C18 unsaturated and saturated fatty acids was tested for clastogenic activity in a chromosome aberration test according to OECD guideline 473 (2001).Chinese hamster ovary (CHO) cells were incubated with S9 mix at concentrations of 5, 10 and 20 µg fatty acids, tall oil/mL and without S9 mix at concentrations of 39, 78 and 156 µg fatty acids, tall oil/mL for 6 hours. Cells were harvested at 24 hours post-treatment. Chromosome aberrations were induced at cytoxic concentrations of 20 µg/mL with S9 mix and 156µg/mL without S9 mix.At these concentrations the cell count was reduced to ≤17% of the mean vehicle control values and there was consistent evidence of changes to cell morphology at 156 µg/mL without S9 mix.No chromosomal aberrations were observed at concentrations that were not cytotoxic.The increase in aberrant cells at the overtly toxic concentrations is considered as artefact and to be not biologically relevant. The positive controls substances cyclophosphamide and methyl methanesulphonate significantly increased the rate of chromosome aberrations indicating the sensitivity of the assay. Therefore, fatty acids, tall oil is considered not to be clastogenic.

 

The following study also indicates, that fatty acids do not have a genotoxic potential although not performed according to current guidelines.

 

Oleic acid (CAS# 112-80-1) was tested for the induction of sister chromatid exchanges in Indian muntjac fibroblasts (Higgins et al., 1999). The cells were incubated with 50 µM oleic acid in ethanol (equivalent to 14.1 µg/mL) for 24 h without metabolic activation. The cells were washed free of substance and cultured in the presence of BrdU for 48 h. Colcemid was added 3 h prior to harvesting. Chromosome preparations were made and stained with fluorescent plus Giemsa. The frequency of SCE was scored as the number of exchanges in 20 metaphases per slide and expressed as the number of SCEs/chromosome. Oleic acid did not induce increases in SCE frequencies above control levels and was therefore not considered genotoxic. No cytotoxicity was observed and the positive controls included showed the expected results.

 

In summary, no structural chromosomal aberrations were observed at non-cytotoxic concentrations for different members of the category and consequently fatty acids are considered not to be clastogenic.

 

Gene mutation in mammalian cells

An in vitro mammalian cell gene mutation assay was performed with decanoic acid (CAS# 334-48-5) under GLP according to OECD guideline 476 (2010). In two independent experiments, mouse lymphoma L5178Y cells were treated with decanoic acid at concentrations up to 1.18 mM without metabolic and up to 1.54 mM with metabolic activation by phenobarbital and beta-naphthoflavone-induced rat liver S9-mix, respectively. The exposure duration was 4 hours and 24 hours in experiments without S9 mix and 4 hours in the experiments with S9 mix. The treatment of cells in all experiments was followed by an expression period of 2 days and a selection period of 11-14 days in the presence of trifluorothymidine. Although cytotoxicity was observed at the highest concentrations tested, all mutant values were found to be within the range of the historical control data of the test facility, so that decanoic acid was regarded not to be mutagenic. In addition, colony sizing was performed for the highest concentrations used to detect potential clastogenic effects and/or chromosomal aberrations. As result, decanoic acid was found not to be clastogenic at all dose groups tested. The positive controls caused a pronounced increase in the mutation frequency demonstrating the sensitivity of the test system.

 

Another mouse lymphoma TK+/-assay was performed with linoleic acid (CAS# 60-33-3) and linolenic acid (CAS# 463-40-1) similar to OECD guideline 476 (Seifried et al., 2006). The applied test concentrations were very low and covered only a small dose range due to the insolubility of the test substances. Mouse lymphoma L5178Y cells were exposed for 4 hours to linoleic acid in a concentration range of 0.005 - 0.024 µL/mLwithout metabolic activation and to linoleic concentrations of 0.01 - 0.006 µL/mL with metabolic activation. Linolenic acid was tested in a concentration range of 0.021 - 0.025 µL/mL without S9 mix and in a range of 0.01 - 0.041 µL/mL with S9 mix. 2 days after treatment, treated cells were plated in soft agar medium containing TFT for 10 – 12 days. Cytotoxicity was observed for linoleic acid at the highest tested concentration with S9 mix (0.006 µL/mL). The relative total growth for linolenic acid was decreased below 50% starting at 0.024 µL/mL without S9 mix and at 0.041 µL/mL with S9 mix. No increase in mutation frequency was observed for linoleic acid and linolenic acid compared to the control. Therefore, both C18 unsaturated fatty acids were considered not to be mutagenic in mammalian cells.

In summary, no gene mutation in mammalian cells was detected for different members of the category and consequently fatty acids are considered to be not genotoxic in vitro.

 

Investigations in humans

De Kok et al. (2003) analysed the oxidative DNA damage of linoleic acid after human dietary supplementation. Female volunteers (10/group), aged between 18 and 25 years, received either a high amount of linoleic acid (15 g linoleic acid/day), an intermediate amount of linoleic acid (7.5 g linoleic acid/day + 7.5 g palmitic acid/day) or a suspension containing only palmitic acid (15 g palmitic acid/day) for a period of 6 weeks. The average plasma linoleic concentration was significantly increased after two weeks and persisted until the end of the study in the two linoleic acid supplemented groups. No significant increase in oxidative DNA damage, measured as relative amount of 8-oxodG in DNA from peripheral lymphocytes, was noted in both high and intermediate linoleic acid-supplemented groups (increase of respectively 13 and 21%; P>0.05) comparing the average level of oxidative DNA damage before and after supplementation. Moreover, the differences between levels of oxidative DNA damage in the high or intermediate linoleic acid-supplemented group and the control group (23% decrease) were not significant. Additionally, no depletion of plasma antioxidants (alpha-tocopherol, retinol and beta-carotene) or total antioxidant status (TEAC) and no increase of plasma malondialdehyde, an important end product of lipid peroxidation were observed after the linoleic acid supplementation. Thus, based on this study, there is no indication of increased oxidative stress or genetic damage as a result of increased dietary intake of linoleic acid.

 

Conclusion

Taking all the results together, the negative results of the available study data with different members of the fatty acids category do not provide any evidence that fatty acids are mutagenic or cytogenic as expected based on their physiological function within the body.

 

 

 References

de Kok, T.M.C.M. et al.(2003). Analysis of oxidative DNA damage after human dietary supplementation with linoleic acid. Food and Chemical Toxicology 41:351-358.

Higgins, S. et al. (1999). Effects of oleic acid, docosahexanoic acid and eicosapentaenoic acid on background and genotoxin-induced frequencies of SCEs in Indian muntjac fibroblasts. Mutagenesis 14(3):335 – 338.

Justification for classification or non-classification

All available data on genetic toxicity of the members of the fatty acids category do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.