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Mutagenesis, Vol. 16, No. 2, 151-154, March 2001
© 2001 UK Environmental Mutagen Society/Oxford University Press

The mutagenic potential of acetonitrile in the bone marrow and peripheral blood of the mouse

Eryl Jones, Virginia Fox, Barry M. Elliott and Nigel P. Moore1,2

Zeneca Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire SK10 4JT and 1 BP Chemicals Ltd, Chertsey Road, Sunbury on Thames, Middlesex TW16 7LN, UK


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Acetonitrile was tested for its ability to induce clastogenic or aneugenic effects through the induction of micronucleated polychromatic erythrocytes (MNPCE) in mouse bone marrow and peripheral blood. Groups of NMRI mice, five males and five females, were administered a single i.p. dose of acetonitrile, corresponding to the maximum tolerated dose (MTD), 100 or 125 mg/kg body wt for males and females, respectively. Bone marrow was sampled at 18, 24 or 36 h after treatment, while peripheral blood was sampled before and 24, 48, 72 and 96 h after treatment. Positive controls were administered cyclophosphamide (65 mg/kg i.p.). Acetonitrile did not increase the incidence of MNPCE in either bone marrow or peripheral blood in male mice or in peripheral blood in females. A small, but statistically significant (P < 0.05), increase was observed in female bone marrow 36 h after administration, but since this was within the range of the control data it is not considered to be of biological significance. Cyclophosphamide increased the incidence of MNPCE in bone marrow and peripheral blood of both sexes. It is concluded that acetonitrile is neither clastogenic nor aneugenic in the bone marrow of the mouse at the MTD.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Acetonitrile (CAS no. 75-05-8) is a widely used industrial and laboratory solvent and reagent. It is acutely toxic due to its metabolism in vivo to cyanide (Willhite and Smith, 1981Go). The mutagenicity of acetonitrile has been investigated in numerous studies in mammalian and sub-mammalian test systems, both in vivo and in vitro, covering a variety of end points.

Acetonitrile showed no evidence of inducing point mutations in either bacteria (Florin et al., 1980Go; Mortelmans et al., 1986Go; Schlegelmilch et al., 1988Go) or cultured mammalian cells (Rudd et al., 1983Go) and no evidence of point mutation or recombination in Saccharomyces cerevisiae (Zimmerman et al., 1985Go; Whittaker et al., 1989Go). Weak or equivocal activity has been noted in in vitro cytogenetic assays with Chinese hamster ovary cells using both sister chromatid exchange and chromosome aberrations as the end point (Galloway et al., 1987Go). Acetonitrile was inactive in rat hepatocyte unscheduled DNA synthesis assays, both in vitro and in vivo (Mirsalis et al., 1983Go). In contrast, there are data to suggest that acetonitrile may induce aneuploidy in sub-mammalian test systems. Aneuploidy induction has been reported in S.cerevisiae (Zimmerman et al., 1985Go; Whittaker et al., 1989Go) and Drosophila melanogaster (Osgood et al., 1991aGo,bGo). Acetonitrile has also been shown to interfere with porcine brain tubulin assembly in an in vitro assay (Gröschel-Stewart et al., 1985Go).

Acetonitrile has demonstrated equivocal activity in two micronucleus studies in mice. An increase in micronucleated normochromatic erythrocytes (MNNCE) was reported in the blood of male B6C3F1 mice exposed to an atmosphere containing 400 p.p.m. acetonitrile for 13 weeks (NTP, 1996Go), although the incidence of micronuclei in all groups was within the range of control values for this strain (Allen et al., 1990Go; Kligerman, 1992; NTP, 1993Go). Intraperitoneal injection of acetonitrile increased the incidence of micronucleated polychromatic erythrocytes (MNPCE) in the bone marrow of NMRI mice within 24 h (Schlegelmilch et al., 1988Go). However, the induction of micronuclei in both of these studies was fairly weak, amounting to less than a 3-fold increase in relation to comparative controls.

