5,9-dimethyldec-4-enal

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19-11-2015 to 08-12-2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: June 2015; signature: September 2015

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(4E)-5,9-dimethyldec-4-enal; (4Z)-5,9-dimethyldec-4-enal

EC Number:

943-495-7

Cas Number:

689-65-6

Molecular formula:

C12H22O

IUPAC Name:

(4E)-5,9-dimethyldec-4-enal; (4Z)-5,9-dimethyldec-4-enal

Test material form:

liquid

Details on test material:

- Physical state: Liquid
- Storage condition of test material: Approximately 4 °C, in the dark
- Other: clear colourless

Method

Species / strain

Species / strain 1

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9

Test concentrations with justification for top dose:

Experiment 1 (pre-incubation method): 0, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Part of the first mutation test was repeated due to excessive toxicity (TA1535 and TA1537 dosed in the absence of S9-mix) employing an amended test item dose range of 0.05 to 150 µg/plate.

Experiment 2 (pre-incubation method): Up to eight test item dose levels were selected in Experiment 2 in order to achieve both a minimum of four non-toxic doses and the toxic/guideline limit of the test item following the change in test methodology. The dose levels were selected based on the results of Experiment 1.

Salmonella strains TA1535 and TA1537 (absence of S9-mix): 0.015, 0.05, 0.15, 0.5, 1.5, 5, 15, 50 µg/plate.
Salmonella strains TA100 and TA98 and E.coli strain WP2uvrA (absence of S9-mix): 0.05, 0.15, 0.5, 1.5, 5, 15, 50, 150 µg/plate.
Salmonella strains TA1535 and TA1537 and E.coli strain WP2uvrA (presence of S9-mix): 0.15, 0.5, 1.5, 5, 15, 50, 150, 500 µg/plate.
Salmonella strain TA100 (presence of S9-mix): 0.5, 1.5, 5, 15, 50, 150, 500, 1500 µg/plate.
Salmonella strain TA98 (presence of S9-mix): 1.5, 5, 15, 50, 150, 500, 1500, 5000 µg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration in solubility checks performed. Dimethyl sulphoxide was selected as the vehicle.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: Experiment 1. in medium; in agar (pre-incubation) ; Experiment 2. in medium; in agar (pre-incubation).
The choice of application was due to the test item to either have unknown volatility or was suspected to be volatile, therefore all testing was performed using the pre-incubation method (20 minutes at 37 ± 3 °C) except for the untreated controls.

DURATION
- Exposure duration:
Experiment 1. All of the plates were pre-incubated in sealed, small volume containers, by application of 0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer OR S9-mix (as appropriate) and 0.1 mL of the test item formulation, vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3 ºC for 20 minutes (with shaking) prior to addition of 2 mL of molten amino-acid supplemented media. All of the plates were sealed in anaerobic jars or bags (one jar/bag for each concentration of test item/vehicle) during the incubation procedure (37 ± 3 ºC for approximately 48 hours) to minimize potential losses of the test item from the plates. After incubation, the plates were scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). A number of manual counts were performed, predominantly due to colonies spreading, colonies on the edge of the plates and artefacts on the plates, thus distorting the actual plate count.

Experiment 2. 0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer OR S9-mix (as appropriate) and 0.1 mL of the test item formulation, vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3 ºC for 20 minutes (with shaking) prior to addition of 2 mL of molten amino-acid supplemented media Subsequently, the procedure for incubation and duration was the same as in Experiment 1.

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response (Cariello and Piegorsch, 1996)).
A test item is considered non-mutagenic (negative) in the test system if the above criteria are not met.
In instances of data prohibiting definitive judgement about test item activity are reported as equivocal.

Statistics:

Statistical methods (Mahon, et al.); as recommended by the UKEMS Subcommittee on Guidelines for Mutagenicity Testing, Report - Part III (1989).

