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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Acetonitrile does not induce gene mutations in bacteria, and has produced only marginal effects in chromosome aberration assays in vitro. A positive response is also reported in a comet assay in vitro. Reliable in vivo micronucleus studies have shown marginal or negative results.

Link to relevant study records

Referenceopen allclose all

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
Qualifier:
according to guideline
Guideline:
other: US EPA TSCA 48 FR 50942, November 4, 1983. Docket OPTS 42019A.
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
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.
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
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).
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).

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

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.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1985-1987
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: US National Toxicology Program
Principles of method if other than guideline:
Galloway et al (1987)
GLP compliance:
not specified
Type of assay:
other: in vitro mammalian chromosome aberration assay
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced male Sprague-Dawley rat liver S9 and cofactor mix
Test concentrations with justification for top dose:
500, 1600, 5000 µg/ml
Untreated negative controls:
yes
Positive controls:
yes
Positive control substance:
other: triethylenemelamine, cyclophosphamide
Details on test system and experimental conditions:
In the Abs test without S9, cells were incubated in McCoy's 5A medium with acetonitrile for 12 hours; Colcemid was added and incubation continued for 2 hours. The cells were then harvested by mitotic shake-off, fixed, and stained with Giemsa.

For the Abs test with S9, cells were treated with acetonitrile and S9 for 2 hours, after which the treatment medium was removed and the cells were incubated for 12 hours in fresh medium, with Colcemid present for the final 2 hours. The cells were then harvested by mitotic shake-off, fixed, and stained with Giemsa.
Evaluation criteria:
Cells were selected for scoring on the basis of good morphology and completeness of karyotype (21 ± 2 chromosomes). All slides were scored blind and those from a single test were read by the same person. One hundred first-division metaphase cells were scored at each dose level. Classes of aberrations included simple (breaks and terminal deletions), complex (rearrangements and translocations), and other (pulverized cells, despiralized chromosomes, and cells containing 10 or more aberrations).

For a single trial, a statistically significant (P<0.05) difference for one dose point and a significant trend (P<0.015) were considered weak evidence for a positive response; significant differences for two or more doses indicated the trial was positive. A positive trend test in the absence of a statistically significant increase at anyone dose resulted in an equivocal call (Galloway et al., 1987). Ultimately, the trial calls were based on a consideration of the statistical analyses as well as the biological information available to the reviewers.
Statistics:
To arrive at a statistical call for a trial, analyses were conducted on both the dose response curve and individual dose points.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Genotoxicity:
other: equivocal
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:

Despite the increase in Abs noted at the high dose in the trial conducted with S9, the trend test was not significant (P>0.015) and the trial results were concluded to be equivocal.
Conclusions:
Acetonitrile was negative without S9. Acetonitrile was a weak inducer of chromosomal aberrations in the presence of S9. The increase was noted at the highest dose tested (5,000 ug/mL). Despite the increase in aberrations noted at the high dose in the trial conducted with S9, the trend test was not significant (P>0.015) and the trial results were concluded to be equivocal.
Executive summary:

In an in vitro Chromosome Aberration test conducted by the US National Toxicology Program, Acetonitrile concentrations up to 5000 ug/mL produced a negative response in CHO cells without S9 activation, and produced an equivocal response with S9 metabolic activation.

Endpoint:
in vitro DNA damage and/or repair study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1985-1987
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5900 - In vitro Sister Chromatid Exchange Assay
Principles of method if other than guideline:
Galloway et al (1987)
GLP compliance:
not specified
Type of assay:
sister chromatid exchange assay in mammalian cells
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced male Sprague-Dawley rat liver S9 and cofactor mix
Test concentrations with justification for top dose:
160, 500, 1600, 5000 µg/ml
Untreated negative controls:
yes
Positive controls:
yes
Positive control substance:
other: triethylenemelamine, cyclophosphamide
Details on test system and experimental conditions:
- In the SCE test without S9, CHO cells were incubated for 26 hours wjth acetonitrile in supplemented McCoy's 5A medium. Bromodeoxyuridine (BrdU) was added 2 hours after culture initiation. After 26 hours, the medium containing acetonitrile was removed and replaced with fresh medium plus BrdU and Colcemid, and incubation was continued for 2 hours. Cells were then harvested by mitotic shake-off, fixed, and stained with Hoechst 33258 and Giemsa.

