dec-1-en-4-yne

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:

03-05-2017 to 22-05-2017

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

Version / remarks:

Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labor and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries (24 November 2000)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: July 2016 ; signature: October 2016

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

dec-1-en-4-yne

EC Number:

813-331-8

Cas Number:

24948-66-1

Molecular formula:

C10H16

IUPAC Name:

dec-1-en-4-yne

Test material form:

liquid

Details on test material:

- Physical state: Liquid
- Storage condition of test material: Approximately 4 °C, in the dark under nitrogen
- Other: Slightly yellow

Method

Target gene:

histidine or tryptophan locus

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:

Type and composition of metabolic activation system: Rat liver S9
- source of S9: prepared in house (dates within full study report) ; S9 Microsomal fraction: Lot No. PB/βNF S9 17/02/2017
- method of preparation of S9 mix: Documented in the full study report. Stored at -196ºC
- concentration or volume of S9 mix and S9 in the final culture medium: 10% S9
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): A Certificate of S9 Efficacy is presented in the full study report. Additionally, concurrent positive control substances all produced marked increases in the number of revertant colonies and the activity of the S9 fraction was found to be satisfactory.

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
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. The dose levels were selected based on the results of Experiment 1.
All strains (absence of S9-mix): 1.5, 5, 15, 50, 150, 500, 1500, 5000 µg/plate.
All strains (presence of S9-mix): 5, 15, 50, 150, 500, 1500, 5000 µg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethylsulphoxide (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 DMSO at the same concentration in solubility checks performed. DMSO was selected as the vehicle.
- Other: Formulated concentrations were adjusted/increased to allow for the stated water/impurity content. See 'Test Material Information' for further details.

Controls

Controls 1

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

Remarks:

Without metabolic activation S9

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-Aminoanthracene

Remarks:

With metabolic activation S9

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 to 72 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). Manual counts may be performed, where automated counting cannot be performed: e.g. 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.

SELECTION AGENT (mutation assays): histidine-deficient agar

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Rationale for test conditions:

In accordance with the OECD TG 471 guidelines.

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. 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)).
5. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
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).
Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Results and discussion

Test results

Test results 1

Key result

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

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

See table 1 and 2

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: The current Positive HCD dataset is presented in the full study report.
- Negative (solvent/vehicle) historical control data: The current background spontaneous revertant counts in concurrent untreated controls and/or or vehicle controls ; historic negative controls are presented in the full study report.

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)	130 102 112	(115) 14.2#	27 16 16	(20) 6.4	22 17 25	(21) 4.0	34 29 27	(30) 3.6	22 24 23	(23) 1.0
	1.5 µg	116 104 116	(112) 6.9	28 18 19	(22) 5.5	20 15 20	(18) 2.9	32 22 27	(27) 5.0	16 15 10	(14) 3.2
	5 µg	120 121 106	(116) 8.4	13 10 9	(11) 2.1	23 27 17	(22) 5.0	19 29 27	(25) 5.3	15 14 23	(17) 4.9
	15 µg	115 77 107	(100) 20.0	9 10 10	(10) 0.6	15 11 11	(12) 2.3	30 14 18	(21) 8.3	15 28 8	(17) 10.1
	50 µg	91 108 101	(100) 8.5	16 14 7	(12) 4.7	11 25 27	(21) 8.7	13 15 16	(15) 1.5	14 9 16	(13) 3.6
	150 µg	96 77 74	(82) 11.9	8 7 12	(9) 2.6	16 13 18	(16) 2.5	25 15 25	(22) 5.8	8 10 9	(9) 1.0
	500 µg	96 85 110	(97) 12.5	22 16 9	(16) 6.5	23 21 26	(23) 2.5	11 13 16	(13) 2.5	19 11 5	(12) 7.0
	1500 µg	103 89 111	(101) 11.1	7 9 8	(8) 1.0	19 25 27	(24) 4.2	29 10 9	(16) 11.3	10 10 4	(8) 3.5
	5000 µg	100 89 65	(85) 17.9	6 5 7	(6) 1.0	11 11 19	(14) 4.6	9 8 13	(10) 2.6	4 3 8	(5) 2.6
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
		560 1005 961	(842) 245.2	1366 1227 1375	(1323) 83.0	532 459 499	(497) 36.6	394 294 325	(338) 51.2	190 169 149	(169) 20.5
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)	115 111 98	(108) 8.9#	19 12 16	(16) 3.5	26 13 31	(23) 9.3	21 18 18	(19) 1.7	7 15 16	(13) 4.9
	1.5 µg	114 116 76	(102) 22.5	13 8 18	(13) 5.0	28 15 26	(23) 7.0	34 24 19	(26) 7.6	8 9 12	(10) 2.1
	5 µg	104 124 119	(116) 10.4	8 9 12	(10) 2.1	30 33 32	(32) 1.5	26 20 19	(22) 3.8	14 14 12	(13) 1.2
	15 µg	120 98 109	(109) 11.0	10 8 8	(9) 1.2	31 31 24	(29) 4.0	17 19 18	(18) 1.0	10 10 10	(10) 0.0
	50 µg	120 95 112	(109) 12.8	8 9 12	(10) 2.1	24 38 24	(29) 8.1	31 21 24	(25) 5.1	8 14 11	(11) 3.0
	150 µg	120 116 113	(116) 3.5	11 9 10	(10) 1.0	34 39 29	(34) 5.0	23 25 24	(24) 1.0	10 9 14	(11) 2.6
	500 µg	98 123 102	(108) 13.4	8 7 10	(8) 1.5	33 18 32	(28) 8.4	28 18 30	(25) 6.4	7 7 14	(9) 4.0
	1500 µg	28 123 116	(89) 52.9	7 8 7	(7) 0.6	29 36 29	(31) 4.0	32 21 24	(26) 5.7	9 14 17	(13) 4.0
	5000 µg	108 112 116	(112) 4.0	10 8 10	(9) 1.2	31 36 25	(31) 5.5	24 25 21	(23) 2.1	11 16 12	(13) 2.6
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
		994 636 997	(876) 207.6	231 151 163	(182) 43.1	1171 892 849	(971) 174.8	141 109 136	(129) 17.2	597 704 554	(618) 77.2

