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other: GLP compliant, guideline study, available as unpublished report. No restrictions, fully adequate for assessment.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

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
- Substance type:
- Physical state: liquid
- Analytical purity: 99% +
- Impurities (identity and concentrations):
- Isomers composition:
- Purity test date: Responsibility of sponsor
- Lot/batch No.: 50218009
- Expiration date of the lot/batch: Responsibility of 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 (e.g. ad libitum): SDS Rat & Mouse no. 1 SQC modified maintenance diet, Special Diet Services, Witham, Essex, UK.
- Water (e.g. ad libitum): Tap water provided in ploypropylene bottles
- Acclimation period: 4 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-23 ºC
- Humidity (%): 40-65 %
- Air changes (per hr): No details. Cages for each test group kept in separate ventilated cabinets.
- Photoperiod (hrs dark / hrs light): 12/12 (0730-1930)

IN-LIFE DATES: From: 12 July 1995 To: 7 February 1996

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Remarks on MMAD:

MMAD / GSD: Vapour - not applicable

Details on inhalation exposure:

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 - 61+ ºC. 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

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of test air were removed at approximate hourly intervals throughout each exposure, via sampling ports at the back of each chamber. Samples were analysed by gas chromatography for acetic anhydride concentrations.
Chamber spatial distribution was measured by rotating the sampling ports.
Nominal chamber concentrations were calculated from the quantity of liquid used over the 6-hour exposure period, the density of acetic anhydride and the exposure mean airflow.

Duration of treatment / exposure:

5 days/week for 13 weeks

Frequency of treatment:

6 h /day

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0, 1, 5, 20 ppm
Basis:
other: target concentrations

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0, 4.18, 20.9, 83.5 mg/m3
Basis:
other: target concentrations

Doses / concentrations 3

Remarks:

Doses / Concentrations:
0, 0.98, 4.96, 20.0 ppm
Basis:
analytical conc.

Doses / concentrations 4

Remarks:

Doses / Concentrations:
0, 1.23, 6.5, 26.3 ppm
Basis:
nominal conc.

No. of animals per sex per dose:

15/sex/group (including 5/sex/group for withdrawal period) - see Table 1.

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale: randomised according to bodyweight
- Rationale for selecting satellite groups: randomised with main study animals
- Post-exposure recovery period in satellite groups: 13 weeks

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: during exposure

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice daily on exposure and non exposure days

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION:
- Food consumption for each cage of rats determined weekly : Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: All animals prestudy, all main study animals in week 13, all withdrawal animals in week 26.
- Dose groups that were examined: All

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Weeks 13 and 26.
- Anaesthetic used for blood collection: Yes (light ether)
- Animals fasted: Yes
- How many animals: All main study animals in week 13 and all withdrawal animals in week 26.
- Parameters examined in week 13 (* - those examined in week 26): packed cell volume* (PCV), haemoglobin* (HB), red cell count* (RBC), total white cell count (WBC), differential white cell count (diff), cell morphology, platelet count (Plts), thrombotest* (TT) and reticulocyte count (retic).
The following indices were calculated : mean cell corpuscular haemoglobin concentration (MCHC), mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCH).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Weeks 13 and 26.
- Anaesthetic used for blood collection: Yes (light ether)
- Animals fasted: Yes
- How many animals: All main study animals in week 13 and all withdrawal animals in week 26.
- Parameters examined in week 13 (* - those examined in week 26): creatinine phosphokinase (CPK), total protein, albumin (Alb), Urea nitrogen* (Urea Nit), Alkaline phosphatase (AP), total bilirubin , creatinine*, sodium (Na), potassium (K), calcium (Ca), inorganic phosphorus (P), chloride (Cl), cholesterol* (Chol), glucose, glutamic-pyruvate transaminase* (GPT), glutamic-oxaloacetic transaminase* (GOT), gamma-glutamyltransferase (GGT).
Globulin was calulated by subtraction (total protein minus albumin).

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:

Sacrifice and pathology:

All main study animals were killed in the day following the completion of 13 weeks of exposure. Withdrawal animals were killed following 13 weeks withdrawal from exposures.
All animals were killed by intraperitoneal injection of pentobarbitone sodium followed by exsanguination.

GROSS PATHOLOGY: Yes

The following organs were weighed from all main study animals : adrenals, brain, kidneys, liver, lungs, ovaries, prostate, seminal vessicles, spleen, testes (with epididymides).

