2-methyldecan-1-al

Genetic toxicity: in vitro

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

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

Test material information

Constituent 1

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 / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

not applicable

Species / strain 2

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 results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

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±2^MR	11±2	11±1^MR	66±4^MR	47±7
		333μg	7±3^MR	10±4	8±2^MR	30±6^MR	41±9
		1000μg	5±2^MR	10±3^R	7±1^MR	20±2^MR	45±4
		2500μg	3±2^MR	4±1^MR	6±1^MR	13±4^MR	47±9
		5000μg	1±0^MR	1±1^MR	3±1^MR	12±2^MR	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±1^MR	25±1	40±6	22±7	47±5
		1000μg	6±1^MR	11±3^MR	12±2^MR	10±2^MR	57±13
		2500μg	3±1^MR	7±1^MR	8±3^MR	7±1^MR	37±10
		5000μg	1±1^MR	4±1^MR	2±1^MR	4±1^MR	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±4^MR	4±1^MR	11±3^MR	48±8^MR	34±4
		333μg	8±2^MR	3±1^MR	10±3^MR	42±3^MR	43±0
		1000μg	3±1^MR	2±1^MR	6±2^MR	21±4^MR	36±7
		2500μg	3±1^MR	1±0^MR	2±1^MR	3±2^MR	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±1^MR	3±1 ^MR	4±1^MR	7±2^MR	57±6
		1000μg	0±1^MR	1±1^MR	0±1^MR	2±1^MR	43±11
		2500μg	0±1^MR	1±1^MR	0±0^MR	1±1^MR	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.