Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD 471): negative in S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvrA and WP2 uvrA pKM 101 with and without metabolic activation

Chromosome aberration test (OECD 473): negative in Chinese hamster lung fibroblasts (V79) with and without metabolic activation

Gene mutation in mammalian cells (OECD 476): negative in Chinese hamster lung fibroblasts (V79) with and without metabolic activation

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
30 Jul - 07 Aug 2007
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
The analytical purity of the test substance was not reported.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted Jul 1997
Deviations:
yes
Remarks:
analytical purity of the test substance was not reported
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
analytical purity of the test substance was not reported
GLP compliance:
yes (incl. certificate)
Remarks:
Direccion General de Farmacia y Productos Sanitarios, Consejera de Sanidad y Consumo, Madrid, Spain
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:
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 rats treated with Aroclor 1254
Test concentrations with justification for top dose:
0.05, 0.15, 0.5, 1.5, 5 µL/plate, with and without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: no data
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
solvent type not reported
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-nitrofluorene, sodium azide, 9-aminoacridine, 4-nitroquinoline-N-oxide, 2-aminoanthracene
Remarks:
See 'Any other information on material and methods incl. tables' for details on positive controls
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); if the first assay result was negative the second assay was performed as a preincubation assay, and if the first assay result was positive the second assay was performed as a plate incorporation assay

DURATION
- Preincubation period: 20 min (second assay)
- Exposure duration: 48 hrs

NUMBER OF REPLICATIONS: 3 replications in 2 independent assays

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
A test substance is considered positive (mutagenic) in the test if:
It induces a dose-resonse in the range tested and/or
a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with ot without mutagenic activation is observed.
Statistics:
Mean values and standard deviation were calculated.
Key result
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no precipitation was observed at any dose level, with and without metabolic activation.

COMPARISON WITH HISTORICAL CONTROL DATA: yes, the negative control and test substance results fell within the historical control data range (see Table 1 under 'Any other information on material and methods incl. tables').

ADDITIONAL INFORMATION ON CYTOTOXICITY: no cytotoxicity was observed up to and including the highest dose level, which is the limit dose level recommended in the OECD 471 guideline.

OTHER: Only 2-amino anthracene was used as a positive control, however, dilutions of the sample S9-mix were tested and confirmed to activate benzo(a)pyrene and 2-amino anthracene.

Table 3. Test results of experiment 1 (plate incorporation method)

With or without S9-Mix

Test substance concentration

(μL/plate)

Mean number of revertant colonies per plate

(average of 3 plates ± Standard deviation)

Base-pair substitution type

Frameshift type

TA 100

TA1535

WP2 uvR

TA98

TA1537

-

Solvent

49.7 ± 26.1

14.7 ± 1.5

76.0 ± 8.2

14.7 ± 2.5

27.3 ± 5.5

-

0.05

41.0 ± 4.0

14.7 ± 3.8

77.3 ± 22.9

21.3 ± 3.8

28.0 ± 8.9

-

0.15

37.7 ± 3.8

11.0 ± 1.0

59.7 ± 7.2

16.0 ± 7.1

29.0 ± 3.0

-

0.5

36.3 ± 6.8

13.3 ± 3.1

59.7 ± 4.9

20.0 ± 4.6

40.0 ± 21.0

-

1.5

42.7 ± 4.7

12.0 ± 2.6

55.7 ± 21.1

16.3 ± 4.9

34.3 ± 9.6

-

5.0

47.3 ± 10.1

10.3 ± 2.1

43.3 ± 18.1

17.0 ± 1.0

37.3 ± 8.6

Positive controls, –S9

Name

NaN3

NaN3

4 -NNO

2-NF

9-AA

Concentrations positive controls

(μg/plate)

10

30

7

90

100

Mean No. of colonies/plate

(average of 3 ± SD)

