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Repeated dose toxicity: inhalation

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chronic toxicity: inhalation
combined repeated dose and carcinogenicity
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, guideline study, available as an unpublished report and a publication in the peer reviewed literature, no restrictions, fully adequate for assessment.
Reason / purpose:
reference to same study

Data source

Referenceopen allclose all

Reference Type:
study report
Reference Type:

Materials and methods

Test guideline
equivalent or similar to
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
GLP compliance:
Limit test:

Test material

Details on test material:
- Name of test material (as cited in study report): vinyl acetate
- Storage condition of test material: In stainless steel drums at ambient temperature in the dark
- Analysis of the bulk material showed that 3 of the 11 batches used had initial acetaldehyde levels higher than the 50 ppm (v/v) specified (52, 58 and 52 ppm (v/v)). These batches (only the first 2 used) were used for months 2 to 7 of the study. Other trace constituents were present at acceptable levels. The analysed levels of hydroquinone of ppm or less demonstrate the absence of added hydroquinone.
- No further details reported

Test animals

other: Crl:CD(SD)BR
Details on test animals and environmental conditions:
- Source: Charles River Uk Ltd, Margate, Kent, UK
- Age at study initiation: Approximately 6 weeks
- Weight at study initiation: 115.5-243.2 g (males), 127.6-189.1 g (females)
- Housing: Individually in stainless steel mesh cages suspended over cardboard-lined trays
- Diet: SQC Rat and Mouse Maintenance Diet No. 1, expanded (Special Diets Services, Witham, UK) ad libitum except during exposure and overnight prior to blood sample collection
- Water: Tap water ad libitum except during exposure and overnight prior to urine collection
- Acclimation period: 17 days

- Temperature: 15-29°C
- Humidity: 30-79%
- Air changes (per hr): At least 15/hr
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 5 January 1984 To: 6 February 1986

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
other: air
Details on inhalation exposure:
- Exposure apparatus: Mild steel and glass, cubic exposure chambers (external dimensions 2.1 x 2.1 x 2.1 m; approximately 8 m3 volume).
- Vapour generation: Liquid vinyl acetate was metered through positive displacement pumps to a nebulizer unit inside the expansion chamber of the generator module. The aerosol produced was mixed with diluent air and passed to the chamber as a vapour. Altering the flow rate of the metering pumps controlled the vinyl acetate concentration in the chamber.
- The chambers reached target concentrations within 15 minutes.
- Method of holding animals in test chamber: in stainless steel wire mesh cages
- Air flow rate: Highest value was 2430 litres/min. The flow rate fell below 1500 litres/min on one occasion only (1446 litres/min).
- The distribution of oxygen in the high dose chamber in the presence of animals gave a coefficient of variation over the whole chamber of 0.8.
- Source and rate of air: Air flow into chambers was drawn from filtered room air through a 6" filter
- Temperature, humidity in chamber: 17-27.3°C, 35.3-100% RH (recorded hourly during exposure)

- Brief description of analytical method used: Fully automatic monitoring system. Sampling and analysis from individual sites in the chambers and exposure room were controlled by a microprocessor. The vapour was analysed using a gas chromatograph fitted with a flame ionisation detector and database.
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
The daily mean concentrations of vinyl acetate in the low, intermediate and high dose chambers were within the following ranges: 43 to 57 ppm (v/v), 180 to 220 ppm(v/v) and 540 to 660 ppm (v/v), respectively, on at least 95% of the occasions.
The daily mean concentration of vinyl acetate in the room and control chamber did not exceed 1.9 ppm (v/v), the lower level of detection was 0.5 ppm (v/v) during the course of the study.
Duration of treatment / exposure:
6 hrs/day
Frequency of treatment:
5 days/wk, 104 weeks
Doses / concentrationsopen allclose all
Doses / Concentrations:
0, 50, 200 or 600 ppm v/v
nominal conc.
Doses / Concentrations:
0, 178, 714 or 2142 mg/m3 (as reported in EU RAR, 2008)
nominal conc.
No. of animals per sex per dose:
60/sex for the main study and 30/sex for satellite groups.
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: Based on the results of an earlier 90-day study.
- Satellite animals were used for laboratory investigations (except at wk 104) and interim kills of 10 males and 10 females from each group after 52 and 82 weeks of exposure.
- Following the results of the 52 week interim kill it was decided by the sponsor to institute a 16 week recovery group. Therefore, in week 70 the remaining satellite animals not designated for sacrifice after 82 weeks of exposure plus any required from the main study to make a total of 10/sex/group underwent a recovery period until being killed in week 85 or 86.


Observations and examinations performed and frequency:
- Time schedule: Immediately before and after exposure for a mortality and morbidity.

- Time schedule: All animals received a detailed examination, including a palpation for masses, once weekly. Any lesions or tissue masses observed at this examination were recorded for each animal and subsequent progress monitored. A record was kept to show the location, size and characteristics of any observed masses.

- Time schedule for examinations: Main study and satellite animals: Individual body weights were recorded pre-dose, at weekly intervals to week 28 and at 4 weekly intervals thereafter.
- Recovery animals: Individual body weights were recorded weekly during weeks 71 to 74 and at intervals of 4 weeks thereafter.




