2-methyldecan-1-al

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

in vitro mutagenicity: Negative (non-mutagenic); OECD 471; Anon, 2014

in vitro gene mutation: Negative (non-mutagenic); OECD 476; Anon., 2010 (read-across to n-undecanal)

in vitro mamalian cell micronucleus: Negative (non-mutagenic); OECD 487; Anon., 2010 (read-across to n-undecanal)

Link to relevant study records

Reference

Reference 1

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.

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

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 / 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.

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.

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

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

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.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Refer to Section 13.2 for read-across justification document.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity observed at 500 μg/mL in the presence of S9 mix

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

The test item did not induce any toxicologically significant or concentration-related increases in mutant frequency per survivor in either the absence or presence of metabolic activation. The test item was therefore considered to be non-mutagenic to V79 cells at the HPRT locus under the conditions of this test.

Executive summary:

In a one-to-one read-across approach, the substance n-undecanal (source substance) is considered appropriate for direct read-across (one-to-one) to 2 -methyldecanal (target substance) for the endpoint in vitro genetic toxicity (in vitro gene mutation in mammalian cells). In conclusion, the test item did not induce any toxicologically significant or concentration-related increases in mutant frequency per survivor in either the absence or presence of metabolic activation. The test item was therefore considered to be non-mutagenic to V79 cells at the HPRT locus under the conditions of this test.. A full justification for the read-across approach is presented in IUCLID Section 13.2.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Refer to Section 13.2 for read-across justification document.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

lymphocytes: Human donor

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Excessive toxicity observed in samples treated at 300 μg/mL after 24 h

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

It is concluded that the test item did not show any evidence of genotoxic activity in this in vitro test for induction of micronuclei when tested in accordance with regulatory guidelines.

Executive summary:

In a one-to-one read-across approach, the substance n-undecanal (source substance) is considered appropriate for direct read-across (one-to-one) to 2 -methyldecanal (target substance) for the endpoint in vitro genetic toxicity (in vitro cytogenicity / micronucleus). In conclusion, the test item did not show any evidence of genotoxic activity when tested in accordance with OECD 487. A full justification for the read-across approach is presented in IUCLID Section 13.2.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 July - 21 September 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was conducted in accordance with international guidelines and in accordance with GLP. All guideline validity criteria were met.

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro mammalian cell gene mutation test using the Hprt and xprt genes (migrated information)

Specific details on test material used for the study:

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: n/a
- Specific activity: n/a
- Locations of the label: n/a
- Expiration date of radiochemical substance: n/a

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature, under inert gas
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: 0.5 % DMSO in culture medium
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: On the day of the experiment (immediately before treatment), the test item was dissolved in DMSO (purity 99.9 %).
- Preliminary purification step (if any): No
- Final dilution of a dissolved solid, stock liquid or gel: 1835.0 µg/mL in DMSO
- Final preparation of a solid: No

FORM AS APPLIED IN THE TEST (if different from that of starting material) Applied as a liquid.

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable) N/A

OTHER SPECIFICS: No

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Laboratory for Mutagenicity Testing; Technical University, 64287 Darmstadt, Germany
- Suitability of cells: The V79 cell line has been used successfully in in vitro experiments for many years. Especially the high proliferation rate (doubling time 12 - 16 h in stock cultures) and a good cloning efficiency of untreated cells (as a rule more than 50 %) both necessary for the appropriate performance of the study, recommend the use of this cell line. The cells have a stable karyotype with a modal chromosome number of 22.
- Cell cycle length, doubling time or proliferation index: Above
- Sex, age and number of blood donors if applicable: Not reported
- Whether whole blood or separated lymphocytes were used if applicable: Not reported
- Number of passages if applicable: Not reported
- Methods for maintenance in cell culture if applicable: Large stocks of the V79 cell line (supplied by Laboratory for Mutagenicity Testing; Technical University, 64287 Darmstadt, Germany) are stored in liquid nitrogen in the cell bank of Harlan CCR allowing the repeated use of the same cell culture batch in experiments. Before freezing, the level of spontaneous mutants was depressed by treatment with HATmedium. Each batch is screened for mycoplasma contamination and checked for karyotype stability and spontaneous mutant frequency. Consequently, the parameters of the experiments remain similar because of the reproducible characteristics of the cells. Thawed stock cultures are propagated at 37 °C in 80 cm2 plastic flasks. About 5×10E5 cells are seeded into each flask with 15 mL of MEM (minimal essential medium) containing Hank’s salts, neomycin (5 Pg/mL) and amphotericin B (1 %). The cells are sub-cultured twice weekly. The cell cultures are incubated at 37 °C in a 1.5 % carbon dioxide atmosphere (98.5 % air). For the selection of mutant cells the medium is supplemented with 11 µg/mL 6-thioguanine (6-TG).
- Modal number of chromosomes: 22
- Normal (negative control) cell cycle time: 12-16 h

