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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:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting 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other: GLP compliant, guideline study, available as unpublished report.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:
1996
Report date:
1996

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Acetic anhydride
EC Number:
203-564-8
EC Name:
Acetic anhydride
Cas Number:
108-24-7
Molecular formula:
C4H6O3
IUPAC Name:
acetic anhydride
Details on test material:
- Name of test material (as cited in study report): Acetic anhydride
- Physical state: Liquid
- Analytical purity: > 99%
- Purity test date: Responsibility of the Sponsor
- Lot/batch No.: 50218009
- Expiration date of the lot/batch: Responsibility of the Sponsor
- Storage condition of test material: Ambient temperature, dry

Test animals

Species:
rat
Strain:
other: CD (Sprague-Dawley)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd, Manston Rd, Margate, Kent, UK
- Age at study initiation: 7-8 wks at randomisation
- Weight at study initiation: 227g (males), 184 g (females)
- Age at start of exposures: 10-11 wks
- Housing: 5/cage (same sex) in suspended stainless steel cages with wire mesh front, back and floor and stainless steel sheet sides
- Diet: SDS Rat & Mouse no. 1 SQC modified maintenance diet, Special Diet Services, Witham, Essex, UK. ad libitum (except during exposure)
- Water: Tap water provided in polypropylene bottles ad libitum (except during exposure)
- Acclimation period: 4 weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 17-23 ºC
- Humidity: 40-65 %
- Air changes (per hr): No details. Cages for each test group kept in separate ventilated cabinets
- Photoperiod: 12 hrs dark / 12 hrs light (07:30-19:30)

IN-LIFE DATES: From: 12 July 1995 To: 10 November 1995

Administration / exposure

Route of administration:
inhalation: vapour
Vehicle:
- Vehicle(s)/solvent(s) used: none
Details on exposure:
TYPE OF INHALATION EXPOSURE: whole body

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: chambers were of stainless steel and glass construction, internal volume of 2.43m3, consisting of a cuboidal main body fitted with pyramidal base and top
- Method of holding animals in test chamber: suspended wire-mesh baskets, each with capacity of hold 10 rats, individually with a stainless steel cover over each basket. 4 such baskets were suspended in the middle portion of the exposure chamber
- System of generating vapour: test substance was metered onto a glass frit contained in a glass vessel and air was passed through at a flow rate of 80 L/min. To facilitate vapourisation, the air to the vapouriser was passed through a copper coil maintained in a water bath at 59-61ºC. The vapour/air mixture then passed into the chamber inlet ducting, where diluting air was added to achieve the appropriate vapour concentrations
- Temperature, humidity, pressure in air chamber: study means; 20.4-20.8 ºC, 56.1-66.7%
- Air flow rate: approximately 650 L/min
- Treatment of exhaust air: chamber extract

TEST ATMOSPHERE
- Brief description of analytical method used: Gas chromatography
- Samples taken from breathing zone: yes

Duration of treatment / exposure:
5 days per week for 13 weeks
Frequency of treatment:
6 hr per day
Post exposure period:
None
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 1, 5, 20 ppm
Basis:
other: target concentration
Remarks:
Doses / Concentrations:
0.98, 4.96, 20.0 ppm
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
1.23, 6.5, 26.3 ppm
Basis:
nominal conc.
No. of animals per sex per dose:
10/sex in air control and acetic anhydride exposure groups. 5/sex in positive control group (see Table 1).
Control animals:
yes, sham-exposed
Positive control(s):
cyclophosphamide;
- Justification for choice of positive control(s): cyclophosamide is known to produce highly significant increases in the frequency of micronucleated immature erythrocytes together with decreases in the proportion of immature erythrocytes
- Route of administration: oral (gastric intubation of a standard dose volume of 10 mL/kg)
- Doses / concentrations: 40 mg/kg cyclophosphamide

Examinations

Tissues and cell types examined:
Bone marrow smears were prepared, stained and examined by light microscopy. The number of micronucleated cells per 1000 immature erythrocytes was determined for each animal and the proportion of immature erythrocytes for each animal was assessed by examination of at least 1000 erythrocytes. A record of the number of micronucleated mature erythrocytes observed during this assessment was also kept.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
The dose levels of acetic anhydride (1, 5 & 20 ppm) were those selected for the 13 week inhalation repeat toxicity study based on the results of a 2 week preliminary inhalation study.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields):
Bone marrow samples were taken from 10 male & 10 female rats in each main study group 24 hours after completion of the final exposure. For the positive control group, samples were taken from 5 males and 5 females 24 hours after dosing with cyclophosphamide.

