Registration Dossier

Administrative data

Description of key information

Repeated inhalation exposure to n-butyl acetate primarily caused reduction in body weight and food consumption in rats besides central sedation. Additionally, local effects in the stomach and respiratory tract occurred. No effects were observed at 500 ppm (2.4 mg/L). Repeated dose toxicity after oral and dermal exposure to n-butyl acetate was not investigated. Repeated exposure of rats to the RA-substance n-butanol caused transient ataxia and hypoactivity (NOAEL 125 mg n-butanol/kg bw/d, corresponding to 196 mg n-butyl acetate/kg bw/d).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline Study (EPA OTS 798.2650), but as read-across from supporting substance maximum reliability is 2 Read-across hypothesis: for details please see read-across report in section 13
Qualifier:
according to guideline
Guideline:
EPA OTS 798.2650 (90-Day Oral Toxicity in Rodents)
Principles of method if other than guideline:
Four groups of male and female rats (30/sex/group) were administered daily by gavage 0, 30, 125 or 500 mg/kgbw/d for either 6 or 13 weeks.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Portage, Michigan, U.S.A.
- Age at study initiation: 36-37 d
- Mean weight at study initiation: males 90 g, females 86 g
- Housing: individually
- Diet (e.g. ad libitum): Purina Certiofied Rodent Laboratory Chow #5002 (pellet)
- Water (e.g. ad libitum): filtered municipal water
- Acclimation period: 7 days before the pretreatment week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 1
- Humidity (%): 48 +/- 9
- Photoperiod (hrs dark / hrs light): 12:12
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Dosing solutions of butanol in deionized water were used.

VEHICLE
- Concentration in vehicle: not specified
- Amount of vehicle (if gavage): 10 ml/kg was the constant dosing volume
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
GC-FI
Duration of treatment / exposure:
6 (interim sacrifice) or 13 weeks
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
0, 30, 125, 500 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
30 (further 10 were sacrificed prior to dosing for determination of clinicopathological baseline levels)
Control animals:
yes, concurrent vehicle
Positive control:
no data
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded weekly


FOOD CONSUMPTION: Yes
-Time schedule: Food consumption was recorded weekly


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmic examination was conducted prior to treatment and during week 13 before final necropsy.
- Dose groups that were examined: no data


HAEMATOLOGY: Yes
- Time schedule for collection of blood: before the start of the study, during week 6 and during week 13
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: 10 males and 10 females
- parameters: hemoglobin ( HGB), hematocrit (PCV), erythrocyte count (RBC), mean cell volume (MCV), mean cell hemoglobin (MCH),mean cell hemoglobin concentration (MCHC) total and differential leucocyte counts (WBC), estimated platelet count (PLT)


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: before the start of the study, during week 6 and during week 13
- Animals fasted: No data
- How many animals: 10 males and 10 females
- parameters: alkaline phosphatase (Alk phos) blood urea nitrogen (BUN), glutamate pyruvate transaminase (SGPT), glutamate oxalacetate transaminase (SGOT), glucose (Gluc), total protein (TP), albumin (Alb), A/G ratio (calculated), globulin (calculated), total bilirubin (Tot. bili.), sodium (Na), potassium (K ), chloride (Cl), calcium (Ca), inorganic phosphate (Phos), carbon dioxide (TCO2), total serum cholesterol (Chol), creatinine.


URINALYSIS: Yes
- Time schedule for collection of urine: before the start of the study, during week 6 and during week 13
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- parameters: pH, specfic gravity, glucose, protein, ketones, bilirubin, urobilinogen, microscopy of sediment