Results and discussion

Test results

Test results 1

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

See table 1 and 2

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

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:

cytotoxicity

Remarks:

See table 1 and 2

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1 : Test Results: Experiment 1 with and without metabolic activation and results of concurrent positive controls

S9-Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains						Frameshift strains
		TA100		TA1535†		WP2uvrA		TA98		TA1537†
	Solvent Control (DMSO)	98 100 103	(100) 2.5#	17 22 16	(18) 3.2	21 19 C	(20) 1.4	20 21 30	(24) 5.5	10 8 14	(11) 3.1
	0.05 µg	N/T		20 12 15	(16) 4.0	N/T		N/T		14 16 5	(12) 5.9
	0.15 µg	N/T		12 11 14	(12) 1.5	N/T		N/T		8 18 13	(13) 5.0
	0.5 µg	N/T		9 14 9	(11) 2.9	N/T		N/T		7 4 21	(11) 9.1
	1.5 µg	98 72 81	(84) 13.2	11 26 18	(18) 7.5	20 22 14	(19) 4.2	29 25 23	(26) 3.1	16 17 10	(14) 3.8
	5 µg	68 64 100	(77) 19.7	16 8 11	(12) 4.0	25 8 20	(18) 8.7	30 30 31	(30) 0.6	13 9 23	(15) 7.2
	15 µg	86 81 69	(79) 8.7	6 S 8 S 6 S	(7) 1.2	26 17 20	(21) 4.6	16 16 20	(17) 2.3	17 S 6 S 3 S	(9) 7.4
	50 µg	82 60 55	(66) 14.4	7 S 3 S 9 S	(6) 3.1	21 13 14	(16) 4.4	25 22 16	(21) 4.6	4 S 3 S 5 S	(4) 1.0
	150 µg	0 V 0 V 0 V	(0) 0.0	0 V 0 V 0 V	(0) 0.0	21 S 13 S 8 S	(14) 6.6	14 S 10 S 16 S	(13) 3.1	0 V 0 V 0 V	(0) 0.0
	500 µg	0 V 0 V 0 V	(0) 0.0	N/T		9 S 7 S 7 S	(8) 1.2	23 S 16 S 17 S	(19) 3.8	N/T
	1500 µg	0 V 0 V 0 V	(0) 0.0	N/T		0 V 0 V 0 V	(0) 0.0	13 S 18 S 17 S	(16) 2.6	N/T
	5000 µg	0 T 0 T 0 T	(0) 0.0	N/T		0 V 0 V 0 V	(0) 0.0	0 V 0 V 0 V	(0) 0.0	N/T
Positive controls S9-Mix (-)	Name Dose Level No. of Revertants	ENNG		ENNG		ENNG		4NQO		9AA
		3 µg		5 µg		2 µg		0.2 µg		80 µg
		300 302 289	(297) 7.0	221 195 139	(185) 41.9	525 955 858	(779) 225.5	330 341 315	(329) 13.1	933 367 346	(549) 333.0
S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains						Frameshift strains
		TA100		TA1535		WP2uvrA		TA98		TA1537
	Solvent Control (DMSO)	115 115 104	(111) 6.4#	8 15 9	(11) 3.8	19 22 27	(23) 4.0	35 17 36	(29) 10.7	12 13 17	(14) 2.6
	1.5 µg	133 129 125	* (129) 4.0	20 7 15	(14) 6.6	36 14 35	(28) 12.4	40 29 33	(34) 5.6	22 10 14	(15) 6.1
	5 µg	100 111 99	(103) 6.7	12 18 18	(16) 3.5	20 21 26	(22) 3.2	29 22 30	(27) 4.4	12 17 20	(16) 4.0
	15 µg	117 116 115	(116) 1.0	14 9 18	(14) 4.5	40 39 23	(34) 9.5	31 29 27	(29) 2.0	22 16 21	(20) 3.2
	50 µg	117 112 112	(114) 2.9	5 9 8	(7) 2.1	34 38 35	(36) 2.1	32 29 31	(31) 1.5	10 14 8	(11) 3.1
	150 µg	107 100 96	(101) 5.6	10 S 12 S 12 S	(11) 1.2	18 13 17	(16) 2.6	25 29 25	(26) 2.3	5 4 10	(6) 3.2
	500 µg	72 68 81	(74) 6.7	0 V 0 V 0 V	(0) 0.0	8 S 16 S 23 S	(16) 7.5	27 21 26	(25) 3.2	5 S 4 S 7 S	(5) 1.5
	1500 µg	63 S 76 S 82 S	(74) 9.7	0 V 0 V 0 V	(0) 0.0	0 V 0 V 0 V	(0) 0.0	17 21 29	(22) 6.1	0 V 0 V 0 V	(0) 0.0
	5000 µg	56 S 45 S 40 S	(47) 8.2	0 V 0 V 0 V	(0) 0.0	0 V 0 V 0 V	(0) 0.0	26 S 18 S 30 S	(25) 6.1	0 V 0 V 0 V	(0) 0.0
Positive controls S9-Mix (+)	Name Dose Level No. of Revertants	2AA		2AA		2AA		BP		2AA
		1 µg		2 µg		10 µg		5 µg		2 µg
		1043 1075 1060	(1059) 16.0	290 276 258	(275) 16.0	171 242 175	(196) 39.9	141 148 158	(149) 8.5	225 275 220	(240) 30.4

