Acetylene

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

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:

74-86-5

IUPAC Name:

74-86-5

Constituent 2

Reference substance name:

Acetylene

EC Number:

200-816-9

EC Name:

Acetylene

Cas Number:

74-86-2

Molecular formula:

C2H2

IUPAC Name:

acetylene

Details on test material:

- Name of test material (as cited in study report): Acetylene
- Physical state: gas
- Analytical purity: 99.88% v/v (see below)
- Purity test date:
- Lot/batch No.: 107316844
- Cylinder number: C2 4341
- Expiration date of the lot/batch: 30 September 2012
- Storage condition of test material: Room temperature in the dark
- Other:

Analytical results:
Sampling date: 09-10-2009

Ethene 0.04 %(v/v)
Ethane <0.01 %(v/v)
Propene 0.08 %(v/v)
Propane <0.01 %(v/v)
Acetylene 99.88 %(v/v)

Method

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 with glutamax-l and 20mMHEPES supplememted with Penicillin, strptomycin, sodium pyruvate and amphotericin giving RO media.
- Properly maintained: yes
- cleansed of homozygous (TK -/-) mutants by culturing in THMG medium for 24 hours.
- Periodically checked for Mycoplasma contamination: yes/no
- Periodically checked for karyotype stability: yes/no
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbitone / ß-napthoflavone induced rat liver S9 (2%)

Test concentrations with justification for top dose:

0.31, 0.63, 1.25, 2.5, 5, 12.5, 25 and 50%

Vehicle / solvent:

Air

Controls

Controls 1

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activiation

Details on test system and experimental conditions:

METHOD OF APPLICATION: 8.5 mL air or acetylene was added to headspace vials containing the cell cultures, using a gas syringe . The treatment vessels were incubated at 37ºC with continuous shaking for 4 or 24 hour exposure.

DURATION
-Two independent experiments were performed.
- Exposure duration: 4 h . In the repeat experiment, the exposure duration was increased to 24 h in the absence of metabolic activation
- Expression time (cells in growth medium): 2 days

SELECTION AGENT (mutation assays): 5-trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: 2

PLATE SCORING: Microtitre plates were scored after 10-14 days inculation. The number of positive wells was recorded with the total number of scorable wells. The numbers of small and large colonies in the TFT mutation plates were also recorded, following incubation with MTT solution.

DETERMINATION OF CYTOTOXICITY
A preliminary toxicity test was conducted using concentrations of acetylene in air of : 2.5, 5, 10, 20, 30, 40 and 50% to determine the dose level that produces 80-90% toxicity using Relative Suspension Growth (% RSG) values

% Relative Suspension Growth ( RSG) values and relative total growth (RTG) values were used to determine the toxicity for each dose level. Cells were cloned for both viability (%V) and mutation frequency (MF).

Evaluation criteria:

The normal range for mutant frequency per survivor is 50-200 x 10-6 for the TK +/- locus in L5178Y cells. Experiments where the vehicle controls are markedly greater than 250 x 10-6 are not normally acceptable.
Positive controls should induce 3-5 fold increases in mutant frequency greater than the corresponding vehicle control.

Statistics:

Plate count data from the viability and mutation frequency plates and the daily cell count data were analysed statistically.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: From the preliminary toxicity test the maximum dose level was set at 50% acetylene in air.

COMPARISON WITH HISTORICAL CONTROL DATA:
The vehicle controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

Any other information on results incl. tables

Mouse Lymphoma Assay on Acetylene:

Experiment 1

Treatment (% Gas)		4-Hours-S9				Treatment (% Gas)		4-Hours+S-9
		%RSG	RTG	MF§				%RSG	RTG	MF§
0		100	1.00	147.69		0		100	1.00	80.06
0.31		99	1.03	160.81		0.31		105	1.32	56.17
0.63		87	0.98	188.04		0.63		112	1.22	94.01
1.25		89	0.98	193.16		1.25		117	1.46	69.99
2.5		91	0.88	208.90		2.5		111	1.35	69.97
5	$$	83	0.88	133.80		5		104	1.26	123.92
12.5		94	1.00	131.52		12.5		105	1.54	90.22
25		94	1.02	183.09		25		93	1.25	103.85
50		92	1.03	128.84		50		101	1.29	125.10
Linear trend				NS		Linear trend				**
EMS						CP
400 µg/ml		65	0.52	1308.82		2 µg/ml		59	0.29	2319.71

Experiment 2

Treatment (% Gas)		24-Hours-S-9				Treatment (% Gas)		4-Hours+S-9
		%RSG	RTG	MF§				%RSG	RTG	MF§
0		100	1.00	68.36		0		100	1.00	88.17
0.31		119	1.10	91.53		0.31		91	1.10	80.11
0.63		130	1.42	72.61		0.63		102	1.25	77.86
1.25		117	1.03	70.17		1.25		106	1.14	61.45
2.5		127	1.09	72.00		2.5		100	1.05	79.45
5		115	1.06	75.15		5		103	1.17	76.50
12.5		108	1.01	98.11		12.5		93	1.04	92.51
25		85	0.87	99.30		25		103	1.27	107.67
50		83	0.82	104.44		50		117	1.37	78.42
Linear trend				*		Linear trend				NS
EMS						CP
150 µg/ml		59	0.29	1289.77		2 µg/ml		62	0.27	645.07

MF§                5‑TFT resistant mutants/10⁶viable cells 2 days after treatment

%RSG            Percent relative survival adjusted by post treatment cell count factor

RTG                Relative total growth adjusted to account for immediate post-treatment toxicity

$$                   Evidence of heterogeneity (poor correlation between A and B viability plates)

NS                  Not significant

*                     Significant at 5%

**                    Significant at 1%

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Acetylene was not mutagenic in the Mouse Lymphoma Mutagenesis Assay.

Executive summary:

Acetylene was assessed for potential mutagenicity on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. Two independent experiments were performed. In Experiment 1,mouse lymphoma cells weretreated with acetylene at dose levels of up to 50% in air, in duplicate, together with vehicle and positive controls using 4-hour exposure groups, both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated using a 4-hour exposure group in the presence of metabolic activation (1% S9) and a 24-hour exposure group in the absence of metabolic activation. Acetylene did not induce any toxicologically significant dose-related increases in the mutant frequency, either with or without metabolic activation. Acetylene was therefore considered to be non-mutagenic to L5178Y cells.