NEUROBEHAVIOURAL EXAMINATION: No data
Sacrifice and pathology:
GROSS PATHOLOGY: Yes: Ten male and ten female rats from each group were necropsied on study days 43 to 44 and the remaining animals on study days 92 to 93. Gross pathology of all animals was assessed and organs from animals necropsied on study days 92 to 93 were weighed.
HISTOPATHOLOGY: Yes: A complete histopathological investigation was made of all animals of the control and high-dose groups. In the low and mid-dose groups, histopathology included the liver, kidney, and heart from all animals and all gross lesions. All animals found dead or killed in extremis were also microscopically examined.
Statistics:
Bartlett's test for homogeneity (body weight, food consumption, clinicopathologic and organ weight); Dunnett's test for homogeneous and Gill's test for non homogeneous data to test for statistical significance
Details on results:
CLINICAL SIGNS AND MORTALITY
Ataxia and hypoactivity (lasting less than 1 h) were observed 2 to 3 minutes after dosing in both sexes of the high-dose group (500 mg/kg bw/d) during the final 6 weeks of dosing. Such ataxia and hypoactivity are typically seen following high oral doses of alcohols. The rapid induction/remission of these effects and the reported increased incidence after the interim kill may be due to the fact that personnel were able to collect post-dose observations more quickly since fewer animals required dosing.
No significant changes between treated groups and controls were observed concerning mortality (three rats were found dead or sacrificed in extremis, but these deaths could not be attributed to the test article.).

BODY WEIGHT AND WEIGHT GAIN
no significant changes between treated groups and controls were observed

FOOD CONSUMPTION
no significant changes between treated groups and controls were observed

OPHTHALMOSCOPIC EXAMINATION
no significant changes between treated groups and controls were observed

HAEMATOLOGY
At the interim clinical pathological evaluation, red blood cell count (RBC) packed cell volume (PCV), and hemoglobin (HGB) averages of the 500 mg/kg/day dose group females were 5% below control averages. Although these differences were statistically significant, they were small and no differences between the parameters were observed in the males of the interim evaluation or between control and treated groups of either sex at the final evaluation. Therefore, even if the lower red blood cell parameters in the 500 mg/kg/day females were an actual treatment-related effect, it was small and transitory and thus not considered as adverse.

CLINICAL CHEMISTRY
no significant changes between treated groups and controls were observed

URINALYSIS
no significant changes between treated groups and controls were observed

ORGAN WEIGHTS
no significant changes between treated groups and controls were observed

GROSS PATHOLOGY
no significant changes between treated groups and controls were observed

HISTOPATHOLOGY: NON-NEOPLASTIC
no significant changes between treated groups and controls were observed
Dose descriptor:
NOAEL
Effect level:
125 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: no effects observed
Dose descriptor:
LOAEL
Effect level:
500 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: clinical signs of CNS depression (ataxia and hypoactivity)
Critical effects observed:
not specified
Conclusions:
Oral application of n-butanol to rats (0, 30, 125, 500 mg/kg bw/d) in a 90-day toxicity study caused CNS effects in the highest dose group (ataxia and hypoactivity). The NOAEL in this study is 125 mg/kg bw/d.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
196 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Results from reliable, GLP conform subchronic toxicity study with RA substance n-butanol in rats are of high reliability (Klimisch score 2).

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
Study period:
From 12 SEP 1994 to 16 AUG 1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline Study (EPA OTS 798.2450)
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
EPA OTS 798.2450 (90-Day Inhalation Toxicity)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Kingston (Stone ridge, NY)
- Age at study initiation: 60 days
- Weight at study initiation: 271 +/- 7 g (males); 215 +/- 8 g (females)
- Fasting period before study: no
- Housing: individually during non expsure periods
- Diet: Certified Rodent Diet (Agway Prolab RMH 3200, ground chow), ad libitum except during exposure
- Water: ad libitum, except during exposure
- Acclimation period: 12 days


ENVIRONMENTAL CONDITIONS
- Temperature: 67-75°F
- Humidity: 46-60%
- Photoperiod (hrs dark / hrs light): 12/12


Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 4200 L stainless-steel and glass inhalation chambers
- Method of holding animals in test chamber: cages
- System of generating vapour: test substance was metered into glass distillation columns packed with glass beads; filtered, compressed air was passed through the glass bead-packed columns to evaporate the test substance; distillation columns were heated to about 50°C to enhance vaporization; the resultant vapour was directed via glass tubing to a tee just upstream of the inhalation chamber where it was mixed with filtered, conditioned outside air
- Temperature, humidity in air chamber: 21.1-24.7°C; 36.7-68.7%
- Air flow rate: 836 to 965 Lpm
- Air change rate: 12 to 14 air changes per hour
- Method of particle size determination: Micro Laser Particle counter (µLPC-301, Particle Measuring Systems, Inc, Coulder, USA); indicating that an aerosol fo the test subsance was not present