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: For seeding and treatment of the cell cultures the complete culture medium was MEM (minimal essential medium) containing Hank’s salts, neomycin (5 Pg/mL) and amphotericin
B (1 %). For the selection of mutant cells the complete medium was supplemented with 11 µg/mL 6-thioguanine. All cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 (98.5 % air).
- 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 specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (prepared in house from 8 - 12 weeks old male Wistar rats (Hsd Cpb: WU, Harlan Laboratories GmbH, 33178 Borchen, Germany

Test concentrations with justification for top dose:

Concentrations based on the results of a preliminary test;

Exp. 1: 0.6, 1.3, 2.5, 5.0, 7.5, 10, 15 µg/mL (without S9 mix) and 7.2, 14.4, 28.8, 57.5, 115, 172.5 and 230 µg/mL (with S9 mix)
Exp. 2: 0.6, 1.3, 2.5, 5, 10, 15, 20, 25 µg/mL (without S9 mix) and 7.8, 15.6, 31.3, 62.5, 125, 187.5, 250, 500 µg/mL (with S9 mix)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to high solubility of test item in solvent.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in suspension
- Cell density at seeding (if applicable): Approximately 1.5×10E6 (single culture) and 5×102 cells (in duplicate) were seeded in MEM with 10 % FBS (complete medium) for the determination of mutation rate and toxicity, respectively.

For seeding and treatment of the cell cultures the complete culture medium was MEM (minimal essential medium) containing Hank’s salts, neomycin (5 Pg/mL) and amphotericin B (1 %). For the selection of mutant cells the complete medium was supplemented with 11 Pg/mL 6-thioguanine. All cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 (98.5 % air).

Two days (experiment I) or three days (experiment II) after sub-cultivation stock cultures were trypsinized at 37 °C for 5 minutes. Then the enzymatic digestion was stopped by adding complete culture medium and a single cell suspension was prepared. The trypsin concentration for all sub-culturing steps was 0.2 % in Ca-Mg-free salt solution (Trypsin: Difco Laboratories, Detroit, USA). The Ca-Mg-free salt solution had the following constituents (per litre):
NaCl 8000 mg
KCl 200 mg
KH2PO4 200 mg
Na2HPO4 150 mg

Prior to the trypsin treatment the cells were rinsed with Ca-Mg-free salt solution containing 200 mg/l EDTA (ethylene diamine tetraacetic acid). The cell suspension was seeded into plastic culture flasks (Greiner, 72632 Frickenhausen,
Germany). Approximately 1.5×106 (single culture) and 5×102 cells (in duplicate) were seeded in MEM with 10 % FBS (complete medium) for the determination of mutation rate and toxicity, respectively.

After 24 hours the medium was replaced with serum-free medium containing the test item, either without S9 mix or with 50 Pl/mL S9 mix. Concurrent solvent and positive controls were treated in parallel. After 4 h this medium was replaced with complete medium following two washing steps with "saline G". In the second experiment the cells were exposed to the test item for 24 h in complete medium, supplemented with 10 % FBS, in the absence of metabolic activation. The "saline G" solution had the following constituents (per litre):
NaCl 8000 mg
KCl 400 mg
Glucose 1100 mg
Na2HPO4×2H2O 192 mg
KH2PO4 150 mg

The pH was adjusted to 7.2

The colonies used to determine the cloning efficiency (survival) were fixed and stained approx. 7 days after treatment as described below. Three (experiment II) or four days (experiment I) after treatment 1.5×106 cells per experimental
point were sub-cultivated in 175 cm² flasks containing 30 mL medium. Following the expression time of 7 days five 80 cm² cell culture flasks were seeded with about 3 - 5×105 cells each in medium containing 6-TG. Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability. The cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 for about 8 days. The colonies were stained with 10 % methylene blue in 0.01 % KOH solution. The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.

