Isopropyl acetate

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:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.29 (Sub-Chronic Inhalation Toxicity:90-Day Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Propyl acetate

EC Number:

203-686-1

EC Name:

Propyl acetate

Cas Number:

109-60-4

IUPAC Name:

propyl acetate

Details on test material:

n-propyl acetate, spectroscopic grade (>99.9%)

Specific details on test material used for the study:

Name of test substance: n-Propyl acetate
Test substance No.: 15/0569-3
Batch identification: 83294456P0
CAS No.: 109-60-4
Purity: n-Propylacetat: 99.9 area-% (Study No. 17L00019)
Homogeneity: given
Storage stability: Expiry date: 10 Dec 2017
The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility.

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Wistar rats, Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH; Sandhofer Weg 7, 97633 Sulzfeld
- Females (if applicable) nulliparous and non-pregnant: [yes]
- Age at study initiation: about 7 weeks, male and female (age when supplied)
- Weight at study initiation: animals of comparable size and weight
- Housing: group-housing (upt o 5 animals/cage)
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 14 days

DETAILS OF FOOD AND WATER QUALITY:
The food used in the study was assayed for chemical as well as for microbiological contaminants.
The drinking water is regularly assayed for chemical contaminants by the municipal authorities of Frankenthal, Germany and the Environmental Analytics Water/Steam Monitoring of BASF SE as well as for the presence of microorganisms by a contract laboratory.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20- 24°C
- Humidity (%): 30-70%
- Air changes (per hr): 15 per hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

air

Remarks on MMAD:

The test item was present as vapor under the current test condition. No particle size measurement was performed.

Details on inhalation exposure:

GENERATION OF THE INHALATION ATMOSPHERES
Generator systems:

• Piston metering pumps (DESAGA; SARSTED AG & Co, Nürnbrecht, Germany)
• Thermostat (JULABO Labortechnik GmbH, Seelbach, Germany)
• Atomization vaporizer (glass) with thermostat (BASF SE, Ludwigshafen, Germany)

Generation procedure:

The test substance was used unchanged.

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 current 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 test pump rate/s /substance flow, air flow/s and evaporation temperature/s were scheduled:

Test group Pump rate (mL/h) Evaporation temperature (°C) Supply air 1 conditioned(m³/h) Supply air 2 compressed (m³/h) Supply air 3 conditioned (partial flow through generator) (m³/h) Exhaust air (m³/h)
0 - - 14.5 – 18.5 - - 15.5 – 19.5
1 5-20 38 - 42 14.5 – 18.5 0.8 -1.2 4.0 – 8.0 15.5 – 19.5
2 30 - 50 38 - 42 14.5 – 18.5 0.8 -1.2 4.0 – 8.0 15.5 – 19.5
3 100 - 150 38 - 42 14.5 – 18.5 0.8 -1.2 4.0 – 8.0 15.5 – 19.5


Conditioned supply air is activated charcoal filtered air conditioned to about 30% to 70% relative humidity and 20°C to 24°C in the control group and 20°C to 26°C for test groups 1 to 3. Compressed air is filtered air pressurized to about 5 bar.

Exposure systems; exposure of the animals
Air conditions
Conditioned air:
The central air conditioning system provides cold air of about 15°C. This cold air passes through an activated charcoal filter, is adjusted to room temperature of 20 to 24°C and passes through a second particle filter (H13 (HEPA) Camfil Farr, Germany). The so generated conditioned air was used to generate inhalation atmospheres.
Compressed air:
Compressed air was produced by an oil-free compressor (HT 6, Josef Mehrer GmbH & Co KG, Germany). For this purpose, air is filtered by an inlet air strainer and introduced into the compressor. After passing through an second ultra filter (SMF 5/3, 108 mm, Donalson), the compressed air (15 bar) is stored in a storage of 1500 or 5000 L. The compressed air is conducted to the laboratories via pipes, where the pressure is reduced to 6 bar. In the laboratory, the compressed air can be taken as required.
Exhaust air:
The exhaust air was filtered and conducted into the exhaust air of the building.

Whole body exposure systems

The animals were kept singly in wire cages located in a glass steel inhalation chamber, volume of 1.1 m³ (BASF SE). The chambers were located in exhaust hoods in an air conditioned room.

Exposures

In order to accustom the animals to the exposure conditions they were exposed to supply air in whole body exposure systems on 2 days before the exposure period (pre-exposure period). Then all test groups were exposed for 6 hours on workdays over a time period of 90 days (90 day study). The number of exposure days was 65.

The animals did not have access to water or feed during the exposures.

A negative pressure was maintained by adjusting the air flow of the exhaust air system. This ensured that the laboratory was not contaminated as the result of any leakage from the inhalation chamber.

For same exposure conditions, the cages with the animals were rotated between the levels within each chamber.


Measurements of the exposure conditions
Recording of exposure parameters was performed according to the following table.
Recording of exposure parameters
Exposure parameters Determination method Recording
Supply air 1 (conditioned) orifice plate with differential pressure measurement automated system
Supply air 2 (compressed) orifice plate with differential pressure measurement automated system
Supply air 3 (conditioned) partial flow through generator Rotameter once per exposure
Exhaust air 1 orifice plate with differential pressure measurement automated system
Chamber pressure differential pressure measurement automated system
Chamber relative humidity Dielectric probe (Testo) automated system
Chamber temperature Thermosensor automated system
Generator temperature Thermosensor automated system
Pump rate Reading from pump display once per exposure
Concentration surveillance Online microGC automated system

 
No surveillance of the oxygen content in the inhalation system was performed. The air change within the inhalation systems was judged to be sufficient to prevent oxygen depletion by the breathing of the animals and the concentrations of the test substance used could not have a substantial influence on oxygen partial pressure.

