Acetonitrile

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:

1984

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1984

Report date:

1984

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: US EPA TSCA 48 FR 50942, November 4, 1983. Docket OPTS 42019A.

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OTS 798.5300 (Detection of Gene Mutations in Somatic Cells in Culture)

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Acetonitrile

EC Number:

200-835-2

EC Name:

Acetonitrile

Cas Number:

75-05-8

Molecular formula:

C2H3N

IUPAC Name:

acetonitrile

Details on test material:

- Name of test material (as cited in study report): Acetonitrile
- Physical state: Clear Liquid
- Analytical purity: 99%
- Lot/batch No.: Fisher Chemical Co. HPLC Grade
- Stability under test conditions: Stable at room temperature
- Storage condition of test material: Room temperature, flammable storage cabinet.

Specific details on test material used for the study:

- Name of test material (as cited in study report): Acetonitrile
- Physical state: Clear Liquid
- Analytical purity: 99%
- Lot/batch No.: Fisher Chemical Co. HPLC Grade
- Stability under test conditions: Stable at room temperature
- Storage condition of test material: Room temperature, flammable storage cabinet.

Method

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Cells used in this assay were obtained from Dr. Samuel Latt at the Children's Hospital Medical School, Boston, MA. Dr. Latt originally obtained the line from Dr. Arthur Pardee at the Sydney Farber Cancer Center, Boston, MA.

Master vials are stored in liquid nitrogen; stock cultures are replaced monthly from the frozen vials. All the frozen cuItures have been prescreened for mycoplasma contamination and the spontaneous background mutation frequency is acceptably low. Working cultures are maintained in cell culture incubators in F10 medium plus 10% fetal calf serum. F12 medium (without hypoxanthine) plus 5% dialyzed fetal calf serum is used for mutation expression and selection.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced male rat liver S9

Test concentrations with justification for top dose:

Experiment 1 without activation: 11 concnetrations ranging from 0.1 - 30 mg/ml
Experiment 2 without activation: 8 cocentrations ranging from 1.0 - 25 mg/ml
Experiment 1 with activation: 8 concentrations ranging from 4-16 mg/ml
Experiment 2 with activation: 8 concentrations ranging from 8-20 mg/ml

Controls

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

other: ethylmethanesulphonate, benzo(a)pyrene

Details on test system and experimental conditions:

Type: HGPRT assay
METHOD OF APPLICATION: The test material was diluted using F10 medium. Initial stock solutions of 200 mg/ml were prepared immediately prior to use and aliquots added to the exposure medium. Further dilutions were made in the exposure medium.

The S9 microsome fraction prepared from the liver of male Sprague-Dawley rats induced with 500 mg/kg Aroclor 1254 was used in the activated assay. The S9 mix consisted of 2 parts rat liver S9 fraction plus 8 parts cofactor mixture (8mM MgCI2, 33mM KCI, 5mM glucose-6-phosphate, 4mM NADP and 100 mM Na 2 HP0 4 degree C, pH 7.4). The S9 mixture was prepared fresh and maintained at 4°C during use for a maximum of up to 5 hours.

The following S9 fraction was used in the study:
Source: Litton Bionetics, Kensington, MD
Lot Batch No.: RFIl15
Expiration Date: 6/86
Storage Conditions: -80°C

DURATION
- Preincubation period: CRO cells were seeded at a density of approximately 5X10^5 in T25 flasks and grown for 24 hours before being exposed to the test sample.
- Exposure duration: 16 hours (nonactivated); 4 hours (activated)
- Expression time: 8 -10 days. The cells were then plated at 2X10 per dish (six dishes per flask) in medium containing 2 ug/ml 6-thioguanine (6TG). Concomitantly with the selection step 200 cells from each flask were plated in medium without 6TG to determine the cloning efficiency of the cells. After 7-10 days the colonies were fixed with methanol and stained with Giemsa. The plates were scored by a trained technician.

NUMBER OF REPLICATIONS: Each test concentration was tested in duplicate flasks.

NUMBER OF CELLS EVALUATED: Data was reported from dishes which received 200 cells/dish except in extreme toxicity. At the highest concentrations, in which no colonies were observed, only the data from dishes receiving 5000 cells is reported.

DETERMINATION OF CYTOTOXICITY: Half a million (5 x 10^5) CHO cells were seeded in plastic T25 flasks. Approximately 24 hours later, the cells were exposed to varying acetonitrile concentrations. Exposure times for activated and nonactivated assays were 4 and 16 hours, respectively. Immediately after the exposure for the nonactivated assay and 18-23 hours after the exposure for the activated assay, 200-5000 cells (total count) were seeded in duplicate petri dishes. After 7-8 days of incubation the cells were fixed, stained and scored.

