(2Z)-2-phenylhex-2-enenitrile

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:

07-12-2005 to 03-02-2006

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:

2006

Report date:

2006

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: August 2005 ; signature: November 2005

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

482-160-5

EC Name:

-

Cas Number:

130786-09-3

Molecular formula:

C12H13N

IUPAC Name:

(2Z)-2-phenylhex-2-enenitrile

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: colourless

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:

Rat liver S9

Test concentrations with justification for top dose:

Preliminary toxicity test: All strains: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate

Experiment 1 (plate incorporation method):
TA100 & TA1535: 5, 15, 50, 150, 500, 1500 µg/plate
TA98 & TA1537: 15, 50, 150, 500, 1500, 5000 µg/plate
WP2uvrA- : 50, 150, 500, 1500, 5000 µg/plate

Experiment 2 (plate incorporation methodd): 0, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
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.

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 (plate incorporation) ; Experiment 2. in medium; in agar (plate incorporation)

DURATION
- Exposure duration:
Experiment 1. All of the plates were incubated at 37 ± 3 ºC for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).

Experiment 2. All of the plates were incubated at 37 ± 3 ºC for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).

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).

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

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 1537

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 3

Key result

Species / strain:

S. typhimurium TA 1535

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 4

Key result

Species / strain:

S. typhimurium 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

Test results 5

Key result

Species / strain:

S. typhimurium TA 98

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

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)	99 97 100	(99) 1.5#	18 21 21	(20) 1.7	29 27 20	(25) 4.7	20 23 23	(22) 1.7	4 9 7	(7) 2.5
	5 µg	86 98 96	(93) 6.4	20 16 9	(15) 5.6	NT		NT		NT
	15 µg	97 99 112	(103) 8.1	15 14 13	(14) 1.0	NT		25 24 21	(23) 2.1	7 10 5	(7) 2.5
	50 µg	102 102 109	(104) 4.0	24 14 15	(18) 5.5	30 19 21	(23) 5.9	20 28 22	(23) 4.2	9 9 10	(9) 0.6
	150 µg	100 99 80	(93) 11.3	14 26 22	(21) 6.1	19 20 15	(18) 2.6	24 18 19	(20) 3.2	13 7 13	(11) 3.5
	500 µg	75 S 82 S 86 S	(81) 5.6	7 S 11 S 8 S	(9) 2.1	24 19 18	(20) 3.2	36 21 29	(29) 7.5	7 8 5	(7) 1.5
	1500 µg	0 S 0 S 0 S	(0) 0.0	0 S 0 S 0 S	(0) 0.0	19 20 19	(0) 0.6	21 27 17	(22) 5.0	10 9 6	(8) 2.1
	5000 µg	NT		NT		20 20 20	(20) 0.0	9 22 8	(13) 7.8	3 S 7 S 4 S	(5) 2.1
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
		543 454 488	(495) 44.9	490 302 421	(404) 95.1	957 969 1005	(977) 25.0	302 301 279	(294) 13.0	1094 1271 1561	(1309) 235.8
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 84	(103) 16.0#	13 14 15	(14) 1.0	32 35 30	(32) 2.5	31 34 32	(32) 1.5	19 24 19	(21) 2.9
	5 µg	85 90 114	(96) 15.5	14 10 10	(11) 2.3	NT		NT
	15 µg	108 104 108	(107) 2.3	11 10 13	(11) 1.5	NT		25 25 22	(24) 1.7	30 20 15	(22) 7.6
	50 µg	104 104 99	(102) 2.9	12 10 9	(10) 1.5	34 33 20	(29) 7.8	29 28 18	(25) 6.1	19 27 20	(22) 4.4
	150 µg	93 78 96	(89) 9.6	12 11 11	(11) 0.6	22 22 20	(21) 1.2	23 29 40	(31) 8.6	20 28 27	(25) 4.4
	500 µg	57 S 57 S 43 S	(52) 8.1	15 S 7 S 8 S	(10) 4.4	21 22 26	(23) 2.6	29 32 29	(30) 1.7	19 19 16	(18) 1.7
	1500 µg	0 S 0 S 0 S	(0) 0.0	0 S 0 S 0 S	(0) 0.0	19 24 24	(22) 2.9	26 37 31	(31) 5.5	11 11 10	(11) 0.6
	5000 µg	NT		NT		24 19 27	(23) 4.0	37 19 18	(25) 10.7	8 S 17 S 14 S	(13) 4.6
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
		955 1002 913	(957) 44.5	288 296 338	(307) 26.9	394 410 443	(416) 25.0	344 340 302	(329) 23.2	393 464 351	(403) 57.1