Rationale for test conditions:

According to the current OECD Guideline for Cell Gene Mutation Test and based on the results of the pre-experiment.

Evaluation criteria:

A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points. A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.

A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.

However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory´s historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity observed at 500 µg/mL in the presence of S9 mix

Vehicle controls validity:

valid

Positive controls validity:

valid

Table 1       Summary of results

	Conc. (µg/mL)	P / PS	S9 mix	Relative cloning efficiency I (%)	Relative cell density (%)	Relative cloning efficiency II (%)	Mutant colonies/10^6cells	Induction factor	Relative cloning efficiency I (%)	Relative cell density (%)	Relative cloning efficiency II (%)	Mutant colonies/ 10^6cells	Induction factor
Experiment I / 4 h				Culture I					Culture II
Solvent control (DMSO)			-	100	100	100	31.1	1	100	100	100	15.6	1
Positive control (EMS)	150		-	90.2	77	84.7	205.7	6.6	86.9	102.2	76.3	167.6	10.7
Test item	0.6		-	100.6	78	96.2	29.9	1	101.2	92.9	90.9	10.4	0.7
Test item	1.3		-	77.3	80.1	94.4	17.3	0.6	95.0	97.1	102.3	17.4	1.1
Test item	2.5		-	90.8	81.2	100	20.3	0.7	100.3	111.3	103.4	20.1	1.3
Test item	5.0		-	0.9	66.4	100.6	11.5	0.4	80.1	106.4	106.9	11.6	0.7
Test item	7.5		-	0	7.6	102.1	8.8	0.3	72.3	104.5	85.5	29.7	1.9
Test item	10.0		-	0	culture was not continued^##				24.9	111.5	89.8	21.8	1.4
Test item	15.0		-	0	culture was not continued^##				32.5	84.8	104	15	1
Solvent control DMSO			+	100	100	100	33.7	1	100	100	100	19.1	1
Positive control (DMBA)	1.1		+	50.1	50.1	84.3	895.1	26.6	68.5	74.7	103.7	633.4	33.1
Test item	7.2		+	105.5	culture was not continued^#				96.2	culture was not continued^#
Test item	14.4		+	107.1	culture was not continued^#				95.7	culture was not continued^#
Test item	28.8		+	110.9	110.9	94.7	39.2	1.2	96.1	103.9	102.9	25.4	1.3
Test item	57.5		+	109.8	109.8	101.9	29.3	0.9	102.3	104.6	104.3	25.5	1.3
Test item	115.0	P	+	108.7	108.7	137.9	20.3	0.6	102.6	87.2	103	17.9	0.9
Test item	172.5	P	+	101.0	101	118.2	22.2	0.7	96	103.9	96.7	12.9	0.7
Test item	230.0	P	+	92.5	92.5	62.5	36.2	1.1	88.2	116.6	90.1	42.5	2.2
Experiment II / 24 h				Culture I					Culture II
Solvent control DMSO			-	100	100	100	6.5	1	100	100	100	11.2	1
Positive control (EMS)	150		-	99.2	79.5	77.6	796	122.1	81.9	84.3	75.3	930.3	83.4
Test item	0.6		-	97.7	culture was not continued^#				99.8	culture was not continued^#
Test item	1.3		-	93.2	culture was not continued^#				82	culture was not continued^#
Test item	2.5		-	95.7	82.2	94.2	11.2	1.7	80.9	92.4	83.8	27.3	2.4
Test item	5.0		-	59.7	85.2	88.2	13.3	2	54	85.3	87.6	30.8	2.8
Test item	10.0		-	59.1	80	95.2	9.6	1.5	46	88.6	89.1	16.4	1.5
Test item	15.0		-	52.3	54.2	90.1	11	1.7	42.2	89.7	86.7	17.7	1.6
Test item	20.0		-	6.3	11.2	90.1	14	2.1	17.1	28.8	85.7	22.4	2
Test item	25.0		-	0	4.7	culture was not continued^##			0	5.4	culture was not continued^##
Experiment II / 4 h				Culture I					Culture II
Solvent control DMSO			+	100	100	100	20.7	1	100	100	100	5.4	1
Positive control (DMBA)	1.1		+	46.4	64.3	78.7	1136.2	55	51.0	45.1	97.7	905.5	167.4
Test item	7.8		+	97.2	culture was not continued^#				97.3	culture was not continued^#
Test item	15.6		+	99.5	culture was not continued^#				91.1	culture was not continued^#
Test item	31.3		+	99.7	113.3	90.2	9.9	0.5	99.7	98	95	13.7	2.5
Test item	62.5		+	102.2	119.6	99.2	22.2	1.1	94.9	93	94.2	15.9	2.9
Test item	125	P	+	100.8	116.6	93.1	22.7	1.1	94.5	96.9	106.4	8.5	1.6
Test item	187.5	P	+	78.2	102.5	87.3	20.6	1	78.4	85.1	109.6	12.5	2.3
Test item	250	P	+	42.2	culture was not continued^###				52.1	culture was not continued^###
Test item	500	P	+	0	20.9	89	14.9	0.7	0	26	81.3	27.3	5