The purpose of the present study was to define the potential for mutagenicity in the mouse micronucleus assay and correlate it with bone marrow cytotoxicity. It was designed to provide a rigorous examination of the in vivo activity of acetonitrile in the micronucleus test, using both bone marrow and peripheral blood sampling. NMRI mice and the i.p. route were used to reproduce the conditions reported in one previous study to provide the largest increases in MNPCE following treatment with acetonitrile. Killing times were chosen close to 24 h, at which the highest incidence of MNPCE was previously reported (Schlegelmilch et al., 1988Go).


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
NMRI mice, supplied by Harlan UK Ltd (Bicester, UK), were maintained on Rat & Mouse No.1 diet (Special Diet Services, Witham, UK) and water ad libitum and kept under controlled conditions (19–25°C, 30–70% relative humidity, 12 h light/dark cycle). Animals were 5–15 weeks old when used for determination of the maximum tolerated dose (MTD) and 6–12 or 6–15 weeks old when used for the micronucleus tests in bone marrow and peripheral blood, respectively. Acetonitrile (HPLC grade) and cyclophosphamide were from Sigma-Aldrich Ltd (Poole, UK).

The micronucleus assay was conducted using a method based on OECD and European Union guidelines (European Union, 1992Go; OECD, 1997Go). Briefly, groups of five male and five female mice were treated with acetonitrile (100 and 125 mg/kg body wt i.p. for males and females, respectively). Corn oil was used as the vehicle for acetonitrile and control animals were treated with the vehicle alone. Positive controls received cyclophosphamide (65 mg/kg i.p.) in water. All doses were administered at 10 ml/kg body wt.

For the determination of bone marrow effects, separate groups of animals were killed at 18, 24 and 36 h after dosing by an overdose of anaesthetic (halothane). All animals receiving acetonitrile underwent gross pathological examination. Femurs were removed and stripped clean of muscle. The iliac end of the femur was removed and bone marrow sampled with a fine brush (rinsed in saline and 6% albumin solution). Two smears were prepared and allowed to air dry, prior to staining with polychrome methylene blue. Slides were coded and scored blind, with 2000 polychromatic erythrocytes (PCE) examined for the presence of micronuclei (Schmid, 1976Go).

For the determination of peripheral blood effects, single groups of animals were placed in a heating box, where necessary, for 10 min. The tip of the tail was cut with a scalpel blade and a blood smear prepared. The smears were allowed to air dry prior to staining with polychrome methylene blue. The slides were coded and scored as for the bone marrow test, with the addition that evidence of cytotoxicity was assessed by determining the ratio of PCE to normochromatic erythrocytes (NCE) in a sample of 1000 erythrocytes.

Data were analysed, separately for males and females, by Student's t-test. Bone marrow samples from treatment groups were compared with the vehicle control group alone, but peripheral blood samples were also compared with the 0 h sampling time for the group. Data for MNPCE were transformed (Freeman and Tukey, 1950Go) prior to analysis. Statistical analysis of micronuclei was conducted with a one-sided test, whereas tests for percentage of PCE were two-sided.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Male mice were more susceptible to the acute toxicity of acetonitrile than females, with MTD values of 100 and 125 mg/kg, respectively. During a preliminary dose range finding experiment, males dosed with 125 mg/kg exhibited severe clinical signs of toxicity (including ataxia, decreased activity, clonic convulsions and shaking) and were humanely killed. Clinical signs observed at the MTD, in both males and females, included piloerection, upward curvature of the spine/hunched posture, urine stains, tiptoe gait, distended abdomen and increased breathing rate. Many of the males appeared ungroomed. In addition, one female and two males were killed prior to their scheduled termination times due to toxic effects of the test substance.

The incidence of MNPCE in male bone marrow and peripheral blood was not affected by treatment with acetonitrile (Figure 1A and BGo). The bone marrow was stressed by treatment (either directly or indirectly), as evidenced by increases in the incidence of PCE in both the bone marrow (Table IGo) and peripheral blood (Table IIGo).