Principles of recording with the automated measuring system:

Each parameter was measured at appropriate measuring points using suitable measuring equipment (sensors, orifice plates etc.). The measurements were standardized (0 20 or 4 20 mA) and transferred to instrumentation consoles. There, the measured values were displayed in an analogous way (where this is provided for) and some were used as actual value for regulating the specific parameter.

In addition, the measured values were scanned every 10 seconds, converted from analog to digital, transferred to a personal computer, displayed on its screen, and saved on hard disk. The computer checked the arriving values against preset threshold values, displayed warnings if violations of thresholds occurred and recorded the start and the end of threshold violations for each measured parameter affected. After the end of each exposure all data gathered during this exposure were backed up on optical media.

Daily protocols were prepared from the recorded values using suitable software. The protocols include start and stop times of exposure and possible threshold violations, and daily means of each parameter. The records saved on optical media and the printed daily records are considered as raw data. Relevant disturbances are reported under “Results”.

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 a calibrated online microGC.

To calibrate the microGC, an appropriate amounts of test substance were weighed into a gas sampling tube. The gas sampling tube was tempered that the test substance was evaporated within the tube. The so prepared atmospheres was sampled by microGC. A calibration line is formed by the pair of substance concentration and peak area. The validity of the calibration was proven every two weeks during the exposure period.
The calibrated microGC samples the atmospheres from each chamber via a steam selector switching from one chamber to another per pre-defined sequence. In order to prevent contamination of the sampling loop, time for flushing out the sampling loop was scheduled prior to each measurement. By doing so, there were 20 or 21 measurement per concentration per exposure day. The daily means were calculated based on the hourly means.

The analyses were carried out at the Laboratory for Inhalation Methods and at the Analytical Chemistry Laboratory of the Experimental Toxicology and Ecology of BASF SE.

Duration of treatment / exposure:

90 days (65 exposures each 6 hours)

Frequency of treatment:

All test groups were exposed for 6 hours on workdays (5 times per week)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm (analytical)

Doses / concentrations 2

Dose / conc.:

148.5 ppm (analytical)

Doses / concentrations 3

Dose / conc.:

505.2 ppm (analytical)

Doses / concentrations 4

Dose / conc.:

1 528.7 ppm (analytical)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
In a previous 14-day range finding study (99I0569/15I087), groups of four male and four female Wistar rats per test group were exposed whole-body to dynamic atmosphere of n-Propyl acetate for 6 hours per day on 14 consecutive days. The target concentrations were 1000 and 3000 ppm. Relevant effects occurring at 3000 ppm comprised multifocal degeneration/regeneration of olfactory epithelium (minimal to severe) of nasal cavity in all male and female animals, reduced mean body weight in males, reduced body weight change in both males and females. Accordingly, the terminal body weight of both males and females were lower than the control. The effect in the nasal cavity was still visible at 1000 ppm, though less severe. Reduced body weight change was observed only in females at the low concentration.
Based on these data, 3000 ppm was considered being too high to expose animals for 90 days. As effects observed at 1000 ppm were not pronounce, we chose 1500 ppm as the high concentration, at which, we expected clear adverse effect. Mid- and low concentration of 500 and 150 ppm, respectively, were selected to establish concentration response relationship.
The low concentration of 150 ppm was the expected no observed adverse effect concentration (NOAEC).

- Rationale for animal assignment: random
- Section schedule rationale: random

Positive control:

N/A

Examinations

Observations and examinations performed and frequency:

CLINICAL EXAMINATIONS
Mortality
The animals were examined for evident signs of toxicity or mortality twice a day (in the morning and in the late afternoon) on working days and once a day (in the morning) on Saturdays, Sundays and public holidays.

Clinical observations
The clinical condition of the test animals was recorded once during the pre-exposure period and on post-exposure observation days and at least 3 times (before, during and after exposure) on exposure days.
During exposure only a group wise examination was possible.

Body weight data
The body weight of the animals was determined prior to the pre-exposure, at the start of the exposure period and then, twice weekly thereafter until one day prior to gross necropsy. As a rule, the animals were weighed at the same time of the day.
The first body weight change was the calculated as the difference between body weights of the first Friday (day 3 for males and day 2 for females) to those of the first exposure day (day 0).
Afterwards, body weight change was calculated as the difference between body weight on the Friday and body weight on the previous Monday. The last body weight change was calculated as the difference of the weight on the last exposure day and that of previous Monday. Group means were derived from the individual differences.
The body weight change of the respective week was calculated as the difference of Friday to the previous Monday.

Food consumption
Food consumption was determined weekly (over a period of five days) and calculated as mean food consumption in grams per animal and day.
The animals were maintained in social-housing cages, with 5 animals per cage, during the whole study period. Therefore, the food consumption was determined cage-wise. The food consumption per animal and day was calculated by dividing food consumption of the day of a respective cage by the 5 animals per cage. As the animals of each test group were housed in only two cages per sex, no statistical evaluation of food consumption is possible.