†: Experimental procedure repeated at a later date (reported in full study report); due to toxicity in the original test

C : Contaminated

#: Standard deviation

* : p≤0.05

 

Table 2 : Test Results: Experiment 2 with and without metabolic activation and results of concurrent positive controls

S9-Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains						Frameshift strains
		TA100		TA1535		WP2uvrA		TA98		TA1537
	Solvent Control (DMSO)	65 77 64	(69) 7.2#	16 19 17	(17) 1.5	15 23 12	(17) 5.7	23 24 32	(26) 4.9	11 3 7	(7) 4.0
	0.015 µg	N/T		12 12 13	(12) 0.6	N/T		N/T		11 7 16	(11) 4.5
	0.05 µg	62 56 55	(58) 3.8	17 17 12	(15) 2.9	23 17 19	(20) 3.1	20 32 18	(23) 7.6	15 7 5	(9) 5.3
	0.15 µg	71 83 57	(70) 13.0	14 7 13	(11) 3.8	6 24 15	(15) 9.0	27 27 25	(26) 1.2	7 14 14	(12) 4.0
	0.5 µg	60 73 68	(67) 6.6	16 15 12	(14) 2.1	14 13 14	(14) 0.6	13 29 32	(25) 10.2	1 6 6	(4) 2.9
	1.5 µg	75 76 74	(75) 1.0	14 16 12	(14) 2.0	16 16 21	(18) 2.9	27 26 11	(21) 9.0	10 5 6	(7) 2.6
	5 µg	58 56 50	(55) 4.2	25 16 17	(19) 4.9	19 8 12	(13) 5.6	16 14 15	(15) 1.0	7 5 8	(7) 1.5
	15 µg	51 55 43	(50) 6.1	14 S 16 S 16 S	(15) 1.2	7 11 16	(11) 4.5	27 24 17	(23) 5.1	5 S 2 S 4 S	(4) 1.5
	50 µg	51 59 56	(55) 4.0	7 S 7 S 9 S	(8) 1.2	14 S 6 S 6 S	(9) 4.6	22 20 14	(19) 4.2	0 V 0 V 0 V	(0) 0.0
	150 µg	44 S 52 S 57 S	(51) 6.6	N/T		0 V 0 V 0 V	(0) 0.0	0 V 0 V 0 V	(0) 0.0	N/T
Positive controls S9-Mix (-)	Name Dose Level No. of Revertants	ENNG		ENNG		ENNG		4NQO		9AA
		3 µg		5 µg		2 µg		0.2 µg		80 µg
		1322 1173 1346	(1280) 93.7	1082 1153 1183	(1139) 51.9	322 347 264	(311) 42.6	227 253 256	(245) 15.9	211 319 624	(385) 214.2
S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains						Frameshift strains
		TA100		TA1535		WP2uvrA		TA98		TA1537
	Solvent Control (DMSO)	69 70 65	(68) 2.6#	8 7 8	(8) 0.6	15 21 17	(18) 3.1	30 22 17	(23) 6.6	13 13 8	(11) 2.9
	0.15 µg	N/T		9 8 7	(8) 1.0	27 16 16	(20) 6.4	N/T		10 3 9	(7) 3.8
	0.5 µg	64 66 69	(66) 2.5	10 8 8	(9) 1.2	24 18 14	(19) 5.0	N/T		12 4 9	(8) 4.0
	1.5 µg	65 69 73	(69) 4.0	5 9 4	(6) 2.6	15 8 23	(15) 7.5	21 18 29	(23) 5.7	7 11 16	(11) 4.5
	5 µg	75 59 60	(65) 9.0	8 11 12	(10) 2.1	18 16 20	(18) 2.0	16 20 28	(21) 6.1	16 6 6	(9) 5.8
	15 µg	45 61 46	(51) 9.0	6 9 5	(7) 2.1	17 14 25	(19) 5.7	17 10 16	(14) 3.8	7 4 14	(8) 5.1
	50 µg	64 65 59	(63) 3.2	14 9 7	(10) 3.6	15 23 24	(21) 4.9	18 13 11	(14) 3.6	4 5 9	(6) 2.6
	150 µg	75 69 58	(67) 8.6	0 V 0 V 0 V	(0) 0.0	20 18 20	(19) 1.2	15 12 18	(15) 3.0	10 S 5 S 7 S	(7) 2.5
	500 µg	71 63 54	(63) 8.5	0 V 0 V 0 V	(0) 0.0	8 S 16 S 9 S	(11) 4.4	12 22 22	(19) 5.8	0 V 0 V 0 V	(0) 0.0
	1500 µg	44 S 52 S 55 S	(50) 5.7	N/T		N/T		19 21 18	(19) 1.5	N/T
	5000 µg	N/T		N/T		N/T		14 S 5 S 7 S	(9) 4.7	N/T
Positive controls S9-Mix (+)	Name Dose Level No. of Revertants	2AA		2AA		2AA		BP		2AA
		1 µg		2 µg		10 µg		5 µg		2 µg
		395 390 387	(391) 4.0	130 129 143	(134) 7.8	110 105 103	(106) 3.6	122 131 107	(120) 12.1	222 198 211	(210) 12.0

ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO: 4-Nitroquinoline-1-oxide

9AA: 9-Aminoacridine

BP: Benzo(a)pyrene

2AA: 2-Aminoanthracene

N/T: Not tested at this dose level

S: Sparse bacterial background lawn

T: Toxic, no bacterial background lawn

V: Very weak bacterial background lawn

#: Standard deviation

All positive and vehicle and negative controls were within laboratory historic values.

Table 3. Spontaneous Mutation Rates (Concurrent Negative Controls): Experiment 1 and 2, respectively

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA		TA98		TA1537
82		8	(16)	25	(25)	30	(32)	12	(11)
108	(97)	18		23		30		8
100		22		27		35		13
		29	(25)†					13	(12)†
		20						10
		26						12
Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA		TA98		TA1537
88	(89)	16	(14)	23	(22)	20	(19)	11	(7)
79		11		27		16		3
101		14		15		21		6

†: Experimental procedure repeated at a later date (without S9-mix) due to toxicity in the original test

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
negative
Under the conditions of this study the test material was considered to be non-mutagenic in the presence and absence of S9 activation.

Executive summary:

The study was performed to the requirements of OECD Guideline 471, EU Method B13/14, US EPA OCSPP 870.5100 and Japanese guidelines for bacterial mutagenicity testing under GLP, to evaluate the potential mutagenicity of the test substance in a bacterial reverse mutation assay using S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2uvrA- in both the presence and absence of S-9 mix. The test strains were treated with the test substance using the Ames pre incubation method at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5000 µg/plate. Part of the first mutation test was repeated due to excessive toxicity (TA1535 and TA1537 dosed in the absence of S9-mix) employing an amended test item dose range of 0.05 to 150 µg/plate. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations. Eight test item dose levels were again selected in Experiment 2 in order to achieve a minimum of four non-toxic dose levels and the toxic limit of the test item. The dose range was amended following the results of Experiment 1 and ranged between 0.015 and 5000 µg/plate, depending on bacterial strain type and presence or absence of S9-mix. The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate or the toxic limit of the test item depending on the strain type and presence of S9-mix.In the presence S9-mix weakened bacterial background lawns were noted from 150 µg/plate (TA1535), 500 µg/plate (TA1537 and WP2uvrA), 1500 µg/plate (TA100) and at 5000 µg/plate (TA98). Consequently for the second mutation test the same maximum dose level or toxic limit was used, depending on bacterial strain type and presence or absence of S9-mix.Results from the second mutation test confirmed the toxicity previously noted with weakened bacterial background lawns noted in the absence of S9-mix from 15 µg/plate (TA1535 and TA1537), 50 µg/plate (WP2uvrA) and 150 µg/plate (TA100 and TA98). In the presence of S9-mix weakened bacterial background lawns were noted from 150 µg/plate (TA1535 and TA1537), 500 µg/plate (WP2uvrA), 1500 µg/plate (TA100) and at 5000 µg/plate (TA98). The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type and exposures with or without S9‑mix. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. There were no toxicologically significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9‑mix) in Experiment 1. Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2.A small, statistically significant increase in TA100 revertant colony frequency was observed in the presence of S9-mix at 1.5 µg/plate in the first mutation test. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant colony counts at 1.5 µg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.2 times the concurrent vehicle control.It was concluded that, under the conditions of this assay, the test substance gave a negative, i.e. non-mutagenic response in S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2uvrA- in the presence and absence of S-9 mix.