556.3 ± 55.2

184.7 ± 46.8

610.0 ± 30.3

399.3 ± 25.0

215.0 ± 1.0

+

Solvent

44.0 ± 2.0

15.7 ± 7.4

71.7 ± 14.7

20.3 ± 4.7

8.7 ± 3.1

+

0.05

52.0 ± 6.1

14.3 ± 7.8

90.7 ± 17.5

27.7 ± 1.5

10.0 ± 5.6

+

0.15

67.0 ± 14.7

15.3 ± 5.1

76.0 ± 39.9

27.0 ± 5.3

5.7 ± 2.1

+

0.5

47.0 ± 10.1

15.7 ± 3.5

87.3 ± 29.9

26.3 ± 5.0

8.7 ± 0.6

+

1.5

49.7 ± 13.3

10.7 ± 1.2

80.7 ± 24.0

21.7 ± 4.5

9.0 ± 2.6

+

5.0

61.3 ± 9.0

10.7 ± 2.5

64.7 ± 29.7

19.7 ± 4.7

7.0 ± 4.6

Positive controls, +S9

Name

2-AA

2-AA

2-AA

2-AA

2-AA

Concentrations

(μL/plate)

10

30

7

10

30

Mean No. of colonies/plate

(average of 3 ± SD)

305.0 ± 41.6

326.0 ± 10.8

320.0 ± 6.2

611.3 ± 108.1

320.0 ± 6.2

2-NF: 2-nitrofluorene

NaN3: sodium azide

9-AA: 9-aminoacridine

4-NNO: 4-nitroquinoline-N-oxide

2-AA: 2-aminoanthracene

 

Table 4. Test results of experiment 2 (preincubation method)

With or without S9-Mix

Test substance concentration

(μL/plate)

Mean number of revertant colonies per plate

(average of 3 plates ± Standard Deviation )

Base-pair substitution type

Frameshift type

TA 100

TA1535

WP2 uvR

TA98

TA1537

-

Solvent

64.3 ± 5.1

9.7 ± 5.5

74.7 ± 19.0

22.7 ± 6.5

9.7 ± 3.2

-

0.05

39.0 ± 1.0

12.3 ± 5.8

113.0 ± 15.7

23.3 ± 3.1

11.3 ± 3.1

-

0.15

44.3 ± 4.2

12.3 ± 5.0

103.7 ± 17.6

24.0 ± 2.0

11.0 ± 3.5

-

0.5

38.7 ± 14.2

9.0 ± 6.9

116.3 ± 10.8

22.7 ± 2.1

10.7 ± 1.5

-

1.5

34.0 ± 3.0

16.7 ± 4.9

107.3 ± 10.1

29.3 ± 7.6

11.7 ± 5.5

-

5.0

43.3 ± 10.7

10.0 ± 2.6

116.7 ± 10.1

19.3 ± 9.9

11.0 ± 5.2

Positive controls, –S9

Name

NaN3

NaN3

4-NNO

2-NF

9-AA

Concentrations

(μL/plate)

10

30

7

90

100

Mean No. of colonies/plate

(average of 3 ± SD)

580.0 ± 8.2

504.0 ± 27.6

810.0 ± 77.3

544.3 ± 417.5

682.7 ± 91.5

+

Solvent

47.0 ± 9.8

17.7 ± 5.5

45.3 ± 5.9

15.0 ± 4.0

19.3 ± 9.1

+

0.05

52.0 ± 6.6

18.0 ± 3.0

74.0 ± 5.6

17.3 ± 3.8

25.7 ± 1.5

+

0.15

51.3 ± 9.0

13.0 ± 7.9

80.0 ± 6.1

18.0 ± 4.4

24.7 ± 8.7

+

0.5

58.7 ± 8.5

16.0 ± 5.0

63.7 ± 14.5

17.7 ± 1.5

18.7 ± 3.1

+

1.5

58.7 ± 4.2

19.0 ± 6.2

76.0 ± 18.0

14.0 ± 3.6

18.3 ± 6.0

+

5.0

56.3 ± 4.1

15.7 ± 3.1

78.7 ± 26.1

16.0 ± 4.4

32.0 ± 6.9

Positive controls, +S9

Name

2-AA

2-AA

2-AA

2-AA

2-AA

Concentrations

(μL/plate)

10

30

7

10

30

Mean No. of colonies/plate

(average of 3 ± SD)

352.7 ± 62.0

284.7 ± 11.7

475.7 ± 84.4

299.7 ± 79.6

161.7 ± 27.5

2-NF: 2-nitrofluorene

NaN3: sodium azide

9-AA: 9-aminoacridine

4-NNO: 4-nitroquinoline-N-oxide

2 -AA: 2 -aminoanthracene

Conclusions:
CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
11 - 20 Mar 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
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:
E. coli WP2 uvr A
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:
10, 33, 100, 333 and 1000 µg/plate, with and without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
sodium azide
methylmethanesulfonate
other: daunomycine, 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 replications in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (reduction of bacterial background lawn, increase in size of the microcolonies, reduction of the revertant colonies)