HISTOPATHOLOGY: Yes
The following tissues were examined from all main study and withdrawal rats : gross abnormalities, larynx, lungs, cervical lymph node, nasal passages, trachea.
The following tissues were examined from main study control and high dose groups only: adrenals, aorta, brain, caecum, colon, duodenum, epidiymides, eyes, heart, ileum, jejunum, kidneys, liver, mesenteric and tracheobronchial lymph nodes, mammary gland, oesophagus, ovaries, pancreas, pharynx, pituitary, prostate, salivary gland, sciatic nerve, seminal vessicles, skeletal muscle, skin, spinal cord, spleen, sternum, stomach, rectum, testes, thymus, thyroids (with parathyroids), urinary bladder, uterus.
The following tissues were submitted but not examined : femur with joint, head, optic nerve, oviduct, spinal column, tongue, ureter, vagina.

Statistics:

Bartlett's test (1937) for heterogeneity of variance between treatments, followed by logarithmic transformation and one-way analysis of variance or by the Kruskal-Wallis analysis of ranks (1952/3).
Except for pre-exposure data, aanalysis of variance was follwed by Student's t-test and Williams test (1971/2) for a dose related response. Kruskall- Wallis analyses were followed by Shirley's test (1977).
Where appropriate the analysis of covariance was used in place of analysis of variance.

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:

not specified

Details on results:

CLINICAL SIGNS AND MORTALITY
Partially closed eyes were observed during the first two exposures in high dose animals. At other times, noisy breathing with occasional instances of red staining around the snout were observed throughout most of the 13 week exposure period in high dose animals. Noisy breathing was observed in 1 male and 3 females in the intermediate group each on a single occasion. During the withdrawal period, noisy breathing was observed in a high proportion of high dose animals, but this progressively diminished and was no longer present in week 26.

BODY WEIGHT AND WEIGHT GAIN
During the 13 week exposure period, the weight gain by the high dose group was statistically significantly lower than that of the controls. There was also a small reduction in weight gain in the intermediate group but this was not statistically significant. During the withdrawal period, the weight gain of the intermediate males and the high dose males and females exceeded that of the controls, indicating a regression of effect.

FOOD CONSUMPTION
During the 13 week exposure period, food consumption by high dose rats was significantly lower than the controls. During the withdrawal period there were no differences from controls.

OPHTHALMOSCOPIC EXAMINATION
During week 13, corneal changes were evident in a number of animals in the intermediate and high doses groups and comprised corneal lack-lustre and corneal opacities (bi-lateral in less than half of the cases). Nasal quadrant keratitis was seen in 1 high dose female. There were no treatment related findings in week 26.

HAEMATOLOGY
Packed cell volume, haemoglobin concentration and total red cell numbers were significantly greater in high dose rats of both sexes than controls during week 13. Other differences were small and considered to be of no toxicological significance.

CLINICAL CHEMISTRY
During week 13, blood cholesterol levels were lower in high dose males and females than controls, but this was statistically significant in males only. Other differences were small and considered to be of no toxicological significance.

ORGAN WEIGHTS
After 13 weeks of exposure, lung weights were statistically significantly greater than controls in high dose males and females. After 13 weeks of withdrawal, the lung weights of male high dose rats were still significantly higher than controls but the difference was smaller than in week 14 and was no longer present in females.

Testes weights were also greater than controls but this was confined to the right testis only and, in the absence of any pathology findings, was considered to be incidental.

GROSS PATHOLOGY
Lungs- remained inflated in 5/10 males and 9/10 females in the high dose group. Areas of consolidation were evident in 2/10 males and 1/10 females in the high dose group, pale foci were seen in 5/10 high dose rats.
Adipose tissue - a reduction in adipose tissue was seen in 9/10 males and 7/10 females in the high dose group.

HISTOPATHOLOGY: NON-NEOPLASTIC - Table 2
A range of treatment-related changes were seen in the respiratory tract of both male and female animals from the high and intermediate dose groups. For the majority of theses lesions, a statistically significant increase in incidence compared to controls was demonstrated. These changes were predominantly localised inflammatory lesions with subsequent areas of epithelial hyperplasia and/or squamous metaplasia. In general, the changes were of minimal to moderate severity in the majority of high dose animals. Changes seen in occasional animals from the intermediate dose group were, with a few exceptions, minimal in degree.
An increased severity, but not incidence of occurence, of medullary plasmocytosis was seen in cervical lymph nodes and was considered to be associated with the inflammatory response and not directly caused by the inhaled material.
Changes seen in other organs were considered to be incidental to treatment with acetic anhydride. No changes were seen to correlated with or account for the ophthalmological or haematological changes and there were no changes that might account for the increased testicular weights at necropsy.
After the 13 week withdrawal period, all changes in the respiratory tract in the high dose group were reduced in incidence and severity. All changes were completely recovered in the intermediate group, with the exception of minor nasal passage lesions in 1 male.