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  		Fig. 1. . The effect of acetonitrile on MNPCE in the bone marrow and peripheral blood. The incidence of MNPCE in the bone marrow (BM) and peripheral blood (PB) of male and female NMRI mice is shown for various sampling times after administration of a single dose of acetonitrile (males, 100 mg/kg i.p.; females, 125 mg/kg i.p.) or cyclophosphamide (65 mg/kg i.p.). Data represent means ± SD for groups of five animals treated with vehicle control ({square}), acetonitrile ({blacksquare}) or cyclophosphamide ({blacksquare}). Statistical analysis was conducted using Student's t-test (one-sided): aP < 0.01 compared with vehicle control at the same time point; bP < 0.05 compared with vehicle control at the same time point; cP < 0.01 compared with the pre-dose time point.

 

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  		Table I. . Incidence of PCE in the bone marrow of male and female NMRI mice at various times after administration of a single dose of acetonitrile (100 or 125 mg/kg i.p., respectively)
 

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  		Table II. . Incidence of PCE in the peripheral blood of male and female NMRI mice at various times after administration of a single dose of acetonitrile (100 or 125 mg/kg i.p., respectively)
 
A small, but statistically significant (P < 0.05), increase in MNPCE was noted in the bone marrow of female mice at 36 h (Figure 1CGo). However, this was within the range of the control data and is therefore not considered to be biologically significant. There was no effect upon the incidence of MNPCE in female peripheral blood (Figure 1DGo). An increase in the incidence of PCE in the peripheral blood indicated that the bone marrow had been stressed by treatment (Table IIGo).

Cyclophosphamide induced substantial and statistically significant increases in the incidence of MNPCE in both bone marrow and peripheral blood in both sexes (Figure 1Go). The increase in peripheral blood followed that in bone marrow by ~24 h. In addition, cyclophosphamide reduced the percentage of PCE in peripheral blood (not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Acetonitrile has been evaluated for its ability to induce MNPCE in the bone marrow and peripheral blood of NMRI mice. The procedures and experimental design employed complied with the recommendations of the OECD and EU guidelines (European Union, 1992Go; OECD, 1997Go) except that a single dose level was used. Acetonitrile was tested at the MTD in both males and females, as shown by adverse clinical reactions to treatment.

There was a statistically significant increase in the incidence of PCE in the bone marrow of acetonitrile-treated males at the 18 h sampling time, compared with the vehicle control. There were also small PCE increases in the peripheral blood of males at the 48 and 72 h sampling times and in females at the 72 h sampling time, compared with the 0 h control. These increases suggest that the bone marrow had been subjected to stress at the MTD of acetonitrile.

The small increase in MNPCE in the bone marrow of females at 36 h was considered not biologically important, since the value (0.7{per thousand}) was very close to the control value observed at 24 h (0.6{per thousand}) and is within the range of control values (0.4–1.9{per thousand}) reported for the NMRI strain (Busk et al., 1984Go; Clemmensen et al., 1984Go; Mohtashamipur et al., 1985Go; Steinheider et al., 1985Go; Schlegelmilch et al., 1988Go). Therefore, a value of 0.7 MNPCE/1000 PCE is clearly not a biologically important increase over controls for this mouse strain. No other statistically significant increases in the incidence of MNPCE, compared with the control values, were seen in the bone marrow or peripheral blood of any acetonitrile-treated mice at any of the sampling times investigated.