OTHER:
the results of the dose range-finding test were included as part of experiment 1
Evaluation criteria:
A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following
criteria:
a) The negative control data (number of spontaneous revertants per plate) should be within the laboratory background historical range for each tester strain (see Table 1)
b) The positive control chemicals should produce responses in all tester strains which are within the laboratory historical range documented for each positive control substance. Furthermore, the mean plate count should be at least two times the concurrent vehicle control group mean
c) The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation
b) The negative response should be reproducible in at least one independently repeated experiment.
A test substance is considered positive (mutagenic) in the test if:
a) It induces at least a 2-fold, dose related increase in the number of revertants compared to the solvent control value in any of the tester strains, either with or without metabolic activation. However, any plate with a mean plate count of less than 20 colonies is considered to be not significant and the result will be disregarded.
b) The positive response should be reproducible in at least one independently repeated experiment.
The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.
Key result
Species / strain:
S. typhimurium, other: 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:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: precipitation was observed in all strains, with and without metabolic activation, from 1000 µg/plate and above.

RANGE-FINDING/SCREENING STUDIES:
A dose range-finding test with strain TA100 and the WP2uvrA strain (both with and without S9- mix) was performed to select suitable doses for the main experiments. Eight concentrations (3, 10, 33, 100, 333, 1000, 3330, 5000 µg/plate) were tested in triplicate. The results were reported as a part of the first experiment of the mutation assay. The highest concentration of the test substance used in the main experiments was the level at which the test substance exhibited limited solubility. Precipitation was observed on the plates from 1000 µg/plate and above in both strains.

COMPARISON WITH HISTORICAL CONTROL DATA: yes, the spontaneous mutation rate of each tester strain per plate were within the characteristic spontaneous mutation range (see Table 1 under 'Any other information on materials and methods incl. tables'), with the exception of TA 100 without metabolic activation. The value was slightly outside the limit of the range, therefore the validity of the test was considered not to be affected.

ADDITIONAL INFORMATION ON CYTOTOXICITY: no cytotoxicity was observed at any concentration level, with or without metabolic activation (see Table 2 and 3).

Table 2: Results of experiment 1 

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates ± SD)

Base-pair substitution type

Frameshift type

TA 100

TA 1535

WP2 uvrA

TA 98

TA 1537

-S9

ethanol

55 ± 3

12 ± 3

14 ± 6

16 ± 2

6 ± 1

-S9

3

55 ± 6

-

11 ± 2

-

-

-S9

10

63 ± 7

10 ± 4

10 ± 3

15 ± 6

7 ± 2

-S9

33

49 ± 7

8 ± 3

11 ± 1

16 ± 1

7 ± 4

-S9

100

59 ± 2

8 ± 1

12 ± 2

15 ± 3

5 ± 3

-S9

333

57 ± 7

7 ± 2

11 ± 6

17 ± 4

5 ± 3

-S9

1000 SP

70 ± 6

10 ± 3

10 ± 2

16 ± 1

4 ± 1

-S9

3300 SP

77 ± 9

-

12 ± 2

-

-

-S9

5000 SP

59 ± 13

-

10 ± 4

-

-

Positive controls, –S9

Name

MMS

SA

4-NQO

DM

9-AC

Concentrations

(μg/plate)

650

1

10

4

60

 

529 ± 23

121 ± 18

291 ± 26

375 ± 36

185 ± 66

 

 

 

 

 

 

 

+S9

ethanol

71 ± 3

8 ± 2

10 ± 1

26 ± 2

8 ± 1

+S9

3

66 ± 10

 

11 ± 4

 

 

+S9

10

62 ± 3

9 ± 2

9 ± 4

25 ± 4

8 ± 3

+S9

33

64 ± 3

9 ± 3

14 ± 3

22 ± 2

6 ± 2

+S9

100

50 ± 8

9 ± 3

15 ± 3

24 ± 5

6 ± 2

+S9

333

45 ± 5

13 ± 4

10 ± 3

18 ± 4

6 ± 4

+S9

1000 SP

54 ± 8

12 ± 4

12 ± 6

23 ± 5

8 ± 1

+S9

3330 SP

54 ± 9

-

12 ± 1

-

-

+S9

5000 SP

51 ± 9

-

12 ± 2

-

-

Positive controls, +S9

Name

2-AA

2-AA

2-AA

2-AA

2-AA

Concentrations

(μg/plate)

