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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
The mutagenic potential of acetonitrile in the bone marrow and peripheral blood of the mouse.
Author:
Jones, E.
Year:
2001
Bibliographic source:
Mutagenesis 16(2): 151-154.
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report Date:
1998

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Acetonitrile
- Physical state: Colourless liquid
- Analytical purity: 99.93 % w/w (not certified)
- CTL test substance reference number: Y00297/003
- Storage condition of test material: Tightly closed in a cool dry place away from heat, sparks and open flame.
- Supplier: Sigma-Aldrich Co. Ltd, Dorset

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan UK Ltd (Bicester, UK)
- Age at study initiation: 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.
- Diet: Rat & Mouse No.1 diet (Special Diet Services, Witham, UK) ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature: 19-25°C
- Humidity: 30-70%
- Photoperiod: 12 h light/12 h dark cycle

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
Details on exposure:
In the Phase II peripheral blood micronucleus test, male and female animals were weighed and given a single intraperitoneal dose of com oil, cyclophosphamide or acetonitrile.

Lighting was switched on once during a period of darkness for approximately 1 hour to check animals and the air conditioning was switched off for 2 hours for maintenance work.

Additional females (animal numbers 203, 204, 210 and 211) were dosed at 125mg/kg and additional males (animal numbers 257-260) were dosed at 100mg/kg. These animals were included to replace any animals dosed at these dose levels that were found dead or killed in extremis prior to their scheduled termination time.

PREPARATION OF DOSING SOLUTIONS: All test and positive control substance dosing preparations were prepared as close to the time of dosing as possible. The test substance, vehicle and positive control substance were dosed at a volume of 10ml/kg bodyweight.

Duration of treatment / exposure:
A single dose.
Frequency of treatment:
Single Dose.
Post exposure period:
Bone Marrow: 18, 24 or 36 hours for test material and vehicle control. 24 hours for positive control.
Peripheral Blood: 24, 48, 72 and 96 hours.
Doses / concentrations
Remarks:
Doses / Concentrations:
100 mg/kg (male); 125 mg/kg (female)
Basis:

No. of animals per sex per dose:
Bone Marrow: 5 males and 5 females per sampling interval.
Peripheral Blood: 5 males and 5 females
Control animals:
yes, concurrent vehicle
Positive control(s):
The positive control, cyclophosphamide, was supplied by Sigma Chemical Company Ltd Fancy Road, Poole, Dorset, UK and prepared as (65 mg/kg i.p.) in water. It was prepared as a solution in sterile double deionised water (CTL test substance reference number Y04517/027).

Examinations

Tissues and cell types examined:
Femurs were removed and stripped clean of muscle. Bone marrow samples were taken 18, 24 or 36 hours after dosing. Peripheral blood samples were taken prior to treatment (1) hour and then at 24, 48, 72 and 96 hours after dosing. Additional females (animal numbers 203, 204, 210 and 211) were dosed at 125mg/kg and additional males (animal numbers 257-260) were dosed at 100mg/kg. These animals were included to replace any animals dosed at these dose levels that were found dead or killed in extremis prior to their scheduled termination time.
Details of tissue and slide preparation:
DETAILS OF SLIDE PREPARATION
- BONE MARROW: The animals were killed by asphyxiation in halothane Ph. Eur. (FLUOTHANE, zeneca Phannaceuticals) followed by cervical dislocation 18, 24 and 36 hours after receiving a single intraperitoneal dose of the test substance.The iliac end of the femur was removed and a fine paint brush was rinsed in saline, wiped to remove the excess and wetted with a solution of albumin (6% w/v in physiological saline). This was then dipped into the marrow canal and two smears were painted on an appropriately labelled clean, dry microscope slide. This procedure was repeated to give four smears of marrow per slide. The slides were allowed to air dry and were stained with polychrome methylene blue and eosin using an automatic staining machine and mounted in DPX.

- PERIPHERAL BLOOD: At each time point, the mice were placed in a heating box, where necessary, for approximately 10 minutes. The tip of the tail was cut using a scalpel blade and a drop of blood was placed on to a labelled microscope slide. The blood was smeared across the slide using a blood smearing apparatus. The slides were air dried, stained with polychrome methylene blue and eosin using an automatic staining machine and mounted in DPX.

