Isopropenyl acetate

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2000-08-17 to 2002-05-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD Guideline study report

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Deviations:

yes

Remarks:

with scope of examinations according to OECD - Guideline 407and parts of 41 3

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Isopropenyl acetate

EC Number:

203-562-7

EC Name:

Isopropenyl acetate

Cas Number:

108-22-5

Molecular formula:

C5H8O2

IUPAC Name:

isopropenyl acetate

Details on test material:

Designation of test substance: 2-Acetoxypropene (Essigsaureisopropenylester)
Chemical name: 1-Propen-2-o1 acetate, Isopropenylacetate
CAS No.: 108-22-5
Aggregate state/ appearance: liquid / colorless
Storage conditions: room temperature, in closed containers

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Male and female Wistar rats (strain: CrIGlxBrIHan:WI) were supplied by Charles River Laboratories, Deutschland, Sandhofer Weg 7,97633 Sulzfeld.
The female animals were nulliparous and non-pregnant. Only animals free from clinical signs of disease were used for the study.
During the period when the rats were not exposed they were housed singly in wire cages (type OK Ill, Becker &Co., Castrop-Rauxel, FRG (floor area about 800 cm2 )). Underneath the cages, waste trays were fixed containing bedding material (type 3/4 dust free embedding,
supplied by SSNIFF, Soest, FRG). The motor activity measurements were conducted in Polycarbonate cages with wire covers from Ehret, Emmendingen, FRG (floor area about 800 cm2 ) and bedding. The animals were kept in fully air-conditioned rooms in which a temperature in the range of
20 - 24°C and relative humidity in the range of 30 - 70% were ensured by means of a central air-conditioning system.
A light/dark rhythm of 12 hours was maintained. At the start of the exposure period (day 0) the overall mean body weight calculated from the
group means and the ranges of individual weights 0 were: male animals 194 (184.7 - 206.7) g; female animals 154 (1 47.5 -167.2) g.

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Remarks on MMAD:

MMAD / GSD: not applicable

Details on inhalation exposure:

Generation of the inhalation atmospheres:
- Piston metering pumps (Sarstedt DESAGA)
-Glass evaporator with thermostat (BASF Aktiengesellschaft)
Generation procedure:
For each concentration, the test substance was supplied to the two-component atomizer of a thermostated vaporizer at a constant rate by means of the metering pump. The vapor / air mixture was generated by spraying the substance with compressed air into a counter cur-rent of conditioned supply air (about 50% ± 20% relative humidity, 22°C ± 2°C). Thereafter it was further mixed with conditioned supply air and passed into the inhalation system. The following values of test pump rates, air flows and evaporation temperatures were sched-uled.

The animals were kept singly in wire cages located in a glass-steel inhalation chamber,
volume of 1.4 m3.
In order to accustom the animals to the exposure conditions they were exposed to sup-ply air in whole-body exposure systems on 2 days before the exposure period (preflow period). Then all test groups were exposed for 6 hours on workdays over a time period of 28 days (subacute study). The number of exposure days was 20. The animals did not have access to water or feed during the exposures.
A positive pressure was chosen for all test groups during the preflow period. Exposure period: Control test group : A positive pressure was maintained by adjusting the air flow of the exhaust air system. This ensured that no laboratory air reached the control ani-mals. Test groups with IPA dosage: A negative pressure was maintained by adjusting the air flow of the exhaust air system. This ensured that the laboratory was not contami-nated as the result of any leakage from the inhalation chamber.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical determination of concentrations
Principle: The concentrations of the inhalation atmospheres were analyzed by gas chroma-tography. Daily means were calculated based on 2 measured samples per concentration and exposure in test groups 1-3 and one sample per week was measured the control group. From the daily mean values of each concentration, mean concentrations and standard de-viations for the entire study were derived.
The constancy of concentrations in each inhalation system (except control test group) was continuously monitored by total hydrocarbon analyzers.