1

2.5

5

2.5

2.5

 

1182 ± 90

240 ± 8

88 ± 10

1047 ± 57

713 ± 149

MMS = methylmethanesulfonate

SA = sodium azide

4-NQO = 4-nitroquinoline-N-oxide

9-AC = 9-aminoacridine

DM = daunomycine

2-AA = 2-aminoanthracene

SP = slight precipitate

 

Table 3: Results of experiment 2

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates ± SD)

Base-pair substitution type

Frameshift type

TA 100

TA 1535

WP2 uvrA

TA 98

TA 1537

-S9

ethanol

88 ± 10

10 ± 2

10 ± 6

22 ± 7

5 ± 2

-S9

10

94 ± 13

9 ± 3

9 ± 2

17 ± 2

7 ± 3

-S9

33

93 ± 3

8 ± 3

12 ± 2

17 ± 3

5 ± 1

-S9

100

90 ± 7

10 ± 3

9 ± 2

14 ± 5

5 ± 3

-S9

333

90 ± 9

6 ± 3

11 ± 3

15 ± 6

5 ± 1

-S9

1000 SP

77 ± 5

6 ± 3

9 ± 4

14 ± 4

7 ± 4

Positive controls, –S9

Name

MMS

SA

4-NQO

DM

9-AC

Concentrations

(μg/plate)

650

1

10

4

60

 

714 ± 49

152 ± 10

936 ± 118

598 ± 16

251 ± 100

 

 

 

 

 

 

 

+S9

ethanol

99 ± 8

8 ± 4

11 ± 7

15 ± 1

7 ± 3

+S9

10

98 ± 10

8 ± 3

7 ± 1

17 ± 3

4 ± 2

+S9

33

103 ± 21

6 ± 3

8 ± 2

27 ± 6

7 ± 1

+S9

100

98 ± 9

8 ± 3

13 ± 7

21 ± 7

6 ± 2

+S9

333

93 ± 6

7 ± 5

8 ± 3

20 ± 5

8 ± 2

+S9

1000 SP

112 ± 2

9 ± 3

8 ± 1

23 ± 1

4 ± 1

Positive controls, +S9

Name

2-AA

2-AA

2-AA

2-AA

2-AA

Concentrations

(μg/plate)

1

2.5

5

2.5

2.5

 

2235 ± 60

322 ± 15

406 ± 20

1732 ± 115

703 ± 41

 MMS = methylmethanesulfonate

SA = sodium azide

4-NQO = 4-nitroquinoline-N-oxide

9-AC = 9-aminoacridine

DM = daunomycine

2-AA = 2-aminoanthracene

SP = slight precipitate

 

Conclusions:
CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
08 Mar - 07 Apr 1994
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)
Version / remarks:
adopted May 1983
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Hessisches Ministerium für Umwelt, Energie und Bundesangelegenheiten, Wiesbaden, Germany
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not applicable
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: minimal essential medium (MEM) supplemented with 10 % fetal calf serum (FCS)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: no
- doubling time of clone V79/T5 in stock cultures: 12 h
Metabolic activation:
with and without
Metabolic activation system:
Co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with 500 mg/kg bw Aroclor 1254 i.p. 5 days prior to sacrifice.
Test concentrations with justification for top dose:
Experiment 1, chromosome aberration and mitotic index:
4 h treatment, 18 h harvest time, without metabolic activation: 10, 60 and 100 μg/mL
4 h treatment, 28 h harvest time, without metabolic activation: 100 μg/mL
18 h treatment, 18 h harvest time, with metabolic activation: 10, 60 and 100 μg/mL
28 h treatment, 28 h harvest time, with metabolic activation: 100 μg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol. The final concentration of ethanol in the culture medium did not exceed 1 % v/v.
- Justification for choice of solvent/vehicle: the solvent was chosen due to its solubility properties and its non-toxicity to the cells.
Untreated negative controls:
yes
Remarks:
culture medium
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
other:
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4h with metabolic activation, 18 and 28 h without metabolic activation
- Expression time (cells in growth medium): Preparation interval was 18 and 28 hours for all experiments. Therefore, for the cells exposed for 4 h with metabolic activation, the medium was changed to complete medium after the exposure for the remaining time up to preparation. The cells treated without metabolic activation were kept for the whole exposure duration of 18 or 28 hours in medium containing the test substance.
- Fixation time (start of exposure up to fixation or harvest of cells): 2.5 h after adding colcemid (at 18 and 28 h)

