Acetic anhydride

Endpoint summary

Description of key information

Local irritation/corrosion effects at the site of first contact (eyes and respiratory system) are the effects of concern for acetic anhydride.  A clear NOAEC of 1 ppm (4.18 mg/m3) was established in a guideline 13-week study (HRC, 1996).
The weight of evidence indicates that acetic acid will be very rapidly and extensively formed in vivo following exposure to acetic anhydride.  There is sufficient evidence from animal and human investigations with acetic acid, and from human studies on acetate intake and removal to conclude that systemic toxicity following exposure to acetic anhydride, at concentrations below those inducing local toxicity, is extremely unlikely.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

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other: GLP compliant, guideline study, available as unpublished report.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.

Reason / purpose for cross-reference:

reference to same study

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

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

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

Remarks:

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

Remarks:

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

Remarks:

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

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

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.

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.

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.

Critical effects observed:

not specified

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

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Study duration:

subchronic

Species:

rat

Quality of whole database:

The key study is a GLP compliant, guideline study of high quality (Klimisch score=1).

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

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.

Reason / purpose for cross-reference:

reference to same study

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

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

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

Remarks:

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

Remarks:

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

Remarks:

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

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

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.

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.

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.

Critical effects observed:

not specified

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

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

4.2 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

The key study is a GLP compliant, guideline study of high quality (Klimisch score=1).

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Inhalation

Non-human data

The key studies are two GLP-compliant and well-conducted rat inhalation studies (HRC, 1994 & 1996). 

Inhalation exposure was only possible for a single 6-hour period due to severe toxicity at the site of first contact at 400 ppm (1670 mg/m3) (HRC, 1994). At a concentration of 100 ppm (418 mg/m3), 6hr/day for up to 7 days, moderate to severe inflammatory damage throughout the respiratory tract and increased mortality were reported (HRC, 1994). At 20 ppm (83.5 mg/m3) hr/day for 13 weeks, histopathological findings indicative of chronic inflammation were reported in the eyes and entire respiratory tract of the majority of animals. The changes were predominantly localized 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 animals exposed to 20 ppm, i.e the high dose group. At the intermediate concentration, 5 ppm, similar but lower-grade (of minimal severity with few exceptions) histopathological findings were restricted to the upper respiratory tract (nasal passages and larynx) and eyes (HRC, 1996). At 1 ppm (4.18 mg/m3), with exposure for 6hr/day for 13 weeks, no clinical or histopathological findings were reported (HRC, 1996). With the rather weak response at 5 ppm and the robust NOAEC at 1 ppm, in practice the threshold for irritation would be expected to be nearer 5 ppm than 1 ppm.

Both studies indicate that the effects of acetic anhydride are restricted to sites of first contact, (eyes and respiratory system) for which a noticeable dose-response relationship was established (see summary table below). A clear NOAEC of 1 ppm (4.18 mg/m3) was reported following inhalation exposure 6hr/day for 13 weeks to rats in a guideline study (HRC, 1996).

No studies are available for the oral and dermal routes of exposure.

Dose-response relationship for local effects induced by acetic anhydride

Concentration ppm (mg/m3)	Time	Effect
400 (1670)	Single 6hr exposure	Severe respiratory effects, increased mortality at 24 hours
100 (418)	6hr/day up to 7 days	Moderate – severe inflammatory damage, throughout respiratory tract and in eyes, increased mortality by day 7
20 (83.5)	6hr/day, 13 weeks	Chronic inflammation entire respiratory tract and in eyes, no mortality
5 (20.9)	6hr/day, 13 weeks	Low-grade inflammatory response restricted to upper respiratory tract and eyes
1 (4.18)	6hr/day, 13 weeks	Clear NOAEC

Systemic effects

In relation to potential systemic effects of repeat dose exposure, any acetic anhydride absorbed would result in rapid formation of acetic acid and absorption and systemic exposure to the acetate ion.

Acetic acid, as an active substance, has been considered under the fourth stage of the re-evaluation programme of the Plant Protection Product Directive 91/414/EEC (provisions of Regulation (EC) No 1112/2002) for use as a herbicide on e. g. pome and stonefruit, ornamental shrubs, paths. The decision has been made that it is included in Annex I of that Directive (Commission Directive 2008/127/EC of 18 December 2008 amending Council Directive 91/414/EECto include several active substances) and therefore the use of acetic acid as a Plant Protection Product it is therefore regarded as registered under REACh.

EFSA supported the recommendations by the rapporteur Member State and the result of the examination in accordance with the provisions of Article 24a of Regulation 2229/2004 stating that there are clear indications that it may be expected that this use of acetic acid does not result in any harmful effects on human health. The Draft Assessment Report (EUDAR, 2008) states that “Long-term toxicity/carcinogenicity studies in animals with oral exposure are not necessary, considering that humans are exposed to orally ingested acetic acid from various food sources and there is no evidence that such exposure is causally related to toxic effects and an increased cancer incidence. Therefore, no new animal toxicity studies conforming to long-term toxicity or carcinogenicity test guidelines are necessary for Annex I inclusion of acetic acid. ”

Considering exposure to acetic anhydride at an atmospheric concentration of 1 ppm (4.18 mg/m3), the proposed NOAEC for local irritation effects (4.2 mg/m3), systemic exposure to acetate at this concentration is insignificant. For an 8 hour day spent at light work, and assuming 100% absorption of acetic anhydride/acid for a worker would be 4.2 mg/m3 * wRV m3/kg bw * [mw acetic anhydride / acetic acid] = 4.2 * 0.144 * [102.09 / 60.05] = 1.03 mg/kg bw. To put this intake into perspective with the known removal of acetate, it has been shown that about ~0.5 mg/kg bw acetate can be removed each minute via endogenous pathways, such as the citric acid cycle, in humans following administration of acetate in a drink (Smith et al., 2007). Daily administration of 40 mg/kg bw/day may be used as a medicinal product (Johnston & Gaas, 2006), and 25 mg/kg bw /day estimated as average human (infant) dietary intake (Ishiwata et al., 2002), with peak excursions up to 240 mg/kg bw /day (EU DAR, 2008).

Human data

No relevant human information available

Citations

European Union Draft Risk Assessment (2008) Initial risk assessment provided by the rapporteur Member State Germany for the existing active substance Acetic acid of the fourth stage of the review programme referred to in Article 8(2)of Council Directive 91/414/EEC.

Ishiwata H et al. (2002). Daily intake of food additives in Japan in five age groups estimated by the market basket method. Eur Food Res Technol 215: 367-374.

Johnston CS & Gaas CA (2006): Vinegar: Medicinal Uses and Antiglycemic Effect. Med Gen Med. 8(2): 61

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
There are 2 key studies for repeat dose inhalation. This study was selected as it had the longest duration of exposure (90 days).

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
This study was selected as it had the longest duration of exposure (90 days) and a clear NOEAL was identified based on histopathology findings.

Justification for classification or non-classification

There is sufficient evidence to conclude that systemic toxicity is very unlikely and hence there is no justification for classification based on systemic toxicity.

The observed effects (local irritation/corrosion) are appropriately covered by corrosivity and STOT-SE classifications.