3-methyl-5-phenylpent-2-enenitrile

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-05-27 to 2015-07-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3-methyl-5-phenylpent-2-enenitrile

EC Number:

299-682-2

EC Name:

3-methyl-5-phenylpent-2-enenitrile

Cas Number:

93893-89-1

Molecular formula:

C12 H13 N

IUPAC Name:

(2E)-3-methyl-5-phenylpent-2-enenitrile

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM (minimal essential medium) containing Hank’s salts supplemented with 10 % foetal bovine serum (FBS), neomycin (5 μg/mL) and amphotericin B (1 %)
- 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 applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

experiment 1
4 h without metabolic activation: 13.8, 27.5, 55.0, 110.0, 220.0, 330.0, 440.0, 660.0 μg/mL
4 h with metabolic activation: 6.9, 13.8, 27.5, 55.0, 110.0, 165.0, 220.0 μg/mL
experiment 2
24 h without metabolic activation: 13.8, 27.5, 55.0, 110.0, 220.0, 330.0, 440.0 μg/mL
4 h with metabolic activation: 55.0, 110.0, 123.8, 137.5, 151.4, 165.0 μg/mL

Vehicle / solvent:

- Solvent used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

(EMS) 0.15 mg/mL = 1.2 mM, without metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

(DMBA) 1.1 μg/mL = 4.3 μM (experiment I) 2.2 μg/mL = 8.6 μM (experiment II), in DMSO, with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 and 24 h
- Expression time: 7 days
- Selection time: 8 days
- Fixation time: fixation 7 days after treatment (colonies for cloning efficiency)

SELECTION AGENT: 11 μg/mL 6-thioguanine
STAIN: 10 % methylene blue in 0.01% KOH solution

NUMBER OF REPLICATIONS: 2 experiments with each 2 culture dishes

NUMBER OF CELLS EVALUATED:
The stained colonies with more than 50 cells were counted.

DETERMINATION OF CYTOTOXICITY
- Method: reduction of cloning efficiency

OTHER:
A pre-experiment was performed with and without metabolic activation for the selection of the test substance concentration. Concentrations between 13.8 μg/mL and 1760 μg/mL were tested. It was checked for cytotocix effects, precipitation or phase seperation, pH and osmolarity.

The dose range of experiment 1 were based on the pre-experiment and of experiment 2 on the pre-experiment (without metabolic activation) and experiment 1 (with metabolic activation).

Evaluation criteria:

Acceptability
The gene mutation assay was considered acceptable if it met the following criteria:
a) the numbers of mutant colonies per 10exp6 cells found in the solvent controls fell within the laboratory historical control data range
b) the positive control substances produced a significant increase in mutant colony frequencies and remained within the historical control range of positive controls
c) the cloning efficiency II (absolute value) of the solvent controls exceeded 50 %.
The study complied with those criteria.

Evaluation
A test item was classified as positive if it induced 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 was considered non-mutagenic in this system.
A positive response was described as follows:
A test item was classified as mutagenic if it reproducibly induced a mutation frequency that was three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.
The test item was classified as mutagenic if there was 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 was not observed.
However, in a case by case evaluation this decision depended on the level of the corresponding solvent control data. If there was 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 had 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, calculated using Sum_neu_v2.xltm, version 2.0) was performed to assess a possible dose dependent increase of mutant frequencies. The numbers of mutant colonies generated with the test item were compared to the solvent control groups. A trend was judged as significant whenever the p-value (probability value) was below 0.05. However, both, biological and statistical significance was considered together.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

experiment 1 at 110 μg/mL (without metabolic activation), experiment 2 ≥ 123.8 μg/mL (with metabolic activation)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no effects observed
- Effects of osmolality: no effects observed
- Other confounding effects: In the pre-experiment phase separation was observed at the two highest concentrations of 880.0 and 1760 μg/mL following 4 hours treatment with and without metabolic activation. After 24 hours treatment without metabolic activation phase separation was noted at 1760 μg/mL.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Cytotoxic effects were observed for experiment 1 at 110 μg/mL (without metabolic activation) and for experiment 2 ≥ 123.8 μg/mL (with metabolic activation). The cell cultures at these concentrations were terminated earlier.