SPINDLE INHIBITOR (cytogenetic assays): 15.5 and 25.5 h after the start of the treatment colcemid was added (0.2 µg/mL)
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicates

NUMBER OF CELLS EVALUATED: at least 100 well spread metaphases per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes, the number of polyploid cells was scored (% polyploid metaphases)
Evaluation criteria:
The test substance is considered as mutagenic, if it includes either a concentration-related increase in the number of structural chromosomal aberrations or a significant positive response for at least one of the test points.
The test substance neither producing a concentration-related increase in the number of structural chromosomal aberrations nor a significant and positive response at any of the test points is considered non-mutagenic in this system.
Statistics:
Chi-square test at five percent level (p < 0.05)
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Due to limited solubility of the test substance higher concentrations than 100 µg/mL for the cytogenetic evaluation were not feasible.
- Precipitation: observed at concentrations higher than 100 µg/mL

RANGE-FINDING/SCREENING STUDIES/ADDITIONAL INFORMATION ON CYTOTOXICITY:
A pre-test was performed to determine the toxicity of the test article, however the result could only be assessed qualitatively. The highest attainable concentration was 100 µg/mL, at which slight precipitation occured. No cytotoxicity was observed.

TEST CONCENTRATIONS
6 test concentrations (1, 3, 10, 30, 60 and 100 µg/mL) were listed in the study report under 'materials and method', while the results of 3 concentrations (10, 60 and 100 µg/mL) were reported.

COMPARISON WITH HISTORICAL CONTROL DATA: yes (data not presented)

In the absence and presence of S9-mix, the mitotic indices were not substantially reduced after treatment with the highest evaluated concentration at each fixation interval. In the cytogenetic experiment, at both fixation intervals in the absence and presence of S9-mix, the test material did not increase the frequency of cells with aberrations. No biologically relevant increase of polyploid metaphases as compared to the rates of the controls was found after treatment with the test material.The positive controls were valid (see Table 1 and 2) .

Table 1: Number of Polyploid Cells and Mitotic Index

 

conc.

per mL

Exposure

duration

(hours)

S9

mix

fixation

interval

mean number

of polyploid cells*

mitotic

index**

(%)

neg. control

 

4

-

18

1.5

100.0

vehicle control

1.0 %

4

-

18

1.5

100.0

EMS

600 μg

4

-

18

3.0

68.9

test substance

10 μg

4

-

18

1.0

83.4

test substance

60 μg

4

-

18

2.5

100.0

test substance

100 μg

4

-

18

2.0

89.7

neg. control

 

18

+

18

2.0

100.0

vehicle control

1.0 %

18

+

18

3.0

100.0

CPA

0.47 μg

18

+

18

3.0

63.0

test substance

10 μg

18

+

18

1.5

127.1

test substance

60 μg

18

+

18

2.0

113.7

test substance

100 μg

18

+

18

2.0

104.3

vehicle control

1.0 %

4

-

28

4.0

100.0

test substance

100 μg

4

-

28

5.0

119.0

vehicle control

1.0 %

28

+

28

2.5

100.0

test substance

100 μg

28

+

28

2.5

91.5

* The number of polyploid cells was determined in 100 cells per culture of each test group.

** The mitotic index was determined in 1000 cells per culture of each test group.

 

Table 2: Structural chromosomal aberrations

 

conc.

per mL

S9

mix

fixation

interval

cells scored

aberrant cells (% mean)

exchanges

incl.

gaps

excl.

gaps

neg. control

 

-

18

200

0.0

0.0

0.0

vehicle control

1.0 %

-

18

200

1.5

0.5

0.0

EMS

600 μg

-

18

200

23.5

22.0

16.5

test substance

10 μg

-

18

200

1.5

0.5

0.0

test substance

60 μg

-

18

200

1.0

1.0

1.0

test substance

100 μg

-

18

200

5.0

3.0

0.0

neg. control

 

