Acetic anhydride

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study with GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

GLP compliance:

yes

Remarks:

Huntingdon Life Sciences

Test material

Test material information

Constituent 1

Reference substance name:

Acetic anhydride

EC Number:

203-564-8

EC Name:

Acetic anhydride

Cas Number:

108-24-7

Molecular formula:

C4H6O3

IUPAC Name:

acetyl acetate

Details on test material:

- Name of test material (as cited in study report): acetic anhydride
- Physical state: liquid
- Analytical purity: >99 %
- Storage condition of test material: ambient temperature, dry

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited
- Age at study initiation: 6-7 weeks
- Mean weight at study initiation: male 227 g, female 184 g
- Housing: 5 per cage
- Diet (e.g. ad libitum): SDS Rat and Mouse No 1 SQC modified maintenance diet
- Water (e.g. ad libitum): tap water


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20±3
- Humidity (%): 55±15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 2.43 m3, stainless steel and glass
- Source and rate of air: Air was passed through the vapouriser at a rate of 80 l/min
- System of generating vapor: The vapor was generated by metering the test substance from a polypropylene syringe mounted on an infusion pump to a glass frit contained in a glass vessel. In order to facilitate production of the vapor, the air to the vaporizer passed through a copper coil immersed in a water bath, maintained at 60 ± 1°C. The vapor/air mixture passed out of the vaporizer into the chamber inlet ducting. The vapor generating equipment was housed in an extracted cabinet. Different chamber concentrations were achieved by varying the syringe pump infusion rate. For all groups the vapor/air mixture produced passed along the chamber inlet ducting to a tangential inlet mounted at the apex of the chamber where it was mixed with additional diluent air at a rate (approximately 570 L/min) sufficient to maintain the total chamber airflow at approximately 650 L/min.
- Temperature, humidity in air chamber: 20.4-20.8 °C, 56.1-66.7 %


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

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6 h/day, 5 days/week

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
1, 5, 20 ppm
Basis:
nominal conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0.98, 4.96, 20.0 ppm
Basis:
analytical conc.

Doses / concentrations 3

Remarks:

Doses / Concentrations:
0.0042, 0.021, 0.085 mg/l
Basis:
other: calculated with conversion factor, 1 ppm = 0.00424 mg/l

No. of animals per sex per dose:

20

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: The target concentrations were selected based on a 2-week inhalation study.
- Post-exposure recovery period in satellite groups: 13 weeks

Examinations

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: clinical signs twice daily


BODY WEIGHT: Yes
- Time schedule for examinations: weekly


FOOD CONSUMPTION:
- Food consumption for each animal determined : Yes, weekly


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: week 13 and 26
- Dose groups that were examined: all groups


HAEMATOLOGY: Yes
- Time schedule for collection of blood: week 13 and 26
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes
- How many animals: all
- Parameters checked: PCV, Hb, RBC, MCHC, MCV, MCH, differential WBC, platelet, thrombotest, reticulocyte count


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: week 13 and 26
- Animals fasted: Yes
- How many animals: all
- Parameters checked: Creatinine phosphokinase (CPK), Total protein, albumin, globulin, urea nitrogen, alkaline phosphatase, total bilirubin, creatinine, sodium, potassium, calcium, inorganic phosphorus, chloride, cholesterol, glucose, glutamic-pyruvic transminase, Glutamic-oxaloacetic transaminase, y-Glutamyltransferase

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
All main group animals were killed on the day following completion of 13 weeks of dosing. Withdrawal group animals were killed following 13 weeks withdrawal from exposures. Animals were killed by intraperitoneal injection of pentobarbitone sodium and exsanguination from the brachial blood vessels. Immediately prior to exsanguination, the terminal body weight of each animal was recorded. All rats were subjected to a detailed macroscopic post mortem examination. The following organs were weighed: adrenals, lungs, seminal vesicles, brain, ovaries, spleen, kidneys, prostate, testes, liver, testes together with epididymides

Statistics:

Bartlett 's test, Student's t test and Williams' test

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

effects observed, treatment-related

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY: during exposure, signs were confined to the high dose. Partially closed eyes were observed during the first two exposures. This is a general, non-specific response to an irritant atmosphere. Subsequent to this, no other clinical signs were seen during exposure. At other times, treatment-related signs were seen in all high dose animals. Signs were principally noisy breathing with occasional instances of red staining around the snout. These sings were observed throughout most of the 13-week exposure period. Noisy breathing was also recorded in 1 male and 3 female from the intermediate dose each on a single occasion. During the withdrawal period, noisy breathing was observed in a proportion of high dose animals only. The incidence progressively diminished through the withdrawal period and was no longer observed during week 26.