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

V           Very weak bacterial background lawn

T            Toxic, no bacterial background lawn

#           Standard deviation

 

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)	99 84 71	(85) 14.0#	31 30 32	(31) 1.0	21 28 25	(25) 3.5	29 21 25	(25) 4.0	12 6 11	(10) 3.2
	1.5 µg	87 64 85	(79) 12.7	22 30 34	(29) 6.1	28 23 25	(25) 2.5	28 29 26	(28) 1.5	8 10 8	(9) 1.2
	5 µg	77 75 75	(76) 1.2	28 26 31	(28) 2.5	28 37 34	(33) 4.6	21 25 25	(24) 2.3	8 9 6	(8) 1.5
	15 µg	61 76 91	(76) 15.0	34 28 30	(31) 3.1	26 42 36	(35) 8.1	22 29 29	(27) 4.0	9 9 4	(7) 2.9
	50 µg	79 72 60	(70) 9.6	29 23 14	(22) 7.5	31 36 21	(29) 7.6	21 13 18	(17) 4.0	6 12 8	(9) 3.1
	150 µg	67 64 64	(65) 1.7	23 29 32	(28) 4.6	36 26 18	(27) 9.0	25 8 17	(17) 8.5	10 7 8	(8) 1.5
	500 µg	60 62 62	(61) 1.2	21 21 32	(25) 6.4	42 43 14	(33) 16.5	29 20 7	(19) 11.1	8 11 6	(8) 2.5
	1500 µg	70 69 73	(71) 2.1	23 31 23	(26) 4.6	26 20 22	(23) 3.1	17 15 21	(18) 3.1	6 S 4 S 4 S	(5) 1.2
	5000 µg	57 S 52 S 66 S	(58) 7.1	22 S 23 S 25 S	(23) 1.5	29 S 24 S 23 S	(25) 3.2	20 S 8 S 23 S	(17) 7.9	6 S 4 S 5 S	(5) 1.0
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
		988 845 884	(906) 73.9	790 722 657	(723) 66.5	386 392 400	(393) 7.0	302 280 223	(268) 40.8	314 369 304	(329) 35.0
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)	71 79 94	(81) 11.7#	16 22 27	(22) 5.5	43 38 34	(38) 4.5	30 32 23	(28) 4.7	18 11 20	(16) 4.7
	5 µg	84 75 89	(83) 7.1	15 30 21	(22) 7.5	38 39 38	(38) 0.6	13 30 25	(23) 8.7	10 25 20	(18) 7.6
	15 µg	75 77 77	(76) 1.2	22 29 18	(23) 5.6	35 36 32	(34) 2.1	31 36 26	(31) 5.0	16 12 20	(16) 4.0
	50 µg	84 66 76	(75) 9.0	23 27 27	(26) 2.3	25 33 30	(29) 4.0	32 21 24	(26) 5.7	14 13 15	(14) 1.0
	150 µg	92 66 69	(76) 14.2	28 27 29	(28) 1.0	22 50 19	(30) 17.1	25 22 15	(21) 5.1	16 9 19	(15) 5.1
	500 µg	76 67 84	(76) 8.5	26 23 21	(23) 2.5	32 32 42	(35) 5.8	13 18 36	(22) 12.1	19 24 12	(18) 6.0
	1500 µg	72 68 76	(72) 4.0	29 19 21	(23) 5.3	29 41 41	(37) 6.9	39 15 16	(23) 13.6	14 21 14	(16) 4.0
	5000 µg	77 S 83 S 71 S	(77) 6.0	22 S 24 S 22 S	(23) 1.2	45 39 32	(39) 6.5	12 S 17 S 15 S	(15) 2.5	9 S 15 S 9 S	(11) 3.5
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
		1237 1193 885	(1105) 191.8	567 539 613	(573) 37.4	209 220 217	(215) 5.7	148 175 158	(160) 13.7	265 241 202	(236) 31.8

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
negative
Under the conditions of this study, the test item 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 item 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 item 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. 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 0.05 and 500 µg/plate, depending on bacterial strain type and presence or absence of S9-mix. The vehicle (DMSO) 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.In the first mutation test (absence of S9-mix), the test item induced substantial reductions in the frequency of revertant colonies (below a factor of 0.5 fold under the concurrent vehicle control) initially from 1500 µg/plate to Salmonella strains TA1535 and TA1537 and at 5000 µg/plate to E.coli strain WP2uvrA and Salmonella strain TA98. A small reduction in TA100 revertant colony frequency was also noted at 5000 µg/plate. In the presence of S9-mix, small reductions in TA1535 revertant colony were noted from 500 µg/plate. Consequently for the second mutation test, the same maximum dose level (5000 µg/plate) was employed. No precipitates were observed at any dose level in either the presence or absence of S9-mix. There were no 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). It was concluded that, under the conditions of this assay, the test item 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.