Evaluation criteria:

A group of cells containing a minimum of 50 cells was counted as a mutant colony. The activated part of the assay was performed by exposing cells in serum-free medium to the test sample in the presence of a metabolic activation system (optimal concentrations of the S9 microsomal fraction mixture). Results were evaluated statistically for differences between the mutation frequencies of the test concentrations and negative
controls.

Statistics:

Analysis of variance was performed on the combined data of the two experiments. No significant differences between the mutation frequencies of the test concentrations and negative controls was observed (alpha = 0.05). Statistical analysis of the mutagenesis assays is based on methods published by Snee and Irr (Mutation Research, 85, 77-93, 1981) and Snedecor and Cochran (Statistical Methods 7th edition, 1980, The Iowa State University Press).

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

WITHOUT METABOLIC ACTIVATION: Approximately 40% of the cells survived 15.4 mg/ml while less than 0.03% survived at 77.1 mg/ml. Based on these results concentrations ranging from 0.1 to 30 mg/ml were selected for the nonactivated mutagenesis assays.

Experiment ONE: seven survived the expression period. Concentrations above 17.6 mg/ml were very toxic while the mean survival at 15.0 mg/ml was approximately 35%. The background mutation frequencies for the medium control were 11.6 and 21.4 X 10. The average of these values is within the acceptable range of 0-20 outlined by the EPA Gene-Tox Program (Mutation Research 86, 192-214, 1981). Only one of the mutation frequencies for cells exposed to acetonitrile is significantly above the average of the negative control value. At 15.0 mg/ml, mutation frequencies of 45.6 and 5.5 x 10 were observed. The average of these two values is only slightly higher than the background values and is not a sufficient increase to indicate that the test substance induced mutations. The positive control, Ethylmethanesulfonate (EMS, 234 ug/ml) induced significantly higher mutation frequencies (714 and 663 X 10).

Experiment TWO: The highest test concentration, 25 mg/ml, resulted in approximately 10% survivors. Two sample concentrations, 25 and 15 mg/ml, had points with mutation frequencies higher than the negative controls (72.1 and 52.0 X 10). These values are most likely the result of fluctuation since the mutation frequencies for the replicate flasks are much lower.

WITH METABOLIC ACTIVATION:
Slightly greater toxicity was seen under activated conditions compared to nonactivated conditions. At 15.4 mg/ml, an average survival of approximately 17% was observed for the activated assay.

Experiment ONE: only slight toxicity was observed. The background mutation frequencies for F10 + 89 were 5.7 and 8.1 X 10. One test sample concentration, 16 mg/ml, had one point with a mutation frequency significantly higher than the negative controls (40.6 X 10). Another test sample concentration (10 mg/ml) had two points with mutation frequencies higher than the negative controls (21.4 and 33.6 x 10). A concentration related response was not observed.

- Positive Control: The positive control, Benzo(a)pyrene (B(a)P, 20 ug/ml) in the presence of 89, induced significantly greater mutation frequencies than the negative controls (59.8 and 91.2 X 10).

Experiment TWO: The highest concentration tested resulted in a mean survival of approximately 39%. The background mutation frequencies for F10 + S9 were 31.8 and 48.4 X 10. One of the test concentrations, 18 mg/ml, had a point with a mutation frequency higher than the elevated background (60.0 X 10).

-Positive Control: The positive control B (a) P in the presence of S9 induced significant increases in mutation frequency at the 6 two concentrations tested 20 and 10 ug/ml (141.8 and 8103 X 10 and 112.4 and 149.5 X 10 , respectively).

Any other information on results incl. tables

Benozpyrene and ethylmethane sulphonate were included as positive controls, and gave a positive response. No mutagenic activity was seen with acetonitrile.

Applicant's summary and conclusion

Conclusions:

The ability of Acetonitrile to induce mutations at the HGPRT gene locus in Chinese hamster ovary cells was evaluated in the presence and absence of a metabolic activation system. Under the conditions of the assay employed, the compound did not exhibit mutagenic activity.

Executive summary:

In a guideline (EPA TSCA) and GLP study, the ability of Acetonitrile to induce mutations at the HGPRT gene locus in Chinese hamster ovary cells in vitro was evaluated in the presence and absence of a metabolic activation system at cytotoxic concentrations (up to 30 mg/mL). Under the conditions of this assay, the acetonitrile did not exhibit mutagenic activity.