BODY WEIGHT AND WEIGHT GAIN: During the 13-week exposure period weight gain by high dose rats was significantly lower than that of controls. A small reduction in rate of gain was also seen in Intermediate dose rats, however, this did not achieve statistical significance. Weight gain by low dose rats was similar to controls. During the withdrawal period, weight gain by intermediate dose males and high dose males and females exceeded that of controls indicating a regression of effect.


FOOD CONSUMPTION: During the 13-week exposure period consumption by High dose rats was significantly lower than that of controls. Consumption by Intermediate and Low dose rats was comparable to that of controls. During the withdrawal period consumption by all groups was similar.


OPHTHALMOSCOPIC EXAMINATION: During Week 13 corneal changes were evident in a number of animals in the intermediate (5 ppm) and high (20 ppm) dose level groups. Corneal lacklustre was recorded in three animals of each sex in the mid group and one male and one female in the high group. Corneal opacities were observed in one male animal in the mid group and in six males and nine females in the high group. Nasal quadrant keratitis was seen in one female animal in the high group. The corneal opacities were mostly pinpoint in size, and close to, or just below, the centre of the cornea. They were observed bilaterally in less than half of the cases. The incidence and distribution of the corneal changes is indicative of a treatment-related effect. Other ophthalmoscopic findings were within normal limits for animals of this age and strain and were considered to be unrelated to treatment. There were no treatment-related findings during the examination at Week 26.


HAEMATOLOGY: Packed cell volume, hemoglobin concentration and total red cell numbers were significantly greater in high dose rats of both sexes in comparison with controls during week 13 of exposure. The differences were considered to be treatment-related and were probably due to impaired gas exchange caused by respiratory tract lesions. Other statistically significant differences from controls were lower lymphocyte and large unstained cell number and a higher thrombotest time in high dose females. These differences were small, inconsistent between the sexes and considered unlikely to be of toxicological importance. During week 26 differences between groups were small and considered to be of no toxicological importance.


CLINICAL CHEMISTRY: During Week 13 of exposure blood cholesterol levels were lower in male and female High dose animals compared with controls. The difference was statistically significant in males only. The difference is considered to be treatment-related in that it probably reflects the reduced food consumption by these animals. Other significant differences from control were lower albumin in high dose males, higher urea nitrogen and creatinine in the high dose of both sexes; higher alkaline phosphatase in high dose females; higher GPT in high dose males; higher sodium levels in high dose females; lower calcium levels in high dose males and higher chloride levels in high dose females. All of these differences were minimal in scale and are considered unlikely to be of any toxicological importance. During Week 26 GPT and GOT levels were significantly greater in high dose females compared with controls. This finding is considered to be incidental. The values of all other parameters measured at week 26 were similar in all groups.


ORGAN WEIGHTS: After 13 weeks of exposure, the lung weights of High dose rats of both sexes were significantly (p<0.01) greater than those of controls. This is considered to be a consequence of exposure to acetic anhydride. Testicular weights in High dose males killed after 13 weeks of exposure were also significantly greater than controls, however, this occurred in the right testis only and since no abnormalities were seen during histopathological examination this finding is probably incidental. Following 13 weeks of withdrawal the lung weights of male High dose rats were significantly (p <0.05) greater than controls, however, the difference was smaller than that seen at week 14 and the difference was no longer present in females.


GROSS PATHOLOGY: The macroscopic examination performed at termination revealed the following changes:
Lungs - The lungs remained inflated when the thoracic cavity was opened (reported as not collapsed) in 5/10 male and 9/10 female rats treated with 20 ppm compared with 0/10 male and female control rats. Areas of consolidation were seen in 2/10 male and 1/10 female rats treated with 20 ppm compared with 0/10 male and female control rats. Pale foci were observed in 5/10 female rats treated with 20 ppm compared with 1/10 female control rats.
Adipose tissue - A reduction in adipose tissue was seen in 9/10 male and 7/10 females treated with 20 ppm compared with 0/10 male and female control rats.
The incidence and distribution of all other findings were considered to fall within the expected backround range of macroscopic changes. Macroscopic examination performed after 13 weeks of withdrawal revealed no changes associated with acetic anhydride.