Ophthalmology
Before the start of the exposure period (day -6/ -5) the eyes of all main group animals, and at the end of the study (day 83 and 84) the eyes of the animals of test group 0 (control group), and test group 3 (high concentration) were examined for any changes in the refracting media with an ophthalmoscope (HEINE Optotechnik, Herrsching, Germany) after administration of a mydriatic (Mydrum, Chauvin ankerpharm GmbH, Berlin, Germany).

Detailed clinical observation (DCO)
All animals will be subjected to detailed clinical observations outside their cages once before the beginning of the administration period (day 0) and subsequently once on study day 43 and once on study day 85, generally in the morning. For observation, the animals will therefore be removed from their cages and placed in a standard arena (50 x 37.5 x 25 cm). The scope of examinations and the scoring of the findings that are observed will be based on the current index of findings in PDS ToxData® and includes but is not limited to the following parameters listed:
1. abnormal behavior during handling
2. fur
3. skin
4. posture
5. salivation
6. respiration
7. activity/arousal level
8. tremors
9. convulsions
10. abnormal movements
11. impairment of gait
12. lacrimation
13. palpebral closure
14. exophthalmus
15. feces (appearance/consistency)
16. urine
17. pupil size
18. other findings


Functional observational battery
A functional observation battery (FOB) was carried out on the assigned animals (see section 3.4.). There is no exposure of the concerning animals as well as the other 5 animals of the same test group.
At least one hour before the start of the FOB the animals will be transferred singly to Polycarbonate cages (floor area about 800 cm2). The cages were placed in the racks in a randomized order (randomization based upon animals number). Drinking water was provided ad libitum whereas no food was offered during the measurements.
The FOB started with passive observations, without disturbing the animals, followed by removal from home cage, and open field observations in a standard arena. Thereafter, sensorimotor tests and reflex tests were conducted. The examinations were carried out by trained technicians which performed positive control studies as part of their training. Another technician documented all findings and values obtained. The findings were ranked according to the degree of severity, if applicable.
(A more detailed description of the method, the ranking and documentation system can be found in Part III (Supplement).)

Home cage observations:
The animals were observed in their closed home cages; any disturbing activities (touch¬ing the cage or rack, noise) were avoided during these examinations in order not to in¬fluence the behavior of the animals. Attention was paid to:

1. posture
2. tremor
3. convulsions
4. abnormal movements
5. impairment of gait
6. other findings

Open field observations:
The animals were transferred to a standard arena (50 x 50 cm with sides of 25 cm high) and observed for at least 2 minutes. Following parameters were examined:

1. behavior when removed from cage
2. fur
3. skin
4. salivation
5. nasal discharge
6. lacrimation
7. eyes/pupil size
8. posture
9. palpebral closure
10. respiration
11. tremors
12. convulsions
13. abnormal movements/ stereotypies
14. impairment of gait
15. activity/arousal level
16. feces (appearance/consistency) within two minutes
17. urine (amount/color) within two minutes
18. number of rearings within two minutes
19. other findings

Sensorimotor Tests/Reflexes:
The animals were removed from the open field and subjected to following sensorimotor or reflex tests:

1. approach response
2. touch response
3. vision ("visual placing response")
4. pupillary reflex
5. pinna reflex
6. audition ("startle response")
7. coordination of movements ("righting response")
8. behavior during "handling"
9. vocalization
10. pain perception ("tail pinch")
11. grip strength of forelimbs
12. grip strength of hindlimbs
13. landing foot-splay test
14. other findings

Motor activity measurements (MA)
Motor activity was measured on the same day and with the same animals as FOB was performed. The measure¬ment was performed in the dark using the TSE Labmaster System (TSE Systems GmbH, Bad Homburg, Germany) with 18 infrared beams per cage. During the measurement the animals were kept in Polycarbonate cages with absorbent material. The animals were put into the cages in a randomized order. The measurements started at about 14:00 p.m. The numbers of beam interrupts were counted over 12 intervals, each lasting 5 minutes. Measurement did not commence at the same instant for all cages; the period of assessment for each animal started when the first beam was interrupted by pushing the cage into the rack (staggered start). Measurements ended exactly 60 minutes thereafter. During the measurements the animals received no food and no water.


CLINICAL PATHOLOGY
In the morning on the day of the necropsy, blood was taken from the retro-bulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane. The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence.
The assays of blood and serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results.
The results of clinical pathology examinations were expressed in Inter¬national System (SI) units.
The following examinations were carried out in all animals per test group and sex at the end of the administration period.
- Leukocyte count (WBC)
- Erythrocyte count (RBC)
- Hemoglobin (HGB)
- Hematocrit (HCT)
- Mean corpuscular volume (MCV)
- Mean corpuscular hemoglobin (MCH)
- Mean corpuscular hemoglobin concentration (MCHC)
- Platelet count (PLT)
- Differential blood count
- Reticulocytes (RET)

Furthermore, blood smears were prepared and stained according to WRIGHT without being evaluated, because of non-ambiguous results of the differential blood cell counts measured by the automated instrument.
Clotting tests were carried out using a ball coagulometer (AMAX destiny plus model; Trinity biotech, Lemgo, Germany).
- Prothrombin time (Hepato Quick’s test) (HQT)