- In the SCE test with S9, cells were incubated with acetonitrile, serum-free medium, and S9 for 2 hours. The medium was then removed and replaced with medium containing serum and BrdU and no acetonitrile, and incubation proceeded for an additional 26 hours, with Colcemid present for the final 2 hours. Cells were then harvested by mitotic shake-off, fixed, and stained with Hoechst 33258 and Giemsa.

- All slides were scored blind and those from a single test were read by the same person. Fifty second division metaphase cells were scored for frequency of SCEs/cell from each dose leveL
Evaluation criteria:
An increase of 20% or greater at any single dose was considered weak evidence of activity; increases at two or more doses resulted in a determination that the trial was positive. A statistically significant trend (P<0.005) in the absence of any responses reaching 20% above background led to a call of equivocal.
Statistics:
Statistical analyses were conducted on the slopes of the dose-response curves and the individual dose points. An SCE frequency 20% above the concurrent solvent control value was chosen as a statistically conservative positive response; The probability of this level of difference occurring by chance at one dose point is less than, 0:01; the probability for such a chance occurrence at two dose points is less than 0.001.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
other: weakly positive at high dose
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Untreated negative controls validity:
valid
Positive controls validity:
valid

Weak positive response (20% increase in SCEs at 5000 µg/ml), only in the absence of S9. No dose response seen. The protocol allowed testing up to toxic concentrations.

Cells described as CHO-W-B1 were used.

Conclusions:
Acetonitrile was a weak inducer of SCEs in the absence of S9, and negative in the presence of S9 metabolic activation. The increase was noted at the highest dose tested (5,000 µg/mL).
Executive summary:

In an in vitro test conducted by the US National Toxicology Program, Acetonitrile produced a weakly positive response for increased Sister Chromatid Exchange frequency in CHO cells without S9 activation at the highest dose tested (5000 µg/mL), and produced a negative response with S9 metabolic activation.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1986
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: US National Toxicology Program
Principles of method if other than guideline:
Preincubation modification of the Ames test (Ames B.N. et al. Mutat. Res. 31, 347 -364, 1975).
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): Acetonitrile
- Analytical purity: >99% purity
Species / strain / cell type:
other: Salmonella typhimurium strains TA97, TA98, TA100, TA1535, TA1537
Metabolic activation:
with and without
Metabolic activation system:
ArocIor 1254-induced male Sprague-Dawley rat or Syrian hamster liver
Test concentrations with justification for top dose:
0, 100, 333, 1000, 3333, 10000 µg/plate
Untreated negative controls:
yes
Positive controls:
yes
Positive control substance:
other: sodium azide, 9-aminoacridine, 4-nitro-o-phenylenediamine, 2-aminoanthracine
Details on test system and experimental conditions:
Ames test: Acetonitrile was evaluated by two testing laboratories as a coded sample.

METHOD OF APPLICATION: Acetonitrile was incubated with the Salmonella typhimurium tester strains (TA97, TA98, TA100, TA1535, and TA1537) either in buffer or S9 mix (metabolic activation enzymes and cofactors from ArocIor 1254-induced male Sprague-Dawley rat or Syrian hamster liver) for 20 minutes at 37° C. Top agar supplemented with I-histidine and d-biotin was added, and the contents of the tubes were mixed and poured onto the surfaces of minimal glucose agar plates.

DURATION
- Exposure duration: 2 days at 37° C.

NUMBER OF REPLICATIONS: All tests were repeated using either the same or different S9 concentrations. Each trial consisted of triplicate plates of concurrent positive and negative controls and at least five doses of acetonitrile.

