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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:
key study
Study period:
24 Jun - 15 July 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted in accordance with international guidelines and in accordance with GLP. All guideline criteria were met.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2-methyldecan-1-al
EC Number:
242-745-6
EC Name:
2-methyldecan-1-al
Cas Number:
19009-56-4
Molecular formula:
C11H22O
IUPAC Name:
2-methyldecanal
Test material form:
liquid
Details on test material:
physical appearance: liquid

Method

Target gene:
S. typhimurium
TA 1537: his C 3076; rfa-; uvrB-
TA 98: his D 3052; rfa-; uvrB-; R-factor
TA 1535: his G 46; rfa-; uvrB-; R-factor
TA 100: his G 46; rfa-; uvrB-

E. coli
WP2 uvrA: trp-; uvrA-
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/β-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Pre-Experiment/Experiment I: Experiment II
(with and without S9 mix) TA1535, TA 1537, TA 98; TA100: WP2 uvrA:
3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
1; 3; 10; 33; 100; 333; 1000; and 2500 μg/plate 33; 100; 333; 1000; 2500; and 5000 μg/plate
Based on the toxic effects observed with the Salmonella strains a different concentration range was tested with 2500 μg/plate as maximum concentration. As no toxic effects were observed in strain WP2 uvrA, six concentration levels were tested and 5000 μg/plate were chosen as maximum concentration.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO (purity > 99 %)
- Justification for choice of solvent/vehicle: Chosen because of its solubility properties and its relative nontoxicity to the bacteria.
The stability of the positive control substances in solution is unknown but a mutagenic response in the expected range is sufficient evidence of biological stability.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine, 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in selective agar
- Cell density at seeding (if applicable):

DURATION
- Preincubation period: 60 minutes
- Exposure duration: not specified
- Expression time (cells in growth medium): At least 48 hours
- Selection time (if incubation with a selection agent): N/A
- Fixation time (start of exposure up to fixation or harvest of cells): N/A

SELECTION AGENT (mutation assays): The plates with selective agar were obtained from E. Merck. A solution of 20 μL ampicillin (25 μg/mL) was added to the strains TA 98 and TA 100. This nutrient medium contains per litre:
8 g Nutrient Broth 5 g NaCl

SPINDLE INHIBITOR (cytogenetic assays): N/A

STAIN (for cytogenetic assays): N/A

NUMBER OF REPLICATIONS: 3

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: N/A

NUMBER OF CELLS EVALUATED: N/A

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): N/A

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: N/A

DETERMINATION OF CYTOTOXICITY: N/A
- Method: mitotic index; cloning efficiency; relative total growth; other:
- Any supplementary information relevant to cytotoxicity:

OTHER EXAMINATIONS: N/A
- Determination of polyploidy:
- Determination of endoreplication:
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable):

- OTHER: N/A
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Tables 1 and 2 show the summary results (revertant colony counts) for Experiments I and II respectively. Table 3 demonstrates the background growth observed at tested concentrations (μg/plate). Table 4 demonstrates the toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5), observed at tested concentrations (μg/plate).

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test substance at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies.

No precipitation of the test item occurred up to the highest investigated dose.

Table 1 . Summary of experiment I

METABOLIC ACTIVATION

TEST GROUP

DOSE LEVEL PER PLATE

REVERTANT COLONY COUNTS (mean±SD)

TA 1535

TA 1537

TA 98

TA 100

WP2uvrA

Without activation

DMSO

 

14±3

10±3

26±6

106±13

45±8

Untreated

 

13±1

10±2

27±6

100±2

47±8

Aldehyd C11 MOA

3μg

16±4

13±1

28±5

99±6

48±4

10μg

15±3

9±5

21±6

99±8

46±7

33μg

12±6

8±1

16±5

76±6

47±6

100μg

7±2MR

11±2

11±1MR

66±4MR

47±7

333μg

7±3MR

10±4

8±2MR

30±6MR

41±9

1000μg

5±2MR

10±3R

7±1MR

20±2MR

45±4

2500μg

3±2MR

4±1MR

6±1MR

13±4MR

47±9

5000μg

1±0MR

1±1MR

3±1MR

12±2MR

32±3

NaN3

10μg

2173±23

-

-

2279±35

-

4-NOPD

10μg

-

-

296±18

-

-

4-NOPD

50μg

-

70±9

-

-

-

MMS

2.0 μL

-

-

-

-

876±39

With activation

DMSO

 

22±2

22±8

50±3

169±9

62±8

 

Untreated

 