Clinical chemistry
An automatic analyzer (Cobas c501; Roche, Mannheim, Germany) was used to examine the clinicochemical parameters:
- Alanine aminotransferase (ALT) (L-alanine: 2-oxoglutarate aminotransferase; EC 2.6.1.2.)
- Aspartate aminotransferase (AST) (L-aspartate: 2-oxoglutarate aminotransferase; EC 2.6.1.1.)
- Alkaline phosphatase (ALP) (orthophosphoric acid monoester phosphohydrolase; EC 3.1.3.1.)
- gamma-Glutamyltransferase (GGT) (gamma -glutamyl) peptide: aminoacid-gamma-glutamyl-transferase; EC 2.3.2.2.)
- Sodium (NA)
- Potassium (K)
- Chloride (CL)
- Inorganic phosphate (INP)
- Calcium (CA)
- Urea (UREA)
- Creatinine (CREA)
- Glucose (GLUC)
- Total bilirubin (TBIL)
- Total protein (TPROT)
- Albumin (ALB)
- Globulins (GLOB)
- Triglycerides (TRIG)
- Cholesterol (CHOL)

Sacrifice and pathology:

PATHOLOGY
All animals were sacrificed under pentobarbital anesthesia. The left and right brachial vessels were opened by deep cuts through the pectoral muscles along both sides of the rib cage. Caution was exercised to avoid destruction of the axillary lymph node. The exsanguinated animals were necropsied and assessed by gross pathology.

Organ weights
The following weights were determined in all animals sacrificed on schedule:

1. Anesthetized animals (terminal body weight)
2. Adrenal glands
3. Brain
4. Epididymides
5. Heart
6. Kidneys
7. Liver
8. Lungs
9. Ovaries
10. Spleen
11. Testes
12. Thymus
13. Thyroid glands
14. Uterus

Organ/tissue fixation
The following organs or tissues were fixed in 4% neutral-buffered formaldehyde solution or in modified Davidson’s solution:

1. All gross lesions
2. Adrenal glands
3. Aorta
4. Bone marrow (femur)
5. Brain with olfactory bulb
6. Cecum
7. Cervix
8. Coagulating glands
9. Colon
10. Duodenum
11. Epididymides,
12. Esophagus
13. Extraorbital lacrimal gland
14. Eyes with optic nerve (modified Davidson’s solution)
15. Eyelids
16. Femur with knee joint
17. Harderian glands
18. Heart
19. Ileum
20. Jejunum
21. Kidneys
22. Larynx
23. Liver
24. Lungs
25. Lymph nodes (tracheobronchial, mediastinal and mesenteric lymph nodes)
26. Mammary gland (male + female)
27. Nose (nasal cavity)
28. Ovaries
29. Pancreas
30. Parathyroid glands
31. Pharynx
32. Pituitary gland
33. Prostate
34. Rectum
35. Salivary glands (mandibular and sublingual glands)
36. Sciatic nerve
37. Seminal vesicles
38. Skeletal muscle
39. Skin
40. Spinal cord (cervical, thoracic and lumbar cord)
41. Spleen
42. Sternum with marrow
43. Stomach (forestomach and glandular stomach)
44. Teeth
45. Testes
46. Thymus
47. Thyroid glands
48. Tongue
49. Trachea
50. Ureter
51. Urethra
52. Urinary bladder
53. Uterus
54. Vagina

Histopathology
Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings according to the table below:

Organs

1. All gross lesions (Hematoxylin and eosin (H&E) stain in all animals affected/test group)
2. Adrenal glands (Hematoxylin and eosin (H&E) stain in all animals/control and high dose group)
3. Aorta (Hematoxylin and eosin (H&E) stain in all animals/control and high dose group)
4. Bone marrow (femur) (Hematoxylin and eosin (H&E) stain in all animals/control and high dose group)
5. Brain (Hematoxylin and eosin (H&E) stain in all animals/control and high dose group)
6. Cecum (Hematoxylin and eosin (H&E) stain in all animals/control and high dose group)
7. Cervix (Hematoxylin and eosin (H&E) stain in all animals/control and high dose group)
8. Coagulating glands (Hematoxylin and eosin (H&E) stain in all animals/control and high dose group)
9. Colon (Hematoxylin and eosin (H&E) stain in all animals/control and high dose group)
10. Duodenum (Hematoxylin and eosin (H&E) stain in all animals/control and high dose group)
11. Epididymides (Hematoxylin and eosin (H&E) stain in all animals/control and high dose group)
12. Esophagus (Hematoxylin and eosin (H&E) stain in all animals/control and high dose group)
13. Eyes with optic nerve (Hematoxylin and eosin (H&E) stain in all animals/control and high dose group)
14. Extraorbital lacrimal gland (Hematoxylin and eosin (H&E) stain in all animals/control and high dose group)
15. Femur with knee joint (Hematoxylin and eosin (H&E) stain in all animals/control and high dose group)
16. Harderian gland (Hematoxylin and eosin (H&E) stain in all animals/control and high dose group)
17. Heart (Hematoxylin and eosin (H&E) stain in all animals/control and high dose group)
18. Ileum (Hematoxylin and eosin (H&E) stain in all animals/control and high dose group)
19. Jejunum (Hematoxylin and eosin (H&E) stain in all animals/control and high dose group)
20. Kidneys (Hematoxylin and eosin (H&E) stain in all animals/control and high dose group)
21. Larynx (3 levels, one level included the base of the epiglottis) (Hematoxylin and eosin (H&E) stain in all animals/control and all dose groups)
22. Liver (Hematoxylin and eosin (H&E) stain in all animals/control and high dose group)
23. Lung (Hematoxylin and eosin (H&E) stain in all animals/control and high dose group)
24. Lymph nodes (tracheobronchial, mediastinal, mesenteric) (Hematoxylin and eosin (H&E) stain in all animals/control and high dose group)
25. Mammary gland (female) (Hematoxylin and eosin (H&E) stain in all animals/control and high dose group)
26. Nasal cavity (4 levels, one level included nasopharyngeal duct; the 4 levels allow adequate examination of the squamous, transitional, respiratory and olfactory epithelium, and the draining lymphatic tissue (NALT) (Hematoxylin and eosin (H&E) stain in all animals/control and all dose groups)
27. Ovaries (female) (Hematoxylin and eosin (H&E) stain in all animals/control and high dose group)
28. Pancreas (Hematoxylin and eosin (H&E) stain in all animals/control and high dose group)
29. Parathyroid glands (Hematoxylin and eosin (H&E) stain in all animals/control and high dose group)
30. Pharynx (Hematoxylin and eosin (H&E) stain in all animals/control and high dose group)
31. Pituitary gland (Hematoxylin and eosin (H&E) stain in all animals/control and high dose group)
32. Prostate (Hematoxylin and eosin (H&E) stain in all animals/control and high dose group)
33. Rectum (Hematoxylin and eosin (H&E) stain in all animals/control and high dose group)
34. Salivary glands(mandibular and sublingual glands) (Hematoxylin and eosin (H&E) stain in all animals/control and high dose group)
35. Sciatic nerve (Hematoxylin and eosin (H&E) stain in all animals/control and high dose group)
36. Seminal vesicles (Hematoxylin and eosin (H&E) stain in all animals/control and high dose group)
37. Skeletal muscle (Hematoxylin and eosin (H&E) stain in all animals/control and high dose group)
38. Skin (Hematoxylin and eosin (H&E) stain in all animals/control and high dose group)
39. Spinal cord (cervical, thoracic and lumbar cord) (Hematoxylin and eosin (H&E) stain in all animals/control and high dose group)
40. Spleen (Hematoxylin and eosin (H&E) stain in all animals/control and high dose group)
41. Sternum with marrow (Hematoxylin and eosin (H&E) stain in all animals/control and high dose group)
42. Stomach (forestomach and glandular stomach) (Hematoxylin and eosin (H&E) stain in all animals/control and high dose group)
43. Teeth (Hematoxylin and eosin (H&E) stain in all animals/control and high dose group)
44. Testes (male) (Hematoxylin and eosin (H&E) stain in all animals/control and high dose group)
45. Thymus (Hematoxylin and eosin (H&E) stain in all animals/control and high dose group)
46. Thyroid glands (Hematoxylin and eosin (H&E) stain in all animals/control and high dose group)
47. Trachea (one transverse section and one longitudinal section through the carina of the bifurcation of the extrapulmonary bronchi) (Hematoxylin and eosin (H&E) stain in all animals/control and high dose group)
48. Urinary bladder (Hematoxylin and eosin (H&E) stain in all animals/control and high dose group)
49. Uterus (female) (Hematoxylin and eosin (H&E) stain in all animals/control and high dose group)
50. Vagina (female) (Hematoxylin and eosin (H&E) stain in all animals/control and high dose group)

The organs were trimmed according to the “Revised guides for organ sampling and trimming in rats and mice” (Ruehl-Fehlert et al 2003; Kittel et al. 2004; Morawietz et al. 2004).
A correlation between gross lesions and histopathological findings was attempted.

Statistics:

Means, medians and standard deviations of each test group were calculated for several parameters. In summary tables, mean values were rounded, but deviations of means versus control means were calculated with not rounded values. Therefore, slight differences may occur when changes were re-calculated with rounded means. In these tables “deviation vs control” means x-fold of controls expressed as percentages minus 100%.
Body weight, body weight change: Comparison of each group with the control group was performed using DUNNETT test (two-sided) for the hypothesis of equal means.
Rearing, grip strength length forelimbs, grip strength length hindlimbs, foot-splay test, motor activity: Non-parametric one-way analysis using the KRUSKAL-WALLIS test (two-sided).If the resulting p-value was equal to or less than 0.05, a pair-wise com¬parison of each dose group with the control group was performed using WILCOXON test (two-sided) for the hypothesis of equal medians.
Blood parameters: For parameters with bidirectional changes:
Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the hypothesis of equal medians
Weight parameters in pathology: Non-parametric one-way analysis using KRUSKAL-WALLIS 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 was performed using WILCOXON-test (two-sided) for the equal medians

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

During the pre-exposure period the animals showed no clinical signs and findings different from normal.
During the exposure period the male (from study day 14 through to 44) and female animals (from study day 13 to 43) of the high concentration (1500 ppm) showed reduced attention during the exposure, which disappeared quickly after cessation of each exposure. This finding was considered being substance-related.