TEST ATMOSPHERE
- Brief description of analytical method used: MIRAN IA infrared gas analyzer (Wilks Foxboro Analytical, South Norwalk, CT) set at a wavelength of 3.38 µM
- Samples taken from breathing zone: no; collection of chamber vapour samples

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- MIRAN IA infrared gas analyzer (Wilks Foxboro Analytical, South Norwalk, CT) set at a wavelength of 3.38 µM
- chamber vapour samples were continuously collected from each chamber throught TEFLON tubing (3/16" i.d.)
- valve position was pepriodically changed to sample from each chamber at least once each hour
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 hours per day, 5 days per week
Remarks:
Doses / Concentrations:
500, 1500, 3000 ppm
Basis:
nominal conc.
No. of animals per sex per dose:
15
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: Range finding study: 2-Weeks repeated exposure in which animals were exposed to 0, 750, 1500 or 3000 ppm n-butyl acetate. The test substance produced concentration-related reductions in general activity levels during exposure periods. Animals appeared to acclimate to the 750 and 1500 ppm concentrations but not to 3000 ppm. Mean body weights for the female 1500 ppm animals and for the 3000 ppm male and female animals were lower than the control group on Days 7 and 14, but no statistically significant differences were noted. 3000 ppm was selected as an exposure concentration that would produce overt signs of toxicity, and 500 ppm was selected as an exposure concentration that was expected to have no effect. An exposure concentration of 1500 ppm was selected as the intermediate exposure concentration.
Positive control:
none
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: during exposure and once per day on weekends
- Cage side observations checked were included.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before and after exposure


BODY WEIGHT: Yes
- Time schedule for examinations: weekly


FOOD CONSUMPTION: YES
- Time schedule for examinations: weekly

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: prior to the start of the study and during the last week of exposure
- Dose groups that were examined: all animals prior to the start of the study; animals from the control and high-concentration group during the last week of exposure


HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 30 and 90 of the study
- Anaesthetic used for blood collection: Yes (Metofane)
- Animals fasted: Yes
- How many animals: 5 per sex per dose group
- Parameters checked:
Whole blood: hemoglobin concentration, red blood cell count, white blood cell count, hematocrit, red blood cell indices, prothrombin time
Blood smears: cellular morphology and differential white blood cell count


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day 30 and 90 of the study
- Anaesthetic used for blood collection: Yes (Metofane)
- Animals fasted: Yes
- How many animals: 5 per sex per dose group
- Parameters checked:
Blood serum: aspartate aminotransferase, sorbitol dehydrogenase, alkaline phosphatase, creatinine, albumin, total protein, total bilirubin, alanine aminotransferase, gamma glutamyltranspeptidase, urea nitrogen, glucose, calcium, phosphorus


URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: Organ weights (liver, kidneys, testes or ovaries, spleen adrenal glands, lungs, brain)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes (embedded in paraffin, sectioned at 5 µm and stained with hematoxylin and eosin; nasal passages were decalcified prior to being embedded and sectioned); all tissues (see below) were examined microscopically from the control and high-concentration groups, in addition, the lungs, nasal passages, thymus (males only), stomach (females only), and gross lesions were examined from the mid- and low concentration group
- tissues examined: nasal passages, trachea, larynx, lungs, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, heart, aorta, adrenal glands, pituitary gland, thymus, pancreas, urinary bladder, thyroid gland, parathyroid gland, spleen, liver, kidneys, mesenteric lymph nodes, sternum (with bone marrow), right testis, right epididymis, male accessory sex glands, ovaries, vagina, uterus, fallopian tubes, salivary glands, gross lesions
Other examinations:
- assessment of sperm morphology and development (reported in section 7.8)
Statistics:
Bartlett's test, one-way analysis of variance, duncan's multiple range test, Kruskal-Wallis H-test and Mann-Whitney U-test
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 specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
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 examined
Details on results:
CLINICAL SIGNS AND MORTALITY
- no spontaneous mortality occurred during the study