Two other mouse micronucleus studies have been conducted, with equivocal results. In one study groups of male and female B6C3F1 mice inhaled an atmosphere containing acetonitrile for 13 weeks. Peripheral blood was sampled at the end of the exposure period and the incidence of MNNCE was determined. There were no clinical signs of toxicity in animals exposed to acetonitrile concentrations up to 200 p.p.m., but exposure to 400 or 800 p.p.m. acetonitrile resulted in female mortality rates of 10 and 40%, respectively. There was a marginal tendency to increased frequencies of MNNCE in both male and female mice, but this was only statistically significant in males. Even then, the only significant pairwise increase in micronucleated cells occurred at 400 p.p.m. acetonitrile (2.36 compared with 1.42{per thousand} in the control) (NTP, 1996Go). However, the incidence of MNNCE reported in all groups was within the range of control values (between 2.4 and 2.67{per thousand}) reported elsewhere in the literature for the B6C3F1 mouse strain (Allen et al., 1990Go; Kligerman, 1992; NTP, 1993Go). Furthermore, since direct measurements of bone marrow cytotoxicity were not undertaken, it is not possible to determine the level of stress within the bone marrow caused by treatment. In another study NMRI mice were treated with a single i.p. injection of acetonitrile, after which the incidence of MNPCE in the bone marrow was determined at regular intervals. At a dose equivalent to 60% of the LD50 (~105 mg/kg) there was a significantly higher incidence of MNPCE compared with controls after 24 h (4.26 and 1.68{per thousand}, respectively). Once more, males appeared to be more severely affected than females, with 3- and 2-fold increases in micronucleus frequencies in comparison with the respective controls. Again, no direct measurements of bone marrow cytotoxicity were undertaken (Schlegelmilch et al., 1988Go). The incidences of micronucleated cells in acetonitrile-treated animals were larger than any reported control value for this strain (Busk et al., 1984Go; Clemmensen et al., 1984Go; Mohtashamipur et al., 1985Go; Steinheider et al., 1985Go).

The hepatic oxidative metabolism of acetonitrile releases cyanide, which is the cause of acute systemic toxicity, since many of the effects of acetonitrile are ameliorated by the administration of sodium thiosulphate (Willhite and Smith, 1981Go). The induction of micronuclei in other studies either accompanied signs of systemic toxicity or occurred at doses that would be expected to cause such toxicity if not observed or reported. In the NTP study, micronuclei were observed only at an exposure which caused mortality in females (NTP, 1996Go) or at a dose equivalent to that which caused signs of toxicity in the current study (Schlegelmilch et al., 1988Go). The binding of cyanide, liberated during acetonitrile metabolism, to haemoglobin reduces the oxygen carrying capacity of the blood and oxygen transport may be sufficiently restricted at high doses of acetonitrile for erythropoiesis to be stimulated.

The stimulation of erythropoiesis results in an elevated frequency of cells bearing micronuclei. A concomitant increase in immature and micronucleated erythrocytes was observed in the blood of BALB/c mice following bleeding, haemolysis or splenectomy (Steinheider et al., 1985Go), and in the blood of hamsters infected with Babesia microti, a parasite which induces severe haemolytic anaemia (Ormiston et al., 1989Go). Furthermore, the administration of erythropoietin to male mice increases the population of micronucleated erythrocytes within the bone marrow. Erythropoietin caused a slight, but not statistically significant, increase in MNPCE in BALB/c mice (Suzuki et al., 1989aGo) and a significant increase in MNNCE in ddY mice (Suzuki et al., 1989bGo). Therefore, weak induction of micronuclei may result from an increased rate of erythrocyte production as a consequence of non-specific stress to the haematopoietic system.

Interestingly, although micronucleus induction following stimulation of erythropoiesis in mice, which does not involve administration of a mutagenic substance, is generally weak, it is of a similar magnitude to that reported following exposure to acetonitrile (Schlegelmilch et al., 1988Go; NTP, 1996Go). Hence, acetonitrile might indirectly increase the incidence of micronucleated cells via cyanide-mediated stimulation of erythropoiesis. However, such effects are minimal and the results presented here indicate that acetonitrile is not a direct acting aneugen or clastogen in mouse bone marrow.


   Acknowledgments
 
The authors are grateful to Dale Strother (BP Chemicals Inc.) for helpful discussions and to Elaine Edge (Zeneca CTL) for technical assistance. This work was sponsored jointly by BP Chemicals Ltd (UK) and EniChem SpA (Italy).


   Notes
 
2 To whom correspondence should be addressed. Tel: +44 1932 764060; Fax: +44 1932 764147; Email: mooren{at}bp.com Back


   References
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 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received on August 18, 2000; accepted on November 6, 2000.


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