In addition, following clinical signs was observed:
Sparse fur: 1 male in test group 0 (control group)
1 female in test group 0 (control group)
4 females in test group 1 (150 ppm)
2 females in test group 3 (1500 ppm)
Discolored fur: 1 female in test group 3
Injury: 1 female in test group 2

These findings were considered not substance-related due to lack of concentration-response relationship. Thus, during the exposure period the animals of the low concentration (150 ppm), and the mid concentration (500 ppm) groups showed no substance-related clinical signs of toxicity.

Mortality:

no mortality observed

Description (incidence):

No deaths were recorded throughout the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean body weights of male and female rats of test groups 1 and 2 were not statistically significantly different from the control group 0.

The mean body weight of the male animals of the high concentration (1500 ppm) was statistically significantly decreased from day 3 (-6.9 %, p ≤ 0.01) through to day 91 (-12.5 %, p ≤ 0.01).
The mean body weight of male animals of test group 2 was slightly lower than the concurrent control group, without statistical significance.

The mean body weight of the female animals of the high concentration (1500 ppm) was repeatedly decreased from study day 5 onward. The deviations were presented as following:
Study day 19: 202.4 g (-6.9 %, p ≤ 0.01)
Study day 23: 205.6 g (-6.1 %, p ≤ 0.05)
Study day 33: 210.6 g (-7.1 %, p ≤ 0.05)
Study day 37: 213.6 g (-7.0 %, p ≤ 0.05)
Study day 47: 217.4 g (-7.5 %, p ≤ 0.05)
Study day 51: 217.8 g (-8.4 %, p ≤ 0.01)
Study day 54: 219.4 g (-8.7 %, p ≤ 0.01)
Study day 58: 219.4 g (-8.9 %, p ≤ 0.01)
Study day 61: 222.6 g (-8.1 %, p ≤ 0.05)
Study day 65: 222.9 g (-8.2 %, p ≤ 0.05)
Study day 68: 222.3 g (-8.3 %, p ≤ 0.05)
Study day 72: 223.2 g (-8.8 %, p ≤ 0.05)
Study day 75: 223.3 g (-10.0 %, p ≤ 0.01)
Study day 79: 221.0 g (-10.0 %, p ≤ 0.01)
Study day 82: 227.8 g (-9.2 %, p ≤ 0.01)
Study day 86: 227.0 g (-9.0 %, p ≤ 0.01)
Study day 89: 228.9 g (-8.7 %, p ≤ 0.05)
Study day 91: 227.7 g (-9.4 %, p ≤ 0.01)

Body weight change:

The body weight change of the male and female animals of the low (150 ppm) and mid (500 ppm) concentration groups was comparable with the concurrent control group during the most time of the study period. Following deviation to the control was found for male animals of test group 2:
From study day 41 to 45: +1.6 g (-70.4 %, p≤ 0.05)

The mean body weight change of the male animals of the high concentration group (1500 ppm) was statistically significantly lower compared to the controls throughout the exposure period. The significant changes were listed as following:

From study day 0 to 3: +1.8 g (-85.5 %, p ≤ 0.01)
From study day 6 to 10: +2.6 g (-81.4 %, p≤ 0.01)
From study day 13 to 17: +2.0 g (-79.6 %, p≤ 0.01)
From study day 27 to 31: +0.1 g (-97.7 %, p≤ 0.01)
From study day 34 to 38: +1.8 g (-73.3 %, p≤ 0.01)
From study day 41 to 45: -1.3 g (-124.8 %, p≤ 0.01)
From study day 55 to 59: +1.1g (-78.3 %, p≤ 0.05)
From study day 62 to 66: -1.1g (-125.2 %, p≤ 0.01)
From study day 83 to 87: -2.1g (-167.0 %, p≤ 0.01)

In addition, following deviation from the control was found in high concentration female animals:
From study day 47 to 51: 0.5 g (-84.8 %, p≤ 0.05)

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Due to social housing, there were only two cages for each concentration group per sex. Thus, no statistic evaluation was performed.
Comparing the mean food consumption of exposed animals with the concurrent control group, the food consumption of male (-9.7 % to -24.4 %) and female animals (-9.9 % to -23.2 %) of the test group 3 (1500 ppm) was obviously lower than the concurrent control throughout the study period. In male and female animals of test group 1 (150 ppm) and 2 (500 ppm) no substance-related changes of food consumption were observed during the whole study period.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

The ophthalmologic examinations did not show any impairment of the refracting media.

Spontaneous findings such as remainders of the pupillary membrane or corneal stippling were observed in several animals of all test groups and the control group without any concentration-response relationship.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes among hematological parameters were observed.

At the end of the administration period, in males of test group 3 (1500 ppm) hemoglobin values were significantly increased and in females of test groups 2 and 3 (500 and 1500 ppm) absolute reticulocyte counts were significantly decreased. However, all values were within historical control ranges (males, hemoglobin 8.6-9.3 mmol/L; females absolute reticulocytes 124.2-199.7 Giga/L). Therefore, these alterations were regarded as incidental and not treatment-related.