DETAILS OF SLIDE PREPARATION:
One femur was dissected from each animal, cleaned of tissue and the contents eluted in 10 mL Hank's balanced salt solution by aspiration using a needle and syringe. The cell suspension was spun at 1000 rpm for 5 minutes. Each cell pellet was then resuspended in 2 mL filtered foetal calf serum before being sedimented out by centrifuge. The final cell pellet was resuspended in a small volume of foetal calf serum to facilitate smearing on a glass microscope slide. Slides were stained using a modified Feulgen staining method. Several slides were prepared from each animal but only 1 slide per animal from the negative control, the high dose group and the positive control groups was examined.

METHOD OF ANALYSIS:
The number of micronucleated cells per 1000 immature erythrocytes was determined for each animal and the proportion of immature erythrocytes for each animal was assessed by examination of at least 1000 erythrocytes. A record of the number of micronucleated mature erythrocytes observed during this assessment was also kept.


Evaluation criteria:
A positive response is indicated by a substantial, statistically significant increase (p <0.01) in the incidence of micronucleated immature erythrocytes compared to the incidence for the concurrent vehicle control group; individual and/or group mean values should exceed the laboratory historical control range.
A negative result is indicated where individual and group mean incidence of micronucleated immature erythrocytes for animals treated with the test substance are not significantly greater that the incidence for the concurrent control group and where these values fall within the historical control range.
An equivocal response is obtained when the results cannot be adequately classified using the criteria for a positive or negative response.

Statistics:
Non-parametric statistical methods, based on rank, were chosen for the analysis of results. Unless there was a substantial difference in response between sexes, results for the two sexes were combined to facilitate interpretation and maximise the power of statistical analysis.
For a comparison of an individual treated group with a concurrent control group, Wilcoxon's sum of ranks test was used.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Remarks:
see Table 2
Toxicity:
yes
Remarks:
noisy breathing, reduced weight gain and reduced food consumption in animals exposed to 20 ppm, during 13 week exposure period.
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Clinical signs were observed during the 13 week exposure period in animals exposed to 20 ppm acetic anhydride. These included partially closed eyes (observed during the first two exposures in high dose animals) and noisy breathing (with occasional instances of red staining around the snout) observed throughout most of the 13 week exposure period in high dose animals. Food consumption and weight gain was also reduced throughout the exposure period in these animals. All these signs resolved during the 13 week withdrawal period.

Any other information on results incl. tables

Table 2 Group totals/mean and results of statistical analysis

Treatment

Exposure Level

Ie / ie+me (mean %)a

Incidence mie (mean)a

Incidence mme (total)

Negative control

-

44

1.9

0.6

Acetic anhydride

20 ppm

41 ns

1.5 ns

0.6

Cyclophosphamide

40 mg/kg

18 **

14.1 **

0.0

ie / ie+me % - percentage of immature erythrocytes

mie - number of micronucleated cells observed in 1000 immature erythrocytes examined.

mme - number of micronucleated cells observed in 1000 mature erythrocytes examined

a - results of statistical analysis using Wilcoxon’s sum of ranks test:

ns - not significant - p> 0.01 {1 sided probabilities},  * - p< 0.01,  ** - p< 0.001}

Table 3 Historical control values

Cummulative total of results for vehicle control animals used in previous, unrelated experiments between September 1990 to November 1993:

Cummulative results for 90 individual animals:

mie

0

1

2

3

4

5

6

7

>7

Frequency

36

33

16

4

1

0

0

0

0

mie  The incidence of micronucleated cells per 1000 immature erythrocytes

Frequency - The number of times that result has been obtained

Applicant's summary and conclusion

Conclusions:
Acetic anhydride did not cause chromosomal damage in rat bone marrow following inhalation exposure at 20 ppm.
Executive summary:

Since the test substance did not cause any substantial increase in the incidence of micronucleated immature erythrocytes or any substantial decrease in the proportion of immature erythrocytes, it is concluded that acetic anhydride did not show any evidence of causing chromosome damage or bone marrow cell toxicity following subchronic inhalation exposure of rats in this in vivo test procedure.

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