NUMBER OF CELLS EVALUATED: Histidine-independent mutant colonies arising on these plates were counted following incubation


Evaluation criteria:
A positive response was defined as a reproducible, dose-related increase in revertant colonies in any one strain/activation combination. A negative response was obtained when no increase in revertant colonies was observed following chemical treatment.
Key result
Species / strain:
other: Salmonella typhimurium strains TA97, TA98, TA100, TA1535, TA1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Positive controls validity:
valid
Additional information on results:
In the absence of toxicity, 10,000 µg/plate was selected as the high dose.

Data from 2 laboratories gave negative results in all strains.

Conclusions:
Acetonitrile was not mutagenic in Salmonella typhimurium strains TA97, TA98, TA100, TA1535, or TA1537, with or without S9 metabolic activation.
Executive summary:

In studies conducted by the US National Toxicology Program, Acetonitrile concentrations up to 10,000 µg/plate were not mutagenic in Salmonella typhimurium strains TA97, TA98, TA100, TA1535, or TA1537, with or without S9 metabolic activation.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Guideline:
other: Parry and Zimmermann, 1976
Principles of method if other than guideline:
A system with the yeast Saccharomyces cerevisiae for detecting the induction of mitotic aneuploidy.
GLP compliance:
not specified
Type of assay:
yeast cytogenetic assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): Acetonitrile
- Analytical purity: >99% purity
Species / strain / cell type:
other: Saccharomyces cerevisiae strain D61.M
Metabolic activation:
without
Test concentrations with justification for top dose:
0.05-4.76% (<= 37.4 mg/ml)
Metabolic activation:
without
Genotoxicity:
positive
Remarks on result:
other: other: Saccharomyces cerevisiae strain D61.M
Remarks:
Migrated from field 'Test system'.

Acetonitrile induced chromosome loss in studies at two laboratories, with or without a cold interruption phase. It is possible that microtubules formed during growth are somehow weakened and function poorly during mitosis resulting in the observed chromosomal malsegregation.

Conclusions:
Acetonitrile was shown to be a good inducer of mitotic aneuploidy in Saccharomyces at relatively high concentrations (close to 5%).
Executive summary:

Zimmermann et al (1985) and Whittaker et al (1989) reported that acetonitrile induced mitotic aneuploidy in Saccharomyces at relatively high test concentrations (close to 5%). It is possible that microtubules formed during growth are somehow weakened and function poorly during mitosis resulting in the observed chromosomal malsegregation.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

The overall data indicate that acetonitrile is not a point mutagen, but that high concentrations can interfere with chromosome segregation. Acetonitrile at concentrations up to 10,000 μg/plate was negative in S. typhimurium) strains TA1535, TA1537, TA97, TA98 and TA100 in the absence of S9 and in the presence of rat or hamster S9 induced with Aroclor 1254 (Mortelmans et al., 1986; NTP, 1996). Negative results were also obtained in a preincubation S. typhimurium assay and a reverse mutation assay in (S. cerevisiae) D7, conducted in the presence and absence of S9 from rats induced with acetonitrile or phenobarbitone (Schlegelmilch et al., 1988), although the bacterial assay was limited by the use of stationary cultures.

The IPH of Japan (1980) reported that acetonitrile was not mutagenic in bacterial strains S. typhimurium TA 100, TA 98, TA 1535, TA 1537, TA 1538 and E. coli WP2uvr A and WP2uvrA/pKM101, when tested at concentrations up to 5000 μg/plate with or without a metabolic activation system.

Mammalian cell point gene mutation was investigated in a mouse lymphoma assay. Under the experimental conditions reported acetonitrile did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation. Two mouse lymphoma studies and one HRPT assay all gave negative results for mamalian cell point mutations.