^#culture was not continued since a minimum of only 4 analysable concentrations is required

^##culture was not continued due to exceedingly strong toxic effects

^###culture not continued to avoid evaluation of too many precipitating concentrations

P precipitation

Conclusions:

The test item did not induce any toxicologically significant or concentration-related increases in mutant frequency per survivor in either the absence or presence of metabolic activation. The test item was therefore considered to be non-mutagenic to V79 cells at the HPRT locus under the conditions of this test.

Executive summary:

OECD 476 (2010) - In a mammalian cell gene mutation assay (HPRT locus), Chinese hamster (V79) cells cultured in vitro were exposed to n-undecanal at at concentrations of 0.6, 1.3, 2.5, 5.0, 10.0, 15.0, 20.0, 25.0 µg/mL and 7.2, 7.8, 14.4, 15.6, 28.8, 31.3, 57.5, 62.5, 115, 125, 172.5, 187.5, 230, 250, 500 µg/mL in the absence and presence of mammalian metabolic activation (S9-mix), respectively.

 

The test item was tested up to cytotoxic and solubility limits. The positive controls did induce the appropriate response. There was no evidence of induced mutant colonies over background levels at the concentrations tested.

 

This study is classified asacceptable. This study satisfies the requirement for Test Guideline OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data.

Reference 5

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 August - 08 October 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was conducted in accordance with international guidelines and in accordance with GLP. All relevant validity criteria were met.

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Refer to Section 13.2 for read-across justification document.

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Kept in a controlled temperature area set to maintain 21°C, under nitrogen gas.
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: Dissolved in DMSO. Assumed stable.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test article was prepared as a stock solution (60 mg/mL) in the chosen vehicle.
- Preliminary purification step (if any): n/a
- Final dilution of a dissolved solid, stock liquid or gel: The test article was prepared as a stock solution (60 mg/mL) in the chosen vehicle
- Final preparation of a solid: n/a

FORM AS APPLIED IN THE TEST (if different from that of starting material) : applied as a liquid

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable) n/a

OTHER SPECIFICS: n/a

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

A peripheral blood sample was taken from a young, healthy, non-smoking, male donor of 33 years of age with no known recent exposures to genotoxic chemicals or radiation by venipuncture. The blood sample Was taken directly into blood collecting tubes containing sodium heparin then held at room temperature for less than 2 hours prior to culture initiation. The blood sample was used for the entire study.

Metabolic activation:

with and without

Test concentrations with justification for top dose:

1.25, 2.5, 5.0, 10.0, 20.0, 40.0, 80.0, 160, 300, 600 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Test item was suitabily soluble.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension

Treatments were performed approximately 48 hours after culture initiation. Appropriate dilutions of test article and positive control solutions were prepared so as to reach the final concentrations indicated in the study design. Cultures tested in the absence of S9 mix were treated as indicated in the study design then returned to the incubator for 4 or 24 hours as appropriate. For cultures tested in the presence of S9 mix, 0.2 mL of S9 mix per mL of culture was added
immediately prior to treatment as indicated in the study design. Cultures were returned to the incubator for 4 hours.

