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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
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
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. QA statement)
Remarks:
Direccion General de Farmacia y Productos Sanitarios, Consejera de Sanidad y Consumo, Madrid, Spain
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2-octyldodecyl myristate
EC Number:
245-205-8
EC Name:
2-octyldodecyl myristate
Cas Number:
22766-83-2
Molecular formula:
C34H68O2
IUPAC Name:
2-octyldodecyl myristate
Test material form:
liquid

Method

Target gene:
his operon
Species / strainopen allclose all
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. Purity of test substance was considered 100%.
Density of liquid test material was 0.854, no cytotoxicity was seen.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: no data
Controls
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.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
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.

Any other information on results incl. tables

Table 1. 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 2. 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

Applicant's summary and conclusion

Conclusions:
CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.