Sister chromatid exchanges (SCEs) in Chinese hamster ovary (CHO) cells were significantly increased in the absence of S9 (only at 5000 μg/mL), but there was no effect in the presence of S9 (Galloway et al., 1987; NTP, 1996). Gene conversions in (S. cerevisiae) were increased in the absence of S9, but not in the presence of S9 (Schlegelmilch et al., 1988). Both of these assays measure repair of DNA damage, rather than persistent DNA damage.

Wu et al. (2009) report a positive response for acetonitrile in a comet assay performed in cultured human lymphocytes and HepG2 cells.

 

Chromosome aberrations were significantly elevated in CHO cells at 5000 μg/mL in the presence of rat S9 but the trend test was borderline (p= 0.016); there was no effect in the absence of S9 (Galloway et al., 1987; NTP, 1996). Although the types of chromosome aberrations were not reported, Galloway et al. (1987) reported that both simple and complex aberrations were elevated at the high dose. The ability of acetonitrile to induce mutations at the HGPRT gene locus in CHO cells in vitro was investigated both in the presence and absence of rat liver S9 (Bioassay Systems Corporation, 1984). There were no significant differences between treated and control cells over a concentration range of 0.1 to 30 mg/mL. Micronucleated normochromatic erythrocytes (NCEs) were slightly increased, without dose response, in the peripheral blood of male mice, but not in females, in a micronucleus assay conducted in conjunction with a 13-week inhalation toxicity experiment (NTP, 1996). Although the micronucleus assay is usually conducted in polychromatic erythrocytes (PCEs), it has been shown that micronucleated peripheral blood PCEs and NCEs are at steady state following dosing for 45-90 days. Schlegelmilch et al. (1988) found that intraperitoneal injection of mice with acetonitrile at 60% of the oral LD50 was weakly clastogenic, with micronucleated polychromatic erythrocytes significantly increased at 24 hours. When the mice were induced by injection of low doses of acetonitrile for 7 days, and then challenged with 60% of the oral LD50, an increase in micronucleated PCEs was not observed until 72 hours after the challenge dose. In a separate study, Jones et al (2001) reported that acetonitrile did not produce significantly increased frequencies of micronucleated polychromatic erythrocytes in the bone marrow or peripheral blood of mice following a single maximum tolerated dose by intraperitoneal injection.

Micronucleus formation can indicate clastogenic activity or interference with chromosome segregation. Acetonitrile (131 ppm for up to 70 minutes) induced aneuploidy (both chromosome gain and chromosome loss) in treated mature oocytes of Drosophila melanogaster females exposed either as larvae or as adults (Osgood et al., 1991a,b). Toxicity and sterility were induced by the 70-minute exposure. When S. cerevisiae was exposed to 5% acetonitrile, it induced mitotic aneuploidy (Zimmerman et al., 1985); the investigators suggested that the induction of aneuploidy by acetonitrile in the absence of point mutations or recombination resulted from interference with tubulin assembly and the formation of microtubules in the spindle apparatus. More recently, Sehgal et al. (1990) obtained in vitro evidence that acetonitrile does inhibit microtubule assembly in taxol-purified Drosophila or mouse microtubules, further indicating that acetonitrile has potential to interfere with chromosome segregation.

 

In summary, acetonitrile is negative in gene mutation assays and produces only marginal effects in chromosome aberration assays. The potential of high doses of acetonitrile to interfere with chromosome segregation both in vivo and in vitro have been demonstrated in D. melanogaster, which has been attributed to inhibition of microtubule assembly.

 

Justification for selection of genetic toxicity endpoint

A weight of evidence approach is taken to this endpoint.

Short description of key information:

A number of standard guideline and non-standard studies in vitro and in vivo are available.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

No classification of acetonitrile is proposed for genetic toxicity. Acetonitrile does not induce gene mutations in bacteria, and has produced only marginal effects in chromosome aberration assays in vitro. A positive response is also reported in a comet assay in vitro. Reliable in vivo micronucleus studies have shown marginal or negative results. The potential of acetonitrile to interfere with chromosome segregration in D. melanogaster has been demonstrated both in vitro and in vivo, which has been attributed to inhibition of microtubule assembly.