- Blood samples collected from satellite animals in weeks 51 and 81 and from main study animals in week 104. On these occasions the 10 male and 10 female animals with the highest identification numbers were used and were subsequently killed and necropsied so that no animal was sampled on more than one occasion.
- Blood samples were collected by orbital sinus puncture under halothane anaesthesia following an overnight period without food. All other satellite animals also had food withdrawn overnight.
- The following parameters were measured on blood collected into EDTA for rats (except PT which was measured on blood collected into 3.13% trisodium citrate): haemoglobin, mean cell volume, red blood cell count and indices: (cell haemoglobin, packed cell volume, cell haemoglobin concentration), total and differential white blood cell count, and prothrombin time.
- Two blood smears were prepared from the sample collected into anticoagulant. One smear was stained with Romanowski stain and examined microscopically for the differential white blood counts. The other smear was allocated for staining with brilliant cresyl blue for examination of reticulocytes.

- Blood samples collected from satellite animals in weeks 51 and 81 and from main study animals in week 104. On these occasions the 10 male and 10 female animals with the highest identification numbers were used and were subsequently killed and necropsied so that no animal was sampled on more than one occasion.
- Blood samples were collected by orbital sinus puncture under halothane anaesthesia following an overnight period without food for either species. All other satellite animals also had food withdrawn overnight.
- The following parameters were measured on blood collected into lithium heparin anticoagulant: glutamate oxaloacetate transaminase, glutamate pyruvate transaminase, alkaline phosphatase, creatinine phosphokinase, sodium, potassium, chloride, calcium, glucose, blood urea nitrogen, total protein, albumin, albumin/globulin ratio.

- Immediately after exposure on the day of sampling, the animals were allowed access to water for approximately 2 hours. They were then placed in individual urine analysis cages without food and water and an overnight urine sample collected. The following parameters were measured: volume, specific gravity, pH, protein, glucose, ketones, bilirubin, blood, urobilinogen, reducing substances, microscopy of spun deposits- Food and water were returned for approximately 2 hours after urine collection before the animals were exposed to the test article.


Sacrifice and pathology:
- A full internal and external examination was made including thoracic abdominal and cranial cavities and all lesions were recorded. All unusual macroscopic abnormalities were photographed.
- The scheduled necropsies (3 interim, 1 terminal) were carried out over periods of 4, 4, 11 and 18 days respectively.

- Adrenals, gonads, spleen, brain, liver, heart, kidneys, lungs, thyroids.

-The following tissues were submitted in appropriate fixatives: bone marrow, brain (medullary, cerebellar and cortical section), duodenum, eyes (and optic nerve), femur, Harderian glands, heart, ileum, jejunum, kidneys, larynx, lungs, mammary gland, muscle (quadriceps), nasal turbinates (4 or 6 levels), ovaries, pituitary, rectum, sciatic nerves, seminal vesicles, spinal cord (high cervical), stomach (glandular and forestomach), thyroids (with parathyroids, where identified), urinary bladder, gross lesions and tissue masses, caecum, colon, +ear canal, fallopian tubes, +ganglia (lumbar, sacral and dorsal), +hind limbs (post-distal portions for examination of tibial and plantar nerves), liver, lymph nodes (mandibular, mediastinal, mesenteric and bronchial), oesophagus, pancreas, prostate, salivary gland (submaxillary), skin, spleen, testes with epididymides, thymus (where present), tongue, trachea, uterus (including cervix), gross lesions and masses.
- All tissues listed, except for those indicated (+) from all animals in the control and high dose groups and from all animals that died or were killed in extremis were routinely processed, stained and examined.
- In addition, as the respiratory tract was identified as being the target organ, these tissues from animals in the low and intermediate dose groups were similarly processed and examined. Additionally gross abnormalities were examined from some animals in these groups. At the terminal kill special attention was given to the lungs with all 5 lung lobes being sectioned longitudinally in the plane of the major airways.

- Initially, 4 sections of the nasal turbinates were taken and examined. The locations of the sections was more closely defined and 2 further sections (making 6 in total) were taken and examined for all rats necropsied to week 52.
Statistical analysis was performed on survival, absolute body weights, body weight gain, necropsy body weight, haematological and blood clinical chemistry parameters and organ weights/organ to body weight ratios, and selected pathology findings.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Details on results:
- Non-specific clinical signs, associated with exposure to vinyl acetate, were rough haircoat and hunched posture seen at all concentrations.
- The incidence of superficial palpable masses in one of 4 categories (large or small and moveable or stationary) showed an increase in the small moveable tissue masses in all treated female rats from week 89 onwards, which reached a maximum in week 102 (5 in controls versus 20 and 22 in low and high dose). There were no other noteworthy differences between controls and treated animals.

- A statistically significant reduction in weight gain was noted for all high dose animals at all intervals examined. By week 104 the absolute weights showed reductions in the region of 10% for high dose male rats and 15% for the female high dose groups in comparison with the controls. There was no consistent pattern of body weight effect at the two lower exposure levels.
- During the recovery phase only high dose males showed a statistically significant improvement in weight gain.

