Reaction mass of (2E)-Tridec-2-enenitrile and (2Z)-Tridec-2-enenitrile and (3E)-Tridec-3-enenitrile and (3Z)-Tridec-3-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 2015-07-28

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro mammalian cell gene mutation

Test material

Test material information

1

Reference substance name:

Reaction mass of (2E)-Tridec-2-enenitrile and (2Z)-Tridec-2-enenitrile and (3E)-Tridec-3-enenitrile and (3Z)-Tridec-3-enenitrile

EC Number:

919-489-5

Molecular formula:

C13H23N

IUPAC Name:

Reaction mass of (2E)-Tridec-2-enenitrile and (2Z)-Tridec-2-enenitrile and (3E)-Tridec-3-enenitrile and (3Z)-Tridec-3-enenitrile

Method

Target gene:

HPRT locus

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

mammalian (rat) microsomal fraction S9 mix, Phenobarbital/β-naphthoflavone induced

Test concentrations with justification for top dose:

Experiment 1:
Exposure period 4 hours, with S9-mix: 1.9, 3.8, 7.5, 15, 30, 60, 120, 240 µg/mL (phase separation was observed from 30 µg/mL onwards)
Exposure period 4 hours, without S9-mix: 0.94, 1.9, 3.8, 7.5, 15, 30, 45, 60 µg/mL

Experiment 2:
Exposure period 4 hours, with S9-mix: 0.94, 1.9, 3.8, 7.5, 15, 30, 45, 60 µg/mL (phase separation was observed from 45 µg/mL onwards)
Exposure period 24 hours, without S9-mix: 0.94, 1.9, 3.8, 7.5, 15, 30, 45, 60 µg/mL

Vehicle / solvent:

- Vehicle/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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours or 24 hours
- Expression time (cells in growth medium): 7 days
- Selection time: 8 days

SELECTION AGENT: 6-TG
STAIN: 10 % methylene blue in 0.01 % KOH solution

NUMBER OF REPLICATIONS: 5 flasks were seeded with the cells after the expression time, two additional flasks were seeded in non-selective medium for determination of the viability

NUMBER OF CELLS EVALUATED: 500 cells were seeded per flask

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

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 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 is judged as significant whenever the p-value (probability value) is 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 I: at 3.8 µg/mL and above without S9-mix, Experiment II: at 15 µg/mL and above without S9-mix, at 45.0 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
Phase separation was noted at the highest evaluated concentration of experiment I and II in the presence of metabolic activation.
Relevant cytotoxic effects indicated by a relative cloning efficiency I or cell density below 50% in both cultures occurred in the first experiment at 3.8 μg/mL and above in the absence of metabolic activation. In the second experiment exceedingly severe cytotoxic effects were noted at 15.0 μg/mL and above without metabolic activation, and at 45.0 μg/mL with metabolic activation.

COMPARISON WITH HISTORICAL CONTROL DATA:
No relevant and reproducible increase in mutant colony numbers/10^6 cells was observed in the main experiments up to the maximum concentration. The mutant frequency did not exceed the historical range of solvent controls. Only in the second culture of experiment I without metabolic activation the range of the historical solvent control data (1.6 - 45.7 mutant colonies/10^6 cells) was slightly exceeded at 7.5 μg/mL (49.6 mutant colonies per 10^6 cells).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The recommended cytotoxic range of approximately 10%-20% relative cloning efficiency or relative cell density was covered without metabolic activation. In the presence of metabolic activation an extremely steep cytotoxic gradient at the onset of phase separation did not permit any coverage of this range.