DURATION
After the 4-hour treatment, cultures were centrifuged and the supernatant replaced with fresh complete medium containing 6 ug Cytochalasin B per mL. Incubation was continued for a further 24 hours prior to harvesting. After the 24-hour treatment in the absence of S9, cultures were centrifuged, and the supernatant replaced with fresh complete medium containing 6 pg Cytochalasin B per mL. Incubation was continued for a further 24 hours until harvesting.

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Cultures were harvested by centrifugation. The cell pellet was resuspended in slightly hypotonic solution (0.075M KCl) at room temperature. Fixative (25:1 v/v methanol: acetic acid) was mixed with the suspended cells and following another centrifugation, the cells were treated with 3 changes of fixative. After the third change of fixative, the cell pellet was resuspended in fixative at an appropriate density for slide preparation. The fixed cells were dropped onto clean slides and air-dried before staining. Two slides were prepared from each culture. Fixed cells not used for slide preparation were discarded. Slides were stained with the fluorescent metachromatic dye, acridine orange.

NUMBER OF CELLS EVALUATED: The CBPI was determined by examination of at least 500 cells (if available) per culture in selected treatment groups, i.e. relevant dose levels not showing extreme toxic effects. Lymphocyte toxicity is nonnally indicated by a decreased CBPI compared to the concurrent control group.

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

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
The diameter of the MN must not exceed 1/3rd of each of the two main nuclei diameter
The micronuclei can touch but must not overlap the two main nuclei
Micronuclei should be large enough to discern morphological characteristics
Micronuclei should possess a generally rounded shape with a clearly defined outline
Micronuclei should be similar in color to the nuclei
Should lie in the same focal plane as the cell
Micronuclei must not be linked to the nuclei by a nucleoplasmic bridge
Micronuclei must be within cytoplasmic boundary
Micronuclei must be non-refractive (staining)

The results obtained for each experimental point are compared with the results obtained for the concurrent vehicle control culture from the same treatment regime. The test article is considered to be non-genotoxic if none of the treatment groups produces an incidence of MBC in excess of the upper 99% observed limit for the negative laboratory historical control range. The test article is considered to have shown genotoxicity if MBC values are beyond the 99% upper limit with a value of at least twice the concurrent control. Normally the increase would be expected to be dose-related, usually with more than one experimental point being affected. If results do not meet the above criteria, they would normally be considered equivocal and additional testing might be considered.

Rationale for test conditions:

The highest dose tested (600 pg/mL) was a level expected to show visible precipitation in the culture medium as recommended by the OECD based on a preliminary solubility test.

Evaluation criteria:

The results obtained for each experimental point are compared with the results obtained for the concurrent vehicle control culture from the same treatment regime. The test article is considered to be non-genotoxic if none of the treatment groups produces an incidence of MBC in excess of the upper 99% observed limit for the negative laboratory historical control range. The test article is considered to have shown genotoxicity if MBC values are beyond the 99% upper limit with a value of at least twice the concurrent control. Normally the increase would be expected to be dose-related, usually with more than one experimental point being affected. If results do not meet the above criteria, they would normally be considered equivocal and additional testing might be considered.

Statistics:

No statistical analysis was performed for this study as incidences of MBC for all test article treated groups were within the negative laboratory historical control range.

Key result

Species / strain:

lymphocytes: Human donor

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Excessive toxicty observed in samples treated at 300 µg/mL after 24 h treatment in the absence of S9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Table 1       Summary of results

 