HISTOPATHOLOGY: NON-NEOPLASTIC
Nasal passages - Lesions were primarily located in the anterior portion of the nose and comprised varying, predominantly minimal, degrees of hyperplasia of the respiratory epithelium, frequently with increased goblet cell prominence; inflammation associated with the respiratory epithelium and the presence of granular eosinophilic inclusions within the respiratory epithelium, possibly representative of globule leucocytes. In the transitional epithelium lining anterior portions of the nasal turbinates, hyperplasia, squamous metaplasia and inflammation, occasionally with areas of erosion, was seen together with the presence of granular eosinophilic inclusions which may represent the presence of globule leucocytes. These latter inclusions were more evident in animals from the intermediate dosage group than in those from the high dosage group where transitional epithelial squamous metaplasia was more prominent. In general, olfactory epithelium was unaffected by the acetic anhydride inhalation. Various amount of exudative inflammatory cell accumulation, predominantly minimal in degree, were seen in the nasal chambers of high dosage group animals and of single intermediate group animals.
Larynx - Inflammatory infiltration, squamous metaplasia of the ventral epithelium and hyperplasia of the epithpelim the ventral epithelium and hyperplasia of the epithelium covering the arytenoid processes, frequently with varying degree of erosion or ulceration, were seen in the majority of animals from the high dosage group as well as occasional animals from the intermediate dosage group. In general these changes were of minimal to moderate severity.
Trachea - Epithelial hyperplasia was seen at various sites, including at the carina, in the majority of animals from the high dosage group and in a proportion of intermediate dosage group animals. Also seen were epithelial squamous metaplasia at the carina; eosinophilic epithelial inclusions and inflammatory infiltration of the lamina propria. These changes were predominantly of a minimal degree of severity.
Lungs - Various changes were seen in a proportion of animals primarily from the high dosage group. These included the presence of occasional perivascular inflammatory cells, increased prominence of bronchus-associated lymphoid tissue (BALT), small foci of fibrosis in the walls of the alveolar ducts and increases prominence of alveolar macrophages. Changes were predominantly of minimal severity.
Cervical lymph nodes - An increased severity, primarily from minimal to moderate, but not incidence of occurrence, was seen in high dosage group animals in respect of parameters indicative of overall activity - specifically an increase in the degree of meullary plasmacytosis. Statistical significance was only achieved in male high dosage group animals. This change is probably associated with the inflammatory lesions of the respiratory tract.
Incidental changes (Termination)- The various other histological changes that were seen in animals from this study killed at termination were within the normal range of spontaneous lesions commonly encountered in animals of this species and age range and were, consequently, not considered to be of any toxicological significance. Some minor perturbation of the splenic white pulp and of the thymic cortex, predominantly seen in occasional high dosage group animals, was considered to be fortuitous and not
associated with treatment. No histological changes were seen to correlate with, or to account for the treatment-related ophthalmoscopical or haematological observations. No changes were seen that might account for the increased testicular weights at necropsy.
Recovery kill:
Treatment-related findings: The treatment-related changes that were seen in the respiratory tract of both male and female animals from the high and intermediate dosage levels at termination of dosing (see above) were diminished in both incidence and severity in the high dosage level group animals after the recovery period. With the exception of minor nasal passage lesions in a single male animal at the intermediate dosage level, regression was complete in this group. The principal findings are described below.
Nasal passages - After a period of recovery nasal passage lesions seen were reduced in both extent and severity and consisted of minor degrees of respiratory epithelium hyperplasia/goblet cell prominence with some transitional epithelial inflammation and localized hyperplasia, statistically significant only in male, high dosage group animals. Of the remaining lesions only respiratory epithelial hyperplasia/goblet cell prominence in males receiving the high dosage level was statistically significantly increased with relation to the control incidence.
Trachea - After a recovery period the treatment-related changes recorded in the trachea at termination were not detected.
Lungs - After a recovery period the lung lesions appeared to have resolved with the sole exception of an area of minor perivascular inflammatory cells in a single male animal previously receiving the high dosage level.
Cervical lymph nodes - After a recovery period there was no evidence of any maintenance of the increased incidence of plasmacytosis seen at termination.
Incidental changes (Recovery): the various other histological changes that were seen in animals from this study killed after a period of recovery were within the normal range of spontaneous lesions commonly encountered in animals of this species and age range and were, consequently, not considered to be of any toxicological significance.

Effect levels

Dose descriptor:

NOAEC

Effect level:

0.004 mg/L air

Sex:

male/female

Basis for effect level:

other: inflammatory, hyperplastic and metaplastic lesions of the respiratory tract in the higher dose groups

Target system / organ toxicity

Critical effects observed:

not specified