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	22.9	1	100	100	100	22.1	1
Positive control (EMS)	150	-	80.8	125	93.3	205.6	9	75.7	127.4	10..1	231.6	10.5
Test item	0.94	-	93.5	112.6	56.1	24.7	1.1	82.3	121.8	66.1	16	0.7
	1.9	-	94.4	98.2	54.6	32.6	1.4	95.4	107.4	61.8	19	0.9
	3.8	-	84.1	18.9	50.7	8.8	0.4	80.7	23.7	27.2	18.6	0.8
	7.5	-	28.2	15.3	42.5	9.8	0.4	25.8	17.6	31.1	49.6	2.2
	15	-	6.8	8.3	culture was not continued 1)			2.22	7.7	culture was not continued 1)
	30	-	0	8.3	culture was not continued 1)			0	0	culture was not continued 1)
	45	-	0	7.4	culture was not continued 1)			0	0	culture was not continued 1)
	60	-	0	7.2	culture was not continued 1)			0	0	culture was not continued 1)
Solvent control (DMSO)		+	100	100	100	20.9	1	100	100	100	7.5	1
Positive control (DMBA)	1.1	+	84.2	71.9	91.6	176.5	8.4	74.4	79.7	116.5	186.6	24.7
Test item	1.9	+	100.3	60.5	103.2	12.4	0.6	102.7	78.2	75.6	21.3	2.8
	3.8	+	95.5	63.6	99.1	19.4	0.9	96	77.9	81.3	38.3	5.1
	7.5	+	102.2	55.3	109.7	24.1	1	85.3	70.6	81.3	21.6	2.9
	15	+	101.5	72.8	103.9	14.4	0.7	84.2	81.1	63.4	30.4	4
	30	+	95.2	64	96.1	13.5	0.6	82.4	73.6	82.2	22.2	2.9
	60	+	0	9	culture was not continued 1)			0	0	culture was not continued 1)
	120	+	0	0	culture was not continued 1)			0	0	culture was not continued 1)
	240	+	0	0	culture was not continued 1)			0	0	culture was not continued 1)
Experiment 2 (24 h treatment)			culture I					culture II
Solvent control (DMSO)		-	100	100	100	8.1	1	100	100	100	20.7	1
Positive control (EMS)	150	-	85.5	87.1	102.9	227.2	28	95.1	107.3	96.6	183.3	8.8
Test item	0.94	-	86.7	94.7	99.6	21.5	2.6	86.7	116.2	101.7	12.3	0.6
	1.9	-	86.2	73.2	102.5	15.5	1.9	88.3	83.2	99.7	22.1	1.1
	3.8	-	81.8	75.2	98.8	9.6	1.2	81.7	96.6	99.6	22	1.1
	7.5	-	86.1	culture was not continued 2)				85.2	culture was not continued 2)
	15	-	7.1	109.7	99.5	17.1	2.1	4.3	97.1	102.6	19.6	0.9
	30	-	0	9.7	101.1	15.8	1.9	0	14.3	102.9	9.3	0.4
	45	-	0	0	culture was not continued 1)			0	0	culture was not continued 1)
	60	-	0	0	culture was not continued 1)			0	0	culture was not continued 1)
Experiment 2 (4 h treatment)			culture I					culture II
Solvent control (DMSO)		+	100	100	100	17	1	100	100	100	19.7	1
Positive control (DMBA)	2.2	+	79.1	98.4	100	329.3	19.4	71.7	104.3	102.3	311.6	15.8
Test item	0.94	+	87.4	culture was not continued 3)				93.5	culture was not continued 3)
	1.9	+	84.7	culture was not continued 3)				85.6	culture was not continued 3)
	3.8	+	79.9	102.8	100	12.8	0.8	88.8	105.1	102.8	20.5	1
	7.5	+	91.3	103.1	102.5	12.8	0.7	80.1	109.2	101.7	32.4	1.6
	15	+	86.2	103.6	98.2	19.8	1.2	79.7	104.5	101.3	24.1	1.2
	30	+	67.9	119.6	99.5	21.7	1.3	72	119.4	103.3	20.6	1
	45	+	0	70.6	99.1	38.5	2.3	0	69.5	103.6	22.4	1.1
	60	+	0	1.2	culture was not continued 1)			0	1.3	culture was not continued 1)

 

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

2) culture was not continued due to a technical error (no cell growth at all in both cultures)

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

Applicant's summary and conclusion

Conclusions:

The test item did not induce gene mutations at the HPRT locus in V79 cells.

Executive summary:

The study according to OECD 476 was conducted to investigate the potential of the test item to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The 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 maximum test item concentration of the pre-experiment (1960 μg/mL) was equal to a molar concentration of about 10 mM. The concentration range of the main experiments was limited by cytotoxic effects. No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments. The tested concentrations in experiment 1 for an exposure period 4 hours, with S9-mix were 1.9, 3.8, 7.5, 15, 30, 60, 120, 240 µg/mL (phase separation was observed from 30 µg/mL onwards) and for an exposure period 4 hours, without S9-mixwere 0.94, 1.9, 3.8, 7.5, 15, 30, 45, 60 µg/mL. In experiment 2 the test concentrations with an exposure period of 4 hours were with S9-mix 0.94, 1.9, 3.8, 7.5, 15, 30, 45, 60 µg/mL (phase separation was observed from 45 µg/mL onwards) and with an exposure period of 24 hours without S9-mix 0.94, 1.9, 3.8, 7.5, 15, 30, 45, 60 µg/mL were used. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test substance is considered to be non-mutagenic in this HPRT assay.