Effect levels

Dose descriptor:

NOEL

Effect level:

1 ppm

Sex:

male/female

Basis for effect level:

other: Dose related histological changes in the respiratory tract after 13 week exposure to acetic anhydride. 1 ppm was a pathological no-effect level.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 2 - Summary of Treatment Related Histopathological Findings (Total severities)

	MALES				FEMALES
Target conc. (ppm)	0	1	5	20	0	1	5	20
Analysed conc. (ppm)	-	0.98	4.96	20.0	-	0.96	4.96	20.0
NASAL PASSAGES:
Exudative inflammation	0	0	1	9**	0	0	0	8**
Respiratory epithelium:
Inflammation	0	0	1	9**	0	0	0	8**
Granular eosinophilic inclusions	0	0	1	5*	0	0	0	3
Hyperplasia/prominent goblet cells	0	0	1	10**	0	0	1	10**
Squamous metaplasia	0	0	0	6**	0	0	0	4*
Transitional epithelium
Inflammation	0	0	3	8**	0	0	0	8**
Erosion	0	0	0	3	0	0	0	6**
Granular eosinophilic inclusions	0	0	7**	1	0	0	9**	3
Hyperplasia	0	0	9**	8**	0	0	8**	9**
Squamous metaplasia	0	0	1	9**	0	0	0	9**
LARYNX:
Subepithelial inflammatory cell infiltration	0	0	1	9**	0	0	3	4*
Ventrolateral squamous metaplasia	0	0	2	10**	0	0	1	10**
Arytenoid hyperplasia	0	1	5*	10**	0	0	0	10**
Arytenoid erosion/ulceration	0	0	0	9**	0	0	0	8**
TRACHEA:
Inflammation	0	0	0	9**	0	0	0	10**
Epithelial hyperplasia	0	0	1	9**	0	0	0	10**
Hyperplasia (carina)	0	0	0	8**	0	0	0	6**
Squamous metaplasia (carina)	0	0	0	6**	0	0	1	4*
LUNGS:
Perivascular inflammatory cells	0	0	0	6**	0	0	0	7**
Prominent BALT	0	0	0	4*	0	0	0	2
Alveolar duct fibrosis	0	0	0	5*	0	0	0	9**
Increased alveolar macrophages	0	0	0	3	0	0	0	5*

*p< 0.05, **p< 0.01 with Fisher’s Exact Text

Applicant's summary and conclusion

Conclusions:

Following subchronic inhalation exposure to vapours of acetic anhydride, 1 ppm was a pathological no effect level in rats.

Executive summary:

Exposure to acetic anhydride by inhalation at target concentrations of 1, 5 and 20 ppm, caused clinical signs, principally noisy breathing, consistent with exposure to an irritant atmosphere and reduced bodyweight and food consumption at 20ppm during the 13 week exposure period. A progressive recovery from these effects was seen during the 13 week withdrawal period. Corneal lesions were observed in most animals exposed to 20 ppm and in a few exposed to 5 ppm after 13 weeks of exposure. These findings were consistent with exposure to an irritant or mildly corrosive atmosphere and were not observed histologically. There were no treatment related changes after 13 weeks of withdrawal.

Histological changes in the respiratory tract after 13 week exposure to acetic anhydride were dose related and consistent with changes typical of a substance acting as a local/contact irritant. These changes were predominantly localised inflammatory lesions with subsequent areas of epithelial hyperplasia and/or squamous metaplasia and regressed during the withdrawal period, indicating that long-term damage had not been caused within the respiratory tract. 1 ppm was a pathological no-effect level.

Haematological changes seen in high dose animals in week 13 (increased packed cell volume, haemoglobin concentration and total red cell numbers, compared to controls) were considered to be an adaptive response to impaired gas exchange resulting from respiratory tract lesions. Lower blood cholesterol levels and reduced adipose tissue (seen macroscopically) were considered to be related to the lower food consumption seen in these animals.

Histological changes in other organs were considered to be incidental to treatment with acetic anhydride. No changes were seen to correlated with or account for the ophthalmological or haematological changes and there were no changes that might account for the increased testicular weights at necropsy. After the 13 week withdrawal period, all changes in the respiratory tract in the high dose group were reduced in incidence and severity. All changes were completely recovered in the intermediate group, with the exception of minor nasal passage lesions in 1 male. 

The no-effect level for this study was considered to be 1 ppm acetic anhydride.