+

18

200

1.0

1.0

0.0

vehicle control

1.0 %

+

18

200

4.0

3.5

0.5

CPA

0.47 μg

+

18

200

23.0

22.0

11.0

test substance

10 μg

+

18

200

3.0

1.0

0.0

test substance

60 μg

+

18

200

2.0

2.0

1.5

test substance

100 μg

+

18

200

3.0

1.5

0.0

vehicle control

1.0 %

-

28

200

4.0

2.0

0.0

test substance

100 μg

-

28

200

3.5

2.0

0.0

vehicle control

1.0 %

+

28

200

4.0

2.5

0.5

test substance

100 μg

+

28

200

4.0

2.0

1.0

EMS = Ethylmethanesulphonate

CPA = Cyclophosphamide

Conclusions:
CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
08 Mar - 15 Apr 1994
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)
Version / remarks:
adopted April 1984
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 OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Hessisches Ministerium für Umwelt, Energie und Bundesangelegenheiten, Wiesbaden, Germany
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT locus
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: minimal essential medium (MEM) supplemented with 10 % fetal calf serum (FCS)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes, by treatment with HAT-medium
- Doubling time 12 - 16 h in stock cultures
Metabolic activation:
with and without
Metabolic activation system:
Co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with 500 mg/kg bw Aroclor 1254 i.p. 5 days prior to sacrifice.
Test concentrations with justification for top dose:
10, 30, 60 and 100 µg/mL, with and without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol. The final concentration of ethanol in the culture medium did not exceed 1 % v/v.
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its non-toxicity to the cells.

Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent):
Cloning efficiency: 7 days
Mutant selection: 9 and 12 days (experiments 2 and 1, respectively)
- Fixation time (start of exposure up to fixation or harvest of cells):
Cloning efficiency: 7 and 14 days
Mutant selection: 16 and 19 days (experiments 2 and 1, respectively)

Counting of colonies: the colonies were stained with 10 % methylene blue in 0.01 % KOH solution. Stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.

SELECTION AGENT (mutation assays): thioguanine, 11 µg/mL medium (Sigma, Deisenhofen, Germany)

NUMBER OF REPLICATIONS: 1 replication in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: determination of concentration-related cloning efficiency on day 7 after start of exposure; determination of cell survival on day 14 after start of exposure (end of selection time)
Evaluation criteria:
The test substance is considered to be positive if it induces either a concentration-related increase of the mutant frequency or a reproducible positive response for one of the test points.
A test material producing neither a concentration-related increase in the mutant frequency nor a reproducible positive response in any of the test points is considered non-mutagenic in this system.
A significant response is:
1) test substance induces reproducibly with one of the concentrations a mutation frequency that is 3 times higher than the spontaneous mutation frequency in the experiment
2) test substance induces reproducible concentration-related increase of the mutation frequency. May also be considered in case a 3-fold increase of the mutant frequency is not observed.

In a case-by-case evaluation the decision depends on the level of the corresponding negative control data.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
precipitation at limit of water solubility at 100 µg/mL observed.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Due to limited solubility of the test substance higher concentrations than 100 µg/mL for the cytogenetic evaluation were not feasible.
- Precipitation: observed at concentrations higher than 100 µg/mL

RANGE-FINDING/SCREENING STUDIES
Data on the pre-test were obtained from the chromosomal aberration study (IUCLID section 7.6.1: key, Emery, 1994, ChrAb, RL1)
A pre-test was performed to determine the toxicity of the test article. The test substance was dissolved in ethanol due to its solubility properties. The highest attainable concentration was 100 µg/mL, at which a slight precipitation was observed. No toxic effects were observed up to and including the highest dose level.

COMPARISON WITH HISTORICAL CONTROL DATA: yes (data not presented)

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In parallel to the mutagenicity testing the cloning efficiency was tested. Without metabolic activation the relative cloning efficiencies were between 64.2 - 91.8 % in treated cells compared to control cells. With metabolic activation the relative cloning efficiencies were between 92.4 - 109.7 % in treated cells compared to control cells.

No statistically significant increase in gene mutations was observed (see Table 1 and 2). The positive controls were shown to be valid.