 

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)	120 102 126	(116) 12.5#	16 16 23	(18) 4.0	26 20 26	(24) 3.5	15 18 18	(17) 1.7	8 10 11	(10) 1.5
	5 µg	135 133 132	(133) 1.5	21 24 19	(21) 2.5	NT		NT		NT
	15 µg	130 113 123	(122) 8.5	25 19 20	(21) 3.2	NT		16 18 21	(18) 2.5	9 18 7	(11) 5.9
	50 µg	133 124 130	(129) 4.6	19 27 30	(25) 5.7	29 29 18	(25) 6.4	10 20 26	(19) 8.1	11 11 8	(10) 1.7
	150 µg	85 115 134	(111) 24.7	11 20 15	(15) 4.5	18 26 14	(19) 6.1	13 13 20	(15) 4.0	12 16 11	(13) 2.6
	500 µg	104 S 104 S 121 S	(110) 9.8	19 S 14 S 11 S	(15) 4.0	21 16 16	(18) 2.9	19 15 5	(13) 7.2	13 13 11	(12) 1.2
	1500 µg	0 S 0 S 0 S	(0) 0.0	0 S 0 S 0 S	(0) 0.0	12 12 9	(11) 1.7	18 19 15	(17) 2.1	8 4 7	(6) 2.1
	5000 µg	NT		NT		16 26 24	(22) 5.3	9 15 19	(14) 5.0	0 S 0 S 0 S	(0) 0.0
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
		541 566 585	(564) 22.1	691 431 475	(532) 139.2	817 789 883	(830) 48.3	223 218 216	(219) 3.6	588 999 1518	(1035) 466.0
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)	81 95 82	(86) 7.8#	12 10 10	(11) 1.2	35 23 15	(24) 10.1	20 33 24	(26) 6.7	23 15 30	(23) 7.5
	5 µg	97 84 95	(92) 7.0	8 7 9	(8) 1.0	NT		NT		NT
	15 µg	100 91 97	(96) 4.6	9 9 11	(10) 1.2	NT		23 19 31	(24) 6.1	16 23 21	(20) 3.6
	50 µg	81 81 84	(82) 1.7	7 4 10	(7) 3.0	44 24 30	(33) 10.3	22 22 29	(24) 4.0	23 21 22	(22) 1.0
	150 µg	62 70 62	(65) 4.6	7 11 15	(11) 4.0	30 30 25	(28) 2.9	22 30 24	(25) 4.2	21 24 22	(22) 1.5
	500 µg	35 S 38 S 43 S	(39) 4.0	9 5 7	(7) 2.0	22 25 20	(22) 2.5	29 31 23	(28) 4.2	12 20 19	(17) 4.4
	1500 µg	0 S 0 S 0 S	(0) 0.0	0 S 0 S 0 S	(0) 0.0	25 21 18	(21) 3.5	24 33 25	(27) 4.9	11 15 16	(14) 2.6
	5000 µg	NT		NT		22 30 35	(26) 4.0	30 21 18	(23) 6.2	10 S 8 S 7 S	(8) 1.5
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
		762 837 873	(824) 56.6	306 261 245	(271) 31.6	560 542 637	(580) 50.5	209 163 180	(184) 23.3	578 522 536	(545) 29.1

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: partial absence of bacterial background lawn

#: Standard deviation

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 B.13/14, 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 both the Ames plate incorporation and pre incubation methods at up to six 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 based on the results of a preliminary toxicity assay. The dose levels were 5 to 1500 µg/plate for Salmonella strains TA100 and TA1535, 15 to 5000 µg/plate for Salmonella strains TA98 and TA1537 and 50 to 5000 µg/plate for E.coli strain WP2uvrA-. The experiment was repeated on a separate day using the same dose range as the range-finding test, fresh cultures of the bacterial strains and fresh test item formulations. 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 based on cytotoxicity. The test item caused a visible reduction in the growth of the bacterial background lawn, both in the presence and absence of S9, to Salmonella strains TA100 and TA1535 from 500 µg/plate and at 5000 µg/plate to TA1537. The test item caused no visible reduction in the growth of the bacterial background lawn to either Salmonella strain TA98 or Escherichia coli strain WP2uvrA-. The sensitivity of the bacterial tester strains to the toxicity of the test item varied between strain type. The test item was tested up to either the maximum recommended dose level of 5000 µg/plate or the toxic limit depending on bacterial strain type. 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 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.