Sampling for gas chromatographic analyses
Equipment:
• Sampling probe (glass), internal diameter: 4 mm
• 3 absorption vessels (glass)
• Gas sampler GS 312 (Desaga)
Sampling:
• Sampling velocity: 1.25 m/s
• Flow rate of sampling: 1 I/min
• Sample volumes: 5 I
Test group 0 - 3
• Sampling site: immediately adjacent to the animals' noses
• Sampling frequency: as a rule, 2 samples per concentration and exposure, and one sam-ple per week was measured the control group.
The samples were drawn through the absorption vessels connected in series, each of which was filled with Dimethylformamide as absorption solvent. After the sampling, the content of the first 2 absorption vessels was eluted and pooled into a 50 ml graduated flask for individ-ual analysis.
After the final sampling on each exposure day, the content of the last absorption vessel was transferred to a 50 ml graduated flask and analyzed separately to check for the absorbing efficiency of sampling.
Equipment:
Gas chromatograph: Hewlett-Packard 5880 A
Column:glass
Length: 2m
Internal diameter: 2mm
Separation phase:10% Ucon LB 550X
Support: Chromosorb W AW DMCS HP 100/120 mesh

The following operating conditions were used:
Carrier gas:He
Carrier gas flow rate:28.1 ml/min
Hydrogen: 31 mUmin
Air: 360 ml/min
Furnace temp. start: 60° C (after 7 minutes 170° C)
Detector: FID
Detector temperature: 200°C
Injector temperature:200°C
Calibration of the gas chromatograph, Method set up:During the set up of the analytical procedure a calibration curve was prepared in the solvent with the test substance to be in-vestigated to show linearity in the suitable concentration range of the samples. The gas chromatograph was calibrated by means of calibration solutions of the test substance in the solvent. Accurately weighed amounts of test substance in concentrations ranging from 31.1 to 50.5 mg/50 ml were used for this purpose. A calibration curve was calculated (curve ad-justed using the least squares method) showing highly linear correlation.
Routine analysis during test period: Calibration curves of the analytical procedure were pre-pared based on the method described above (details are available in the raw data).

Duration of treatment / exposure:

6 hours on workdays over a time period of 28 days (subacute study). The number of exposure days was 20.

Frequency of treatment:

6 hours on workdays over a time period of 28 days (subacute study). The number of exposure days was 20.

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 207.5, 830 and 2075 mg/m3 (0, 50, 200, and 500 pp)
Basis:
nominal conc.

No. of animals per sex per dose:

5

Control animals:

yes

Details on study design:

Exposure to the high concentration is expected to cause signs of some systemic toxicity and distinct irritation of the respiratory tract in a 28-day study. The intermediate concentration may still lead to some irritation in the upper respiratory tract but is expected to represent the systemic NOAEL. The low concentration represents the expected overall NOAEL for a 28-day inhalation study.

Positive control:

not applicable

Examinations

Observations and examinations performed and frequency:

An observation of the general state of health of the animals as well as a check for dead or moribund animals was performed twice a day on working days or once a day on weekends or public holidays, respectively. Clinical examinations of the test animals were carried out on workdays at least 3 times on exposure days and, as a rule, once during the preflow pe-riod and the post-exposure day. During exposure only a group wise examination was possi-ble. Detailed clinical observations (open field observations) were performed on study days -1,6, 13 and 20. The findings were ranked according to the degree of severity, if applicable. The animals were transferred to a standard arena (50 x 37,5 cm with sides of 25 cm high). The following parameters were examined: Behaviour when removed from cage, Fur, Skin, Posture, Salivation, Respiration, Activity/arousal level, Tremors, Convulsions, Abnormal movements, Impairment of gait, Lacrimation, Palpebral closure, Exophthalmus, Feces (ap-pearance/consistency), Urine, Pupillary size
The body weight of the animals was determined at the start of the preflow, at the start of the exposure period and then, as a ru le, once a week. The animals which underwent neuro-functional testing were additionally weighed on the days of examination. As a rule, the ani-mals were weighed at the same time of the day. Body weight change was calculated as the difference between body weight on the respective exposure day and body weight on the day of the first exposure. Group means were derived from the individual differences.
Before the start of the exposure period (day -2) the eyes of all animals, and towards the end of the study (day 25) the eyes of the animals of test group 0 (control group) and of test group 3 (high concentration) were examined with an ophthalmoscope (HEINE Optotechnik, Herrsching, FRG)) for any changes in the refracting media.
A functional observation battery (FOB) was performed in all animals at the end of the expo-sure period (day 28), starting in the morning. The FOB consisted of 4 parts, starting with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The ani-mals were examined in randomized order.
The animals were observed in their closed home cages; any disturbing activities (touching
the cage or rack, noise) were avoided during these examinations in order not to influence the behavior of the animals. Attention was paid to: Posture, Tremors, Convulsions, Abnor-mal movements, Impairment of gait, General observations (all other findings) .
The animals were transferred to a standard arena (50 x 50 cm with sides of 25 cm high) andobserved for at least 2 minutes. Following parameters were examined: Behaviour when removed from cage, Fur, Skin, Posture, Salivation, Respiration, Activity/arousal level, Tremors, Convulsions, Abnormal movements, Impairment of gait, Lacrimation, Palpebral closure, Exophthalmus, Feces (number of fecal pellets/appearance/consistency) within two minutes, Urine (volume/ color) within two minutes, Number of rearings within 2 minutes.
The animals were removed from the open field and subjected to following sensorimotor o reflex tests: Approach response, Touch response, Vision ("visual placing response"), Pupil-lary reflex, Winking reflex, Pinna reflex, Audition ("startle response"), Olfaction, Examination of catalepsy (descending from box), Coordination of movements ("righting response"), Be-haviour during "handling", Vocalization, Pain perception ("tail pinch"), Grip strength of fore-limbs, Grip strength of hindlimbs, Landing foot-splay test, General observations (all other findings).
Motor activity (MA) was measured on the day when FOB was performed. The measurement was performed in the dark using the Multi-Varimex-System (Columbus Instruments Int. Corp. , Ohio, USA) with 4 infrared beams per cage. During the measurement the animals were kept in Polycarbonate cages with absorbent material.The number of beam interrupts were counted over 12 intervals, each lasting 5 minutes. Measurements were carried out during late morning. The animals were put into the measurement unit in a randomized order at about 11 a.m. Measurement did not commence at the same instant for all cages; the pe-riod of assessment for each animal started when the first beam was interrupted by pushing the cage into the rack (staggered start). Measurements ended exactly 50 minutes thereaf-ter. During the measurements the animals received no food and no water. The cages were cleaned prior to each use.

Sacrifice and pathology:

The animals were killed under narcoren® anesthesia by exsanguination from the abdominal aorta and vena cava. The animals were necropsied and macroscopically assessed.

Other examinations:

not reported

Statistics:

Non-parametric one-way analysis using KRUSKAL-WALLlS test (two-sided).If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group blood count was performed using Wilcoxon-test (two-sided) for the equal medians.
Means and standard deviations of each test group were calculated for the variables of terminal body weight and of absolute and relative organ weights (related to terminal body weight) of the animals in each test group.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

Test group 3 (2075 mg/m3 (500ppm)):
• Low arousal during exposure
• (multi)focal hyperplasia of the transitional epithelium in level I of the nasal cavity
• (multi)focal degeneration of the olfactory epithelium in levels 11 and III of the nasal cavity
• (multi)focal inflammatory cell infiltration In levels I and 11 of the nasal cavity
Test group 2 (830 mg/m3 (200 ppm))
• (multi)focal inflammatory cell infiltration in level I of the nasal cavity
Test group 1 (207.5 mg/m3 (50 ppm))
• (multi)focal inflammatory cell infiltration in level I of the nasal cavity