METHOD OF ANALYSIS: Slides from the male and female bone marrow and the peripheral blood micronucleus tests were coded in separate batches and scored blind. In the bone marrow test, two thousand (2 x 1000) polychromatic erythrocytes were examined for the presence of micronuclei for each animal. In the peripheral blood test, two thousand (2 x 1000) polychromatic erythrocytes, where possible, were examined for the presence of micronuclei for each animal at each sampling time. The slides were also examined for evidence of cytotoxicity, which may be manifest by alterations in the ratio of different cell types in the bone marrow. This was assessed by counting the ratio of polychromatic to normochromatic erythrocytes in a sample of 100 erythrocytes.
Evaluation criteria:
Criteria for identification of micronuclei are as described by Schmid (1976): Spherical (or rounded) with well-defined edges. Diameters of not less than approximately 1/20 of a polychromatic erythrocyte diameter. Dark purple/dark blue staining. Lie in the same plane as the polychromatic erythrocyte in which it is contained (determined by focusing).
Statistics:
The data for the incidence of micronucleated polychromatic erythrocytes per thousand polychromatic erythrocytes were transformed using a square root transformation, prior to analysis. The data for the percentages of polychromatic erythrocytes were transformed using the double arcsine transformation of Freeman and Tukey (1950) prior to analysis. Analyses were carried out separately for the acetonitrile treated groups and positive control group using the GLM procedure in SAS (1989) for both the bone marrow and peripheral blood samples- Each treatment group mean was compared with the control group mean at the corresponding sampling time using Student's t-test based on the error mean square in the analysis. For the peripheral blood samples, analyses were also carried out separately for each sampling time, within each group, using the GLM procedure in SAS (1989). Each sampling time mean was compared with the 1 hour sampling time mean for the group using a one-sided Student's t-test, based on the error mean square in the analysis. The statistical tests for micronucleated polychromatic erythrocytes were one-sided looking for increases. The rests for percentage of polychromatic erythrocytes were two-sided looking for increases and decreases.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
DETERMINATION OF MAXIMUM DOSE
Based on mortalities the maximum tolerated dose was initially selected as 125 mg/kg for males and females. Theis dose level was administered to males and females in the Phase II bone marrow study and subsequently to females in the peripheral blood study. Further dose ranging was conducted in males to re-determine the maximum tolerated dose. In Group 4 two different batches of males were used to investigate whether there was any difference between males used in Phase II and a new batch of animals. As lethalities had been seen at 125mg/kg and there were no obvious differences between the two batches of animals, 100mg/kg was taken to represent the maximum tolerated dose for males. This dose level was administered to males in the peripheral blood study and subsequently in a repeat male bone marrow study.

CLINICAL OBSERVATIONS AND POST-MORTEM FINDINGS
Adverse reactions to treatment were observed for males and females dosed with acetonitrile. Clinical signs observed for males dosed at 100mg/kg and females dosed at 125mg/kg included piloerection, upward curvature of the spine/hunched posture, urine stains, tip-toe gait, distended abdomen and increased breathing rate. Many of the males appeared ungroomed and one showed signs of salivation. One female showed signs of ataxia and pinched in sides. In addition, one female and two males were killed prior to their scheduled termination times due to toxic effects of the test substance.

MICRONUCLEUS TEST
No statistically or biologically significant increases in the incidence of micronucleated polychromatic erythrocytes, over the vehicle control values, were seen in the bone marrow of males at any of the sampling times investigated. A small, but statistically significant increase in the incidence of micronucleated polychromatic erythrocytes, over the vehicle controls value, was observed in the bone marrow of females at the 36 hour sampling time. The value observed (0.7 MPE/1000 PE) was very close to the control value seen at the 24 hour sampling time (0.6 MPE/1000 PE) and is therefore considered clearly to be of no biological significance. These increases suggest that the bone marrow has been subjected to stress at maximally tolerated doses of acetonitrile. No statistically or biologically significant increases in the incidence of micronucleated polychromatic erythrocytes, over the vehicle control values or over the 0 hour control values, were seen in the peripheral blood of males or females at any of the sampling times investigated. Comparison of the percentage of polychromatic erythrocytes showed a statistically significant increase in the bone marrow of males treated with acetonitrile at the 18 hour sampling time. Small increases in the percentage of polychromaic erythrocytes, over the 0 hour controls, were also observed in the peripheral blood of males at 48 and 72 hours after treatment and in females at 72 hours after treatment. The test system positive control, cyclophosphamide, induced statistically significant and biologically meaningful increases in micronucleatcd polychromatic erythrocytes compared to the vehicle control values, at 24 hours in the bone marrow and peaking at 48 hours in the peripheral blood, thus demonstrating the sensitivity of the test system to a known clastogen. Significant reductions in polychromatic erythrocytes were also observed at a number of sampling times when compared to either the vehicle control or the 1 hour control.

Any other information on results incl. tables

The sensitivity of the test system was clearly demonstrated by the marked increases in the frequencies of micronucleated polychromatic erythrocytes induced by the positive control substance, cyclophosphamide, at the expected times after dosing. Reductions in the percentage of polychromatic erythrocytes were observed in the peripheral blood of cyclophosphamide treated animals. This is a response which is expected from a known cytotoxic drug.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Under the conditions of this test, acetonitrile was not clastogenic in the mouse bone marrow micronucleus test.
Executive summary:

In a guideline (OECD 474) and GLP study, 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 (100 mg/kg in males; 125 mg/kg in females).

Comparison of the percentage of polychromatic erythrocytes showed statistically significant increases, compared to the vehicle control values, in the bone marrow of acetonitrile treated males at the 18 hour sampling time and small increases, compared to the 0 hour control values, in the peripheral blood of males at 48 and 72 hour sampling times and in females at the 72 hour sampling time. These increases suggest that the bone marrow has been subjected to stress at maximally tolerated doses of acetonitrile.