Treatment	Conc. (µg/mL)	Average CBPI	Cytotoxicity (%)^a	Total No. of BC examined	Total number of MBC	% MBC
4 h treatment in the absence of S9 mix
Vehicle	-	1.9	0	2000	12	0.6
Test Item	80	1.8	10	2000	14	0.7
	160	1.9	6	2000	10	0.5
	300	1.8	14	2000	8	0.4
	600	1.5	45	1698^h	8	0.5
MMC	0.3	1.6	30	2000	134	6.7*
4 h treatment in the presence of S9 mix
Vehicle	-	1.9	0	2000	8	0.4
Test Item	160	1.8	11	2000	16	08
	300	1.7	24	2000	9	0.5
	600	1.6	30	2000	12	0.6
CP	10	1.5	41	2000	91	4.6*
24 h treatment in the absence of S9 mix
Vehicle	-	2.0	0	2000	3	02
Test Item	40	1.9	10	2000	4	0.2
	80	1.7	25	2000	10	0.5
	160	1.5	44	2000	15	0.8
	300	1.2	84	NR	NR	NR
MMC	0.2	1.6	42	2000	334	16.7*

CBPI: cytokinesis block proliferation index

BC, MBC: binucleated cells, micronucleated binucleated cells (% MBC calculated based on rounded values)

NR: Not reported as considered excessively toxic (cytoxicity > 60 % relative to vehicle controls)

* substantial positive response (at least twice the concurrent vehicle control in terms of % MBC)

^acytotoxicity calculation based on rounded values

^bnot enough cells available

 

Conclusions:

It is concluded that n-Undecanal did not show any evidence of genotoxic activity in this in vitro test for induction of micronuclei when tested in accordance with regulatory guidelines.

Executive summary:

In a mammalian cell micronucleus assay human lymphocyte cells cultured in vitro were exposed to n-Undecanal at concentrations 40-600 µg/mL in the presence and absence of mammalian metabolic activation (S9-mix).

 

The test item was tested up to insoluble concentrations. The positive controls induced the appropriate response. There was no evidence (or a concentration related positive response) of induced mutant colonies over background.

 

This study is classified asacceptable. This study satisfies the requirement for Test Guideline 487 for in vitro mutagenicity.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

Not applicable, no mutagenic activity observed in available studies.

Additional information

Ames Test (OECD 471)

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.

In Vitro Gene Mutation Assay (OECD 476)

In a mammalian cell gene mutation assay (HPRT locus), Chinese hamster (V79) cells cultured in vitro were exposed to the test item (n-undecanal) at concentrations of 0.6, 1.3, 2.5, 5.0, 10.0, 15.0, 20.0, 25.0 µg/mL and 7.2, 7.8, 14.4, 15.6, 28.8, 31.3, 57.5, 62.5, 115, 125, 172.5, 187.5, 230, 250, 500 µg/mL in the absence and presence of mammalian metabolic activation (S9-mix), respectively (Anon., 2010).  The test item was tested up to cytotoxic and solubility limits. The positive controls did induce the appropriate response. There was no evidence of induced mutant colonies over background levels at the concentrations tested.  In a one-to-one read-across approach, the substance n-undecanal (source substance) is considered appropriate for direct read-across (one-to-one) to 2 -methyldecanal (target substance) for the endpoint in vitro genetic toxicity (in vitro gene mutation in mammalian cells). In conclusion, the test item did not induce any toxicologically significant or concentration-related increases in mutant frequency per survivor in either the absence or presence of metabolic activation. The test item was therefore considered to be non-mutagenic to V79 cells at the HPRT locus under the conditions of this test.. A full justification for the read-across approach is presented in IUCLID Section 13.2.

In Vitro Mammalian Cell Micronucleus Test (OECD 487)

In a mammalian cell micronucleus assay human lymphocyte cells cultured in vitro were exposed to the test item (n-undecanal) at concentrations 40-600 µg/mL in the presence and absence of mammalian metabolic activation (S9-mix).  The test item was tested up to insoluble concentrations. The positive controls induced the appropriate response. There was no evidence (or a concentration related positive response) of induced mutant colonies over background.  In a one-to-one read-across approach, the substance n-undecanal (source substance) is considered appropriate for direct read-across (one-to-one) to 2 -methyldecanal (target substance) for the endpoint in vitro genetic toxicity (in vitro cytogenicity / micronucleus). In conclusion, the test item did not show any evidence of genotoxic activity when tested in accordance with OECD 487. A full justification for the read-across approach is presented in IUCLID Section 13.2.

Justification for classification or non-classification

The substance did not meet the criteria for classification in accordance with Regulation (EC) No 1272/2008 (CLP).