18±3

19±3

51±2

172±2

56±8

Aldehyd C11 MOA

3μg

17±2

22±2

48±3

166±13

55±4

10μg

18±3

23±4

46±5

167±17

60±4

33μg

25±2

25±2

49±7

160±15

55±6

100μg

12±0

23±2

51±6

159±16

61±5

333μg

7±1MR

25±1

40±6

22±7

47±5

1000μg

6±1MR

11±3MR

12±2MR

10±2MR

57±13

2500μg

3±1MR

7±1MR

8±3MR

7±1MR

37±10

5000μg

1±1MR

4±1MR

2±1MR

4±1MR

32±7

2-AA

2.5μg

470±50

543±26

2294±10

4315±26

-

2-AA

10μg

-

-

-

-

335±18

NaN3: sodium azide
2-AA: 2-aminoanthracene
4-NOPD: 4-nitro-o-phenylene-diamine

MMS: methyl methane sulfonate

M: Manual count
R: Reduced background growth

Table 2. Summary of experiment II

METABOLIC ACTIVATION

TEST GROUP

DOSE LEVEL PER PLATE

REVERTANT COLONY COUNTS (mean±SD)

TA 1535

TA 1537

TA 98

TA 100

WP2uvrA

Without activation

DMSO

 

14±2

9±3

21±1

91±7

41±12

Untreated

 

14±2

10±4

31±5

106±8

52±8

Aldehyd C11 MOA

1μg

12±2

10±4

27±4

86±7

-

3μg

17±4

10±2

24±3

87±11

-

10μg

15±6

9±2

21±7

63±6

-

33μg

9±2

10±2

19±5

67±10

35±7

100μg

12±4MR

4±1MR

11±3MR

48±8MR

34±4

333μg

8±2MR

3±1MR

10±3MR

42±3MR

43±0

1000μg

3±1MR

2±1MR

6±2MR

21±4MR

36±7

2500μg

3±1MR

1±0MR

2±1MR

3±2MR

39±9

5000μg

-

-

-

-

28±4

NaN3

10μg

1832±106

-

-

2012±189

-

4-NOPD

10μg

-

-

326±24

-

 

4-NOPD

50μg

-

71±6

-

-

-

MMS

2.0 μg

-

-

-

-

457±22

With activation

DMSO

 

17±3

18±3

44±4

128±20

57±9

 

Untreated

 

18±5

21±10

40±8

132±22

69±2

Aldehyd C11 MOA

1μg

13±1

19±7

35±2

143±12

-

3μg

17±1

21±4

49±9

134±4

-

10μg

13±3

15±3

41±7

133±18

-

33μg

22±3

16±7

37±7

136±29

67±3

100μg

14±7

19±3

38±9

87±13

59±10

333μg

2±1MR

3±1 MR

4±1MR

7±2MR

57±6

1000μg

0±1MR

1±1MR

0±1MR

2±1MR

43±11

2500μg

0±1MR

1±1MR

0±0MR

1±1MR

37±7

5000μg

-

-

-

-

34±7

2-AA

2.5μg

387±24

332±19

2584±187

3546±307

-

2-AA

10μg

-

-

-

-

524±37

NaN3: sodium azide
2-AA: 2-aminoanthracene
4-NOPD: 4-nitro-o-phenylene-diamine

MMS: methyl methane sulfonate

M: Manual count
R: Reduced background growth

Table 3. Reduced background growth at tested concentrations (μg/plate)

STRAIN

EXPERIMENT I

EXPERIMENT II

 

 without S9 mix

with S9 mix

 without S9 mix

  with S9 mix

TA 1535

100 - 5000

333 - 5000

100 - 2500

333 - 2500

TA 1537

1000 - 5000

1000 - 5000

100 - 2500

333 - 2500

TA 98

100 - 5000

1000 - 5000

100 - 2500

333 - 2500

TA 100

100 - 5000

333 - 5000

100 - 2500 

333 - 2500

WP2 uvrA

/

/

/

/

/ = no reduced background growth observed

Table 4. Toxic effects at tested concentrations (μg/plate)

STRAIN

EXPERIMENT I

EXPERIMENT II

 

without S9 mix

with S9 mix

without S9 mix

with S9 mix

TA 1535

1000 - 5000

333 - 5000

1000 - 2500

333 - 2500

TA 1537

2500, 5000

2500, 5000

100 - 2500

333 - 2500

TA 98

100 - 5000

1000 - 5000

1000 - 2500

333 - 2500

TA 100

333 - 5000

333 - 5000

1000 - 2500

333 - 2500

WP2 uvrA

/

/

/

/

/ = no toxic effects observed

Applicant's summary and conclusion

Conclusions:
During the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Therefore, the substance can be considered non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

This study on genetic toxicity was performed to the requirements of OECD Guideline 471 (21 July 2007) and EU Method B.13/14, to investigate the potential of the test substance to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate.

Reduced background growth was observed at the higher concentrations with metabolic activation (S9 mix) in experiment I and with and without metabolic activation in both experiments.

Distinct toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed at higher concentrations with and without metabolic activation both experiments.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test substance at any concentration level, neither in the presence nor absence of metabolic activation. There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

The test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used, resulting in a non-mutagenic classification.

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