In males of test group 1 (150 ppm) absolute and relative monocyte counts and relative neutrophil counts were significantly decreased whereas relative lymphocyte counts were significantly increased. In females of the same test group hemoglobin and hematocrit values were significantly decreased. However, the mentioned alterations were not dose-dependent and therefore, they were regarded as incidental and not treatment-related.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes among clinical chemistry parameters were observed.

At the end of the inhalation period in females of test groups 2 and 3 (500 and 1500 ppm) inorganic phosphate levels were significantly higher compared to controls, but the means were within the historical control range, whereas that one of the study controls was below this range (females, inorganic phosphate 1.13-1.57 mmol/L). Therefore, this change was regarded as incidental and not treatment-related.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

On the day of the performance of the Functional Observation Battery, the animals were not exposed to the test substances.
Deviations from "zero values" were obtained in several animals. However, as most findings were equally distributed between treated groups and controls, were without a dose-response relationship or occurred in single animals only, these observations were considered to have been incidental.

The following findings were assessed individually:
Observations on day 78:

Quantitative parameters:
No substance-related findings were observed.
Home cage observations:
No substance-related findings were observed.
Open field observations:
No substance-related findings were observed.
Sensorimotor tests/reflexes:
No substance-related findings were observed.

Motor activity
Overall motor activity (summation of all intervals):
There were no statistically significant deviations of overall motor activity from the control group 0.

Single intervals:
Comparing the single intervals with the control group, the following statistically significant increases were seen:
Males, test group 1, interval 2 (p≤ 0.05)
Males, test group 2, interval 2 (p≤ 0.05)
Females, test group 1, interval 11 (p≤ 0.05)
Females, test group 2, interval 5 (p≤ 0.05)
Females, test group 3, interval 5 (p≤ 0.05)
Females, test group 3, interval 6 (p≤ 0.01)
Females, test group 3, interval 11 (p≤ 0.01)
The increased motor activities in male and female animals were considered not substancerelated because of their transient nature and the missing dose-response relationship.

Immunological findings:

no effects observed

Description (incidence and severity):

No substance-related findings were observed.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Absolute organ weights
When compared with the control group 0 (=100%), the following mean absolute weight parameters were significantly increased or decreased in one or more test groups (statistically significant changes printed in bold): see "Any other information on results incl. tables"
All other mean absolute weight parameters did not show significant differences when compared to the control group 0.

Relative organ weights
When compared with control group 0 (=100%), the following mean relative organ weights were significantly increased in one or more test groups (statistically significant changes printed in bold): see "Any other information on results incl. tables"

All other mean relative weight parameters did not show significant differences when compared to the control group 0.

The terminal body weight was significantly decreased in males of test group 2 (500 ppm) and in males and females of test group 3 (1500 ppm) resulting secondarily in significantly
• decreased mean absolute kidney and liver weights and increased mean relative weights of adrenal glands in males of test group 3.
• increased mean relative brain and testes weights in males of test groups 2 and 3.
• decreased mean absolute uterus weight and increased mean relative brain and kidney weights in females of test group 3.
• increased mean relative lung weights in males and females of test group 3.
• increased mean relative heart weight in males of test group 2.

In all these organs, there were no treatment-related histopathological findings.

The reduced terminal body weight was regarded to be treatment-related.

Because there was no concentration-response relationship, the increased mean absolute ovarian weight in females of test group 1 (150 ppm) was considered to be incidental.

Gross pathological findings:

no effects observed

Description (incidence and severity):

All findings were considered to be incidental or spontaneous in origin and without any relation to treatment.

Neuropathological findings:

no effects observed

Description (incidence and severity):

No substance-related findings were observed.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related findings were observed in the nasal cavity.

Nasal cavity:
In the olfactory epithelium at different levels of the nasal cavity, a degeneration and/or regeneration was observed in each 6 males and females of test group 2 (500 ppm) and in all males and females of test group 3 (1500 ppm). The degeneration/ regeneration of the olfactory epithelium was characterized by loss of sustentacular cells, increased intercellular spaces, irregular epithelial architecture, reduction of epithelial height, necrotic epithelium and/or increased nuclear/cytoplasmic ratio. The finding occurred focal or multifocal and was located at the dorsal part of septum, nasoturbinate and/or ethmoid turbinate. The severity of degeneration/ regeneration of the olfactory epithelium varied from few small or single larger areas with only a minimal loss of the organization of the cell layers (diagnosed as grade 1, minimal) up to large areas with clear irregular epithelial architecture (diagnosed as grade 4, severe). The incidence and grading are given in the following table: see "Any other information on results incl. tables"

Larynx
In the larynx (level II), a minimal or slight focal inflammation was observed in 3 males of test group 3 (1500 ppm) that was caused by foreign bodies. Hairs within the inflammation were seen in two of these males. The occurrence of this finding was assessed to be incidental.


All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

N/A

Details on results:

During the exposure period, the target concentrations were maintained as constant and stable as could be provided with vapor generation techniques in the concentration range tested.

During the whole study period, reduced attention was observed in both male and female animals during exposure to high concentration (1500 ppm) of n-Propyl acetate. The effect was observed in males from day 14 to 44, in females from day 13 to 43 during exposure, and disappeared shortly after the termination of the exposure. This transient finding was probably due to narcotic effect of the test substance. No other substance-related clinical signs of toxicity were observed during daily clinical observation and detailed clinical observation. Ophthalmological examination did not reveal any abnormalities in exposed animals. Neither were there any abnormalities during examinations of FOB and MA.