Effect levels

1

Dose descriptor:

NOAEC

Remarks:

for systemic toxicity

Effect level:

2 075 mg/m³ air

Sex:

male/female

Basis for effect level:

other: overall effects

2

Dose descriptor:

NOAEC

Remarks:

for local effects

Effect level:

830 mg/m³ air

Sex:

male/female

Basis for effect level:

other: overall effects

3

Dose descriptor:

NOAEC

Remarks:

for slight irritancy

Effect level:

< 200 mg/m³ air

Sex:

male/female

Basis for effect level:

other: NOAEC for slight irritancy leading to nasal epithelial inflammatory cell infiltration in some of the animals could not be established

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The NOAEC for systemic toxicity under the condition of this study was 2075 mg/m3 (500 ppm). The NOAEC for hyperplasia of nasal
transitional and degeneration of olfactory epithelium was 830 mg/m3 (200 ppm).

Executive summary:

Five male and five female Wistar rats per test group were whole body exposed to dynamic at-mospheres of 2-Acetoxypropene (Essigsaeureisopropenylester) vapours for 6 hours per working day for about 28 days (20 exposures). The target concentrations were 207.5, 830 and 2075 mg/m3 (about 50; 200 and 500 ppm. A concurrent control group was exposed to clean air. On exposure days clinical examination was performed before, during and after exposure. During preflow period and on post exposure day clinical findings were recorded once each working day. Additionally the general state of health was controlled twice on workdays and once on weekends or holidays. Body weight of the animals was determined weekly. Ophthalmology was carried out prior to and at the end of exposure period. Detailed clinical examinations (open field observa-tion; OFO) were performed before start of exposure period and three times during exposure pe-riod in about weekly intervals. Neurofunctional tests comprising functional observational battery and motor activity measurements were performed at the end of exposure period. Hematological and clinicochemical examination of numerous parameters as well as urinalysis was performed at the end of the study as required by the corresponding test guidelines. A complete necropsy in-cluding weighing of selected organs and gross pathological evaluation was performed. Several organs and tissues were examined histopathologically as required by the corresponding test guidelines (OECD - Guideline method 412 with scope of examinations according to OECD - Guideline 407 and parts of 413).

The following study means of concentrations were determined analytically using gravimetry: 209.1 ± 12.3 (50.4); 809.0 ± 22.4 (195); 2011 ± 65.3 (485) mg/m3 (ppm).

The following substance related effects were produced by inhalation of the test substance:

Test group 3 (2075 mg/m3(500ppm)): Low arousal during exposure, (multi)focal hyperplasia of the transitional epithelium in level I of the nasal cavity, (multi)focal degeneration of the olfactory epithelium in levels II and III of the nasal cavity, (multi)focal inflammatory cell infiltration in levels I and II of the nasal cavity

Test group 2 (830 mg/m3(200 ppm)): (multi)focal inflammatory cell infiltration in level I of the nasal cavity

Test group 1 (207.5 mg/m3(50ppm)): (multi)focal inflammatory cell infiltration in level I of the nasal cavity

Inhalation exposure of Wistar rats to 2075 mg/m3 (500 ppm) of the test substance vapours re-sulted in clear upper respiratory tract irritation as demonstrated by the presence of epithelial hy-perplasia or degeneration. The finding of inflammatory cell infiltration in the mid and low con-centration are also interpreted as mild reaction to the irritant properties of the inhaled vapours. No signs of systemic toxicity occurred at any concentration.

Thus the No Observed Adverse Effect Concentration (NOAEC) for systemic toxicity under the condition of this study was 2075 mg/m3 (500 ppm). The NOAEC for hyperplasia of nasal transitional and degeneration of olfactory epithelium was 830 mg/m3 (200 ppm). A NOAEC for slight irritancy leading to nasal epithelial inflammatory cell infiltration in some of the animals could not be established.

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