Table 1: Mutagenicity data, experiment 1

 

Concentration (µg/mL)

Metabolic activation

No. of mutant colonies after plating in TG medium*

(mean of 5 flasks)

% cell survival

No. of mutant colonies per 106 surviving cells

% cloning efficiency

Negative control

-

-

2.2

54

9.4

 100.0

Vehicle control

-

-

1.0

66

3.9

 100.0

EMS

600

-

89.6

50

416.4

 73.8

Test substance

10

-

1.0

72

3.3

 91.8

Test substance

30

-

2.0

72

6.8

 72.0

Test substance

60

-

0.2

81

0.6

 68.1

Test substance

100

-

2.8

65

10.2

 64.2

Negative control

-

+

2.0

69

7.3

 100.0

Vehicle control

-

+

3.2

68

11.4

 100.0

DMBA

3.85

+

112.0

57

496.2

 53.1

Test substance

10

+

5.4

78

16.1

 92.4

Test substance

30

+

0.2

65

0.8

 95.6

Test substance

60

+

1.4

67

5.4

 97.3

Test substance

100

+

0.2

74

0.7

 109.7

*only colonies with more than 50 cells 8 days after seeding in TG selection medium were scored

** ration of mean number of cells found after 7 days in normal growth medium / cell number seeded per flask

TG = Thioguanine

EMS = Ethylmethanesulphonate

DMBA = 7,12-dimethylbenz(a)anthracene

 

Table 2: mutagenicity data, experiment 2

 

Concentration (µg/mL)

Metabolic activation

No. of mutant colonies after plating in TG medium*

(mean of 5 flasks)

% cell survival

No. of mutant colonies per 106 surviving cells

% cloning efficiency

Negative control

-

-

2.0

69

6.6

 100.0

Vehicle control

-

-

1.8

64

6.4

 100.0

EMS

600

-

145.8

60

496.9

 61.5

Test substance

10

-

4.2

62

18.4

 93.6

Test substance

30

-

1.0

63

4.1

 107.8

Test substance

60

-

3.2

60

13.5

 56.4

Test substance

100

-

0.8

59

3.5

 61.4

Negative control

-

+

2.4

60

10.3

 100.0

Vehicle control

-

+

2.2

61

9.0

 100.0

DMBA

3.85

+

141.6

64

550.4

 50.3

Test substance

10

+

1.4

61

5.7

 104.8

Test substance

30

+

2.2

72

7.5

 106.1

Test substance

60

+

0.6

69

2.1

 102.8

Test substance

100

+

1.4

79

4.4

 107.7

*only colonies with more than 50 cells 8 days after seeding in TG selection medium were scored

** ratio of mean number of cells found after 7 days in normal growth medium / cell number seeded per flask

TG = Thioguanine

EMS = Ethylmethanesulphonate

DMBA = 7,12-dimethylbenz(a)anthracene

Conclusions:
CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Analogue justification

No data on the genetic toxicity of Fatty acids, C16 and C18-22 - unsatd., C16-18 and C18 unsatd. alkyl ester (CAS 90990-29-7)are available. The assessment was therefore based on studies conducted with analogue substances as part of a read across approach, which is in accordance with Regulation (EC) No. 1907/2006, Annex XI, 1.5. For each specific endpoint the source substance(s) structurally closest to the target substance is/are chosen for read-across, with due regard to the requirements of adequacy and reliability of the available data. Structural similarities and similarities in properties and/or activities of the source and target substance are the basis of read-across. A detailed justification for the analogue read-across approach is provided in the technical dossier (see IUCLID Section 13).

Genetic toxicity (mutagenicity) in bacteria in vitro

CAS 22766-83-2

The in vitro genetic toxicity of 2-octyldodecyl myristate (CAS 22766-83-2) was assessed in a bacterial reverse mutation assay (Ames test) (Ames, 2007). The study was performed according to OECD guideline 471 and under GLP conditions. The plate incorporation method was applied in the first experiment and the preincubation method in the second experiment, using S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, and E. coli WP2 uvrA pKM 101. All the strains were tested at concentrations up to the limit value of 5 µL/plate. The test substance did not induce an increase in reversions in the S. typhimurium and E.coli strains, with or without metabolic activation. No cytotoxicity was observed, with and without metabolic activation. The vehicle and positive controls were valid.