Mean body weight of male and female animals of the high concentration group (1500 ppm) were significantly lower than the controls, though not always statistically significant. Same trends were apparent in body weight changes. The effects were more pronounced in male animals than in female animals. From the very beginning of the exposure period, the food consumption of high concentration males appeared to be lower than the controls, though without statistical evaluation due to small number of fodder racks per group and sex. The lowered mean body weight, mean body weight gains were consistent with the reduced food consumption. Same tendency was observed in male animals of test group 2 (500 ppm), however without statistical significance during the in-life phase of the study. The mean terminal body weight of male animals, which was the weight of narcotized animal after overnight fasting, were significantly lower than the concurrent control. In this way, the lowered body weight of test group 2 became more apparent. These findings were concentration-related and considered treatment-related. They were likely secondary to the local effects observed in nasal cavity. As there were no adverse histological findings in other organs and tissue beside nasal cavity, the effects with regards of body weight and food consumption were considered not adverse.

Concerning clinical pathology no treatment-related, adverse effects were observed up to a dose of the compound of 1500 ppm.

In pathology, the terminal body weight was significantly decreased in males (-7%) of test group 2 (500 ppm) and in males (-14%) and females (-9%) of test group 3 (1500 ppm) resulting secondarily in weight changes of different organs (kidney, liver, adrenal glands, uterus, brain, testes, lungs, and heart).

Regarding pathology, the target organ was the nasal cavity. In the nasal cavity, 6 male and female animals of test group 2 (500 ppm) and all males and females of test group 3 (1500 ppm) showed a degeneration and/ or regeneration in the olfactory epithelium at different levels (II, III, IV) of the nasal cavity. The degeneration/ regeneration occurred focal or multifocal and was located at the dorsal part of septum, nasoturbinate, and/or ethmoid turbinate. The severity ranged from minimal to severe and was concentration-related increased. The occurrence of degeneration/ regeneration of the olfactory epithelium was considered to be treatment-related and adverse.

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Effect levels

1

Key result

Dose descriptor:

NOAEC

Remarks:

specific systemic target organ toxicity

Effect level:

>= 6 478.3 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no specific systemic target organ toxicity observed up to the highest dose level tested

2

Key result

Dose descriptor:

NOAEC

Remarks:

local irritant effects

Effect level:

>= 629.3 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no local irritant effects observed at this dose level

3

Key result

Dose descriptor:

LOAEC

Remarks:

local irritant effects

Effect level:

2 140.9 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

2 140.9 mg/m³ air (analytical)

System:

respiratory system: upper respiratory tract

Organ:

nasal cavity

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Any other information on results incl. tables

Absolute weights	Males			Females
Test group (ppm)	1 (150)	2 (500)	3 (1500)	1 (150)	2 (500)	3 (1500)
Terminal body weight	100%	93%**	86%**	103%	98%	91%**
Kidneys	103%	96%	89%*
Liver	101%	89%	83%**
Ovaries				118%*	114%	102%
Uterus				109%	66%	61%**

*: p <= 0.05; **:<= 0.01

Relative weights	Males			Females
Test group (ppm)	1 (150)	2 (500)	3 (1500)	1 (150)	2 (500)	3 (1500)
Adrenal glands	106%	112%	133%**
Brain	98%	108%**	117%**	100%	103%	111%**
Heart	99%	109%*	109%
Kidneys				104%	106%	112%**
Lungs	101%	109%	116%*	101%	105%	113%**
Testes	99%	111%**	114%**

*: p <= 0.05; **:<= 0.01

	Male animals				Female animals
Test group (ppm)	0	1 (150)	2 (500)	3 (1500)	0	1 (150)	2 (500)	3 (1500)
Organs examined	10	10	10	10	10	10	10	10
Affected animals	0	0	6	10	0	0	6	10
Nasal cavity (level II)
Degeneration/ regeneration, olfactory epithelium	0	0	5	10	0	0	6	10
Grade 1			1				3
Grade 2			2	2			2	1
Grade 3			2	5				4
Grade 4				3			1	5
Nasal cavity (level III)
Degeneration/ regeneration, olfactory epithelium	0	0	4	9	0	0	2	8
Grade 1			3	3			1	1
Grade 2			1	3			1	5
Grade 3				3				2
Nasal cavity (level IV)
Degeneration/ regeneration, olfactory epithelium	0	0	1	4	0	0	3	9
Grade 1			1	4			3	7
Grade 2								2

Applicant's summary and conclusion

Executive summary:

In a guideline and GLP study, inhalation exposure of 150, 500 and 1500 ppm n-Propyl acetate for 90 days (65 exposures) caused clear treatment-related adverse histological changes in the nasal cavity in animals of test groups 2 (500 ppm) and 3 (1500 ppm). Impaired body weight development, reduced food consumption and terminal body weight were observed at 1500 ppm. Such changes are typical from exposure to acetate esters and the changes occur due to local hydrolysis and irritation from the liberated acetic acid. There was however, no specific systemic target organ toxicity observed up to the highest concentration (1500 ppm) tested.

The No Observed Adverse Effect Concentration (NOAEC) was 150 ppm based on local effects in the nasal cavity.