- There were no treatment related changes in haematological parameters.
- Statistically significantly lower glucose levels were noted in high dose female rats at weeks 51, 81 and 104.

- A statistically significant reduction in urine volume was noted for all high dose rats at weeks 81 and 104, and high dose males at week 51. This was associated with increased specific gravity and decreased pH, which did not always reach levels of statistical significance. Similar effects (although not always statistically significant) were seen in the lower dose groups. This effect was thought to be related to reduced food and water consumption and not to be toxicologically significant.

-There was a statistically significant increase in lung weight in high dose animals.
-There were no other statistically significant effects on other organ weights.
- Recovery animals showed no differences in absolute or relative organ weights between the groups.

-Treatment-related respiratory tract lesions were seen in the intermediate and high dose groups.
- Olfactory atrophy was observed in the nasal cavity from week 53 onwards but showed little progression, and was considered to be associated with chronic irritation. Areas of olfactory atrophy showed re-epithelialisation, by either multilayered unspecialised cells or by respiratory epithelium. The respiratory epithelium was considered to represent in-growth from areas of the nasal cavity normally covered by respiratory epithelium. The unspecialised, multilayered epithelium may be derived from cells of the submucosal glands or their ducts proliferating to cover the damaged sites.
- Nasal cavity tumours were seen in eleven high dose rats. Eight of these tumours were considered to be benign and were diagnosed as papillomas. These tumours arose in a variety of sites within the nasal cavity and displayed a variety of histological patterns. There was no obvious correlation between site and histological appearance. Three nasal cavity tumours in high dose animals were diagnosed as squamous carcinomas, although one showed apparent malignant transformation of the stroma and could be regarded as a carcino-sarcoma. This latter tumour was considered to have caused the demise of the animal. All other nasal cavity tumours in high dose animals occurred in terminal kill animals.
- The nasal cavity tumours were considered to represent the end stage in a series of reactions to chronic irritation in the nasal cavity rather than direct genotoxicity. Predisposing lesions identified were atrophy of the olfactory epithelium, with re-epithelialisation by undifferentiated cells. These cells may be derived from the submucosal glands or their ducts, and the adenoma-like structure of some of the benign nasal cavity tumours may indicate that the tumours arose from cells of glandular origin. Nasal epithelial abnormalities were already present at 53 weeks, and tumour development was considered to be the result of continuing irritation of an already abnormal epithelium over the latter 12 months exposure.
- High dose animals also showed lung lesions considered to be due to exposure to the test article. Bronchioles showed focal exfoliation of the epithelium and the presence of structures termed fibroepithelial tags. The fibroepithelial tags were microscopic structures projecting into the bronchial lumen, comprising a thin fibrous septum covered by respiratory epithelium. They showed no progression, and may represent an end-stage lesion, possibly healed focal inflammation. Pigmented histiocyte accumulations were seen in lung parenchyma in high dose males, possibly representing the accumulation of dust-laden histiocytes due to impaired mucociliary clearance. The exposure-related lung lesions showed a relatively late onset, becoming apparent at the second interim kill, and were considered to represent chronic airways irritation.
- Two animals showed respiratory tract lesions of an uncertain relationship to exposure to the test substance. These lesions were a squamous carcinoma in larynx in a high dose sporadic, and a nasal cavity papilloma in an intermediate dose sporadic.
- The low incidence of mammary and pituitary tumours and associated better survival in high dose females, and the low incidence of glomerulonephropathy in high dose animals of both sexes reaching termination were considered to be due to reduced food consumption and body weight gain.

Effect levels

open allclose all
Dose descriptor:
Effect level:
50 ppm (nominal)
Basis for effect level:
other: (176 mg/m3 air). Non-neoplastic lesions in regions of nasal cavity lined by respiratory and olfactory epithelium; statistically significant at 200 and 600 ppm. (The conversion of ppm to mg/m3 is based on Rm of 86.09, 25 deg C, 1 atmosphere.)
Dose descriptor:
Effect level:
200 ppm (nominal)
Basis for effect level:
other: (704 mg/m3 air). Body weight depression at 600 ppm. (The conversion of ppm to mg/m3 is based on Rm of 86.09, 25 deg C, 1 atmosphere.)

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Following inhalation exposure to vinyl acetate vapours for 2 years, the NOEC in rats of both sexes was considered to be 50 ppm v/v, equivalent to 178 mg/m3 in air.
Executive summary:

In a 104 week inhalation toxicity study, 4 groups of rats were exposed for 6 hours per day, 5 days/week, to vinyl acetate vapour at concentrations of 0, 50, 200 and 600 ppm v/v. A significant reduction in body weight gain was seen throughout the study in males and females at 600 ppm. There was no evidence of systemic toxicity at 200 or 50 ppm. Rats exposed to 600 ppm or 200 ppm v/v vinyl acetate developed treatment-related lesions in the lungs, trachea and nasal epithelium, indicative of respiratory irritation. At 50 ppm (v/v) there were no microscopic changes to indicate respiratory tract irritation. 50 ppm is considered to be a clear no observable effect concentration (NOEC), equivalent to 176 mg/m³ in air.