Any other information on results incl. tables

	concentration [µg/mL]	S9 mix	relative cloning efficiency I [%]	relative cell density [%]	relative cloning efficiency II [%]	mutant colonies/10exp6 cells	induction factor	relative cloning efficiency I [%]	relative cell density [%]	relative cloning efficiency II [%]	mutant colonies/10exp6 cells	induction factor
Experiment 1 (4 h treatment)			culture I					culture II
Solvent control (DMSO)		-	100	100	100	12.9	1	100	100	100	16.2	1
Positive control (EMS)	150	-	93	72.7	92.1	181.4	14.1	95.9	100.7	97.5	168.7	10.4
Test item	13.8	-	99.7	131.3	92.6	23.3	1.8	105.2	130.3	101.6	9.8	0.6
	27.5	-	95	109.2	90.9	14.4	1.1	98.7	86.3	100	15.1	0.9
	55	-	88.8	108.1	96.1	14	1.1	90.8	131.5	99.1	12.1	0.7
	110	-	17.6	22.1	95.5	18.2	1.4	12.8	19.8	99.6	13.6	0.8
	220	-	0	culture was not continued 1)				0	culture was not continued 1)
	330	-	0	culture was not continued 1)				0	culture was not continued 1)
	440	-	0	culture was not continued 1)				0	culture was not continued 1)
	660	-	0	culture was not continued 1)				0	culture was not continued 1)
Solvent control (DMSO)		+	100	100	100	7.6	1	100	100	100	16.4	1
Positive control (DMBA)	1.1	+	102.9	85.7	103.1	221.7	29	98.7	107.9	99.9	291.5	17.8
Test item	3.4	+	98.7	culture was not continued 2)				102.4	culture was not continued 2)
	6.9	+	103.1	89.4	101.7	19.5	2.6	99.8	109.2	104	11.8	0.7
	13.8	+	100.8	55.3	102.8	18	2.4	103.4	125.8	103.7	14.2	0.9
	27.5	+	98.6	66	101.6	18.8	2.5	97.4	106.5	103.3	22	1.3
	55	+	93.4	72.6	102.5	17.6	2.3	91.5	111.7	104.7	13	0.8
	110	+	53.8	49.8	102.9	20.1	2.6	60.5	72.6	103.4	31.1	1.9
	165	+	0	culture was not continued 1)				0	culture was not continued 1)
	220	+	0	culture was not continued 1)				0	culture was not continued 1)
Experiment 2 (24 h treatment)			culture I					culture II
Solvent control (DMSO)		-	100	100	100	14	1	100	100	100	9.6	1
Positive control (EMS)	150	-	93.1	80.3	69.8	370.1	26.4	72	79.5	99.2	350.9	36.6
Test item	6.9	-		culture was not continued 2)				culture was not continued 2)
	13.8	-	99.4	110	76.3	18.9	1.3	97.5	111.5	95.7	21.9	2.3
	27.5	-	93.4	92.6	90.5	11.9	0.9	95.1	96.3	101.3	9.9	1
	55	-	94.5	80.5	83.5	17.1	1.2	84.4	82.1	99.4	28.6	3
	110	-	96.1	76.7	73.2	23.1	1.6	70.8	70.1	99.1	10.4	1.1
	220	-	0	8.4	culture was not continued 1)			0	8.8	culture was not continued 1)
	330	-	0	0	culture was not continued 1)			0	3.9	culture was not continued 1)
	440	-	culture was not continued 1)					culture was not continued 1)
Experiment 2 (4 h treatment)			culture I					culture II
Solvent control (DMSO)		+	100	100	100	24.5	1	100	100	100	11	1
Positive control (DMBA)	2.2	+	79.9	85.7	104.2	236.3	9.6	98.3	97.2	78.8	258.8	23.6
Test item	13.8	+	culture was not continued 2)					culture was not continued 2)
	27.5	+	84.9	culture was not continued 2)				88.6	culture was not continued 2)
	55	+	80.2	81.2	102.3	25	1	86.9	135.8	92.5	7.2	0.7
	110	+	55.6	101.7	101.5	21.4	0.9	63.2	114.5	84.8	13.5	1.2
	123.8	+	19.6	93.6	100.9	25.9	1.1	29	78.2	93.9	18.9	1.7
	137.5	+	7.4	85.8	99.1	26.7	1.1	6.1	59.4	77.8	24.1	2.2
	151.4	+	5.3	59.3	101.6	25.8	1.1	3.8	54	76.2	32.3	2.9
	165	+	culture was not continued 2)					0	culture was not continued 2)

1) culture was not continued due to exceedingly severe cytotoxic effects

2) culture was not continued since a minimum of only four analysable concentrations is required

Applicant's summary and conclusion

Conclusions:

An in vitro gene mutation study (HPRT) was conducted in V79 cells of the Chinese hamster. It was concluded that the test substance did not induce gene mutations at the HPRT locus and therefore was considered to be non-mutagenic.

Executive summary:

An in vitro gene mutation study (HPRT) according to OECD Guideline 476 was conducted in V79 cells of the Chinese hamster. A pre-experiment was performed with and without metabolic activation for the selection of the test substance concentration. Concentrations between 13.8 μg/mL and 1760 μg/mL were tested. For the main experiments a concentration range between 6.9 and 660 μg/mL was used. The main assay was performed in two independent experiments. The cells were exposed to the test item for 4 hours in the first experiment with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The used concentration range of the experiments was limited by cytotoxic effects. Cytotoxic effects were observed for experiment 1 at 110 μg/mL (without metabolic activation) and for experiment 2 ≥ 123.8 μg/mL (with metabolic activation). No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments. It was concluded that the test substance did not induce gene mutations at the HPRT locus and therefore was considered to be non-mutagenic.