CAS 93803-87-3

The in vitro genetic toxicity of 2-octyldodecyl isooctadecanoate (CAS 93803-87-3) was assessed in a bacterial reverse mutation assay (Ames test), performed according to OECD 471 and in compliance with GLP (Ames, 1998). S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, and E. coli WP2 uvrA were exposed to the test substance at concentrations up to 1000 µg/plate (TA 1535, TA 98 and TA 1537) and 5000 µg/plate (TA 100 and E.coli WP2 uvrA), using the plate incorporation method in two independent experiments. Precipitation was observed in the medium at 1000 µg/plate and above in all strains, with and without metabolic activation. The positive and solvent controls were shown to be valid. The test substance did not induce reversions in the S. typhimurium strains or E. coli strain, with or without metabolic activation.

Genetic toxicity (cytogenicity) in mammalian cells in vitro

CAS 3687-45-4

The potential of Oleyl oleate (CAS 3687-45-4) to induce chromosomal aberrations was assessed using Chinese hamster lung fibroblast (V79) cells, in a study performed according to OECD 473 and under GLP conditions (CA, 1994). The V79-cells were exposed to Oleyl oleate at concentrations up to 100 µg/mL, with and without metabolic activation (S9-mix). One experiment with duplicate replications was performed. A 4-h treatment was performed without metabolic activation, using 10, 60 and 100 µg/mL concentration levels with 18-h fixation time and a 100 µg/mL concentration level with a 28-hour fixation time. The treatment with metabolic activation was performed at concentrations of 10, 60 and 100 µg/mL with an 18-hour treatment time, and 18-h fixation time and at 100 µg/mL with a 28-h treatment time and 28-h fixation time, respectively. The test material did not induce a statistically significant increase in the frequency of cells with chromosome aberrations, with or without metabolic activation. Precipitation was observed at concentrations from 100 µg/mL, while no cytotoxicity was noted at any concentration. The vehicle and positive controls were valid.

 

Genetic toxicity (mutagenicity) in mammalian cells in vitro

CAS 3687-45-4

An in vitro mammalian cell gene mutation assay was performed using Oleyl oleate (CAS 3687-45-4), according to OECD 476 and under GLP conditions (HPRT, 1994). Two separate experiments were performed. Chinese hamster lung fibroblast (V79) cells were treated with Oleyl oleate at concentrations of up to 100 µg/mL for 4 hours, with and without metabolic activation. After an expression time of 7 days in growth medium, cells were incubated for 9 or 12 days with 6 -thioguanine as selection agent for forward mutation at the HPRT locus. Precipitation was seen at concentrations of 100 µg/mL and higher. The positive and negative controls were valid and within the range of historical control data. No cytotoxicity was observed. No significant increase in mutation frequency was observed, with and without metabolic activation.

Overall conclusion for genetic toxicity

There are no available studies on the in vitro genetic toxicity in bacteria and mammalian cells of the target substance Fatty acids, C16 and C18-22 - unsatd., C16-18 and C18 unsatd. alkyl ester (CAS 90990-29-7).Therefore analogue read-across from 3 source substances was applied from gene mutation assays in bacteria, and from in vitro studies on cytogenicity and gene mutation in mammalian cells. The results of the available in vitro studies on source substances were consistently negative. Based on the available data on target and source substances, and following the analogue approach,Fatty acids, C16 and C18-22 - unsatd., C16-18 and C18 unsatd. alkyl ester (CAS 90990-29-7) is not expected to be mutagenic and clastogenic in vitro.

Justification for classification or non-classification

According to Article 13 of Regulation (EC) No. 1907/2006 "General Requirements for Generation of Information on Intrinsic Properties of substances", information on intrinsic properties of substances may be generated by means other than tests e.g. from information from structurally related substances (grouping or read-across), provided that conditions set out in Annex XI are met. Annex XI, "General rules for adaptation of this standard testing regime set out in Annexes VII to X” states that “substances whose physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity may be considered as a group, or ‘category’ of substances. This avoids the need to test every substance for every endpoint". Since the analogue concept is applied to Fatty acids, C16 and C18-22 - unsatd., C16-18 and C18 unsatd. alkyl ester (CAS 90990-29-7), data will be generated from data for reference source substance(s) to avoid unnecessary animal testing. Additionally, once the analogue read-across concept is applied, substances will be classified and labelled on this basis.

Therefore, based on the analogue read-across approach, the available data on genetic toxicity do not meet the classification criteria according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.