3000 ppm group:
- reduced activity levels of generally minor severity during exposure (less movement, decreased alertness, slower response to tapping on the chamber wall)
- Sialorrhea was observed in three animals for no more than one or two exposure days
- red discoloration of the chin hair occurred in several females for no more than one or two exposure days

1500 ppm group:
- reduced activity of generally minimal severity during exposure periods (except the first 5 hours of Day 0 and the first hour or two of Days 1 and 2)

Animals in all groups had prophyrin nasal discharges and dried porphyrin stains around the nose, which occasionally persisted till the next morning. Animals from all groups exhibited discolored-red facial hair, usually the chin hair (slightly higher in the 3000 ppm group than in other groups)

BODY WEIGHT AND WEIGHT GAIN

3000 ppm group:
- significantly reduced mean body weights and body weight gains in males and females; overall weight gains were 62% and 78% of weight gains for the control group (males and females, respectively)

1500 ppm group:
- significantly reduced mean body weights and body weight gains in males and females; overall weight gains were 77% and 70% of weight gains for the control group (males and females, respectively)

500 ppm group:
- body weights and body weight gains were not affected; overall weight gains were 90% and 107% of weight gains for the control group (males and females, respectively)

FOOD CONSUMPTION

3000 ppm group:
- significantly lower than for the control group
- mean weekly feed consumption values were 14-25% or 6-16% lower than the control group in male and female rats, respectively

1500 ppm group:
- significantly lower than for the control group
- mean weekly feed consumption values were 4-17% or 10-15% lower than the control group in male and female rats, respectively
500 ppm group:
- significantly (p - mean weekly feed consumption values were 3-12% lower than the control group in male rats and from 2% higher to 7% lower than the control group for female rats
FOOD EFFICIENCY
- no data

WATER CONSUMPTION
- no data

OPHTHALMOSCOPIC EXAMINATION
- no effects

HAEMATOLOGY
No significant differences in hematologic parameters were seen after 30 days on test. Significantly higher (p Evaluation of blood cell morphology did not suggest any compound-related effects. After 30 days on test, spherocytosis and poikilocytosis were seen in blood smears of animals from most groups. Increased polychromasia was observed in one male control rat (# 613), while decreased polychromasia was see in two female 3000 ppm rats (# 719 and 720). Howell-Jolly bodies in the blood and anisocytosis were also noted for Rat # 720. After 90 days on test, poikilocytosis was seen in animals from all groups. Anisocytosis was noted in animals from the control, 500, and 1500 ppm groups. Microcytosis was seen in one male 1500 ppm rat (# 632) and in one male 500 ppm rat (#624) and spherocytosis was seen in two male 1500 ppm rats (# 631 and 638).

CLINICAL CHEMISTRY
After 30 days on test, mean sodium concentrations for the male and female 3000 ppm groups were significantly lower (p After 90 days on test, mean albumin and total protein concentrations for the 3000 ppm female group were significantly lower (p
URINALYSIS
- not examined

NEUROBEHAVIOUR
- not examined

ORGAN WEIGHTS
Mean terminal body weights measured after exsanguination were significantly lower (p kidney weights for the 1500 female and 3000 ppm male and female groups were also significantly lower (p

GROSS PATHOLOGY
- no exposure related effects were observe in male rats.
- Two females of the 3000 ppm group showed hemorrhage involving the glandular stomach (minimal severity). White discoloration in the non-glandular stomach was also observe for these animals.
- No changes were seen in female rats from the 1500 and 500 ppm groups.

HISTOPATHOLOGY: NON-NEOPLASTIC
- exposure related changes were observed in the nasal passages and stomach of 1500 and 3000 ppm rats
- all male and female 3000 ppm rats and 4/10 male and 6/10 female 1500 ppm rats had necrosis of the olfactory epithelium (mild to moderate for the 3000 ppm group, mild for the 1500 ppm group).
- no lesions were observed in the nasal passages of the 500 ppm group
- 3/10 female rats of the 3000 ppm group had inflammation of the stomach mucosa (glandular or forestomach) of mild to minimal severity; the pathologist concluded that this effect was probably due to stress
- no effects were seen in the low- and mid concentration group
- thymus atrophy was observed in one male of the 3000 ppm group, this lesion was attributed to stress by the pathologist

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
- not examined


OTHER FINDINGS
- No exposure-related effect on epidydimidal or testicular sperm count was observed. Effects on testicular staging or spermatogenic were not evaluated due to the unacceptable condition of tissue.
Dose descriptor:
NOAEC
Effect level:
500 ppm
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Critical effects observed:
not specified

- overall time-weighted average analytical concentrations:

548.4; 1487.5, 3009.6 ppm for males

547.9, 1487.6, 3008.8 ppm for females

- target analytical concentration for the 500 ppm group was increased to 550 ppm to ensure that exposure concentration was at least 500 ppm at all locations of the eposure chamber

Conclusions:
Subchronic exposure of rats to n-butyl acetate vapour resulted in acute, transient signs of reduced activity levels during exposure to 1500 and 3000 ppm. Decreased body weight and feed consumption were noted for the 1500 and 3000 ppm groups, but there was no systemic or organ-specific toxicity. Signs of upper respiratory tract irritation were seen in the nasal passages of 1500 and 3000 ppm animals, but there was no evidence of pulmonary toxicity. The no-observed-adverse-effect concentration (NOAEC) for this study is considered to be 500 ppm (2.4 mg/L).
Executive summary:

Male and female Sprague-Dawley rats (15 animals/sex/dose group) were exposed to nominal concentrations of 0, 500, 1500 or 3000 ppm of n-butyl acetate for 6 hours per day, 5 days per week for 13 consecutive weeks. The time-weighted average analytical concentrations were within 10% of the target concentrations. Transient signs of sedation were observed during exposure to the 1500 and 3000 ppm concentrations. Body weights were significantly reduced in the mid and high concentration groups. Feed consumption was significantly lower in the 1500 and 3000 ppm group in comparison to the control group. Organ weights affected: weights of liver, kidneys and spleen were significantly lower for the males of the highest concentration goup. Testes and adrenal gland weights for the mid and high concentration groups and the lung weights for the 3000 ppm males were significantly higher than for the control group. Additionally, effects on the stomach (probably stress related) and pulmonary system were observed: Females of the highest concentration group showed signs of irritation of the glandular stomach and necrosis in the non-glandular stomach. Some rats of the 1500 and 3000 ppm group showed degeneration of the olfactory epithelium along the dorsal medial meatus and ethomtubinates of the nasal passages. The severity was mild to moderate for the 3000 ppm group and minimal to mild for the 1500 ppm group. There was no systemic, organ specific toxicity. The no-observed-adverse-effect concentration (NOAEC) for this study is 500 ppm (2.4 mg/L) (Bernard and David, 1996; David et al., 2001).

The study is reliable without restriction (RL1).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
2 400 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
Results from reliable, GLP conform subchronic toxicity study with rats are of high reliability (Klimisch score 1) and are supported by findings from the dose-range finding study as well as another less reliable subacute toxicity study with cats.

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
Study period:
From 12 SEP 1994 to 16 AUG 1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline Study (EPA OTS 798.2450)
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
EPA OTS 798.2450 (90-Day Inhalation Toxicity)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Kingston (Stone ridge, NY)
- Age at study initiation: 60 days
- Weight at study initiation: 271 +/- 7 g (males); 215 +/- 8 g (females)
- Fasting period before study: no
- Housing: individually during non expsure periods
- Diet: Certified Rodent Diet (Agway Prolab RMH 3200, ground chow), ad libitum except during exposure
- Water: ad libitum, except during exposure
- Acclimation period: 12 days


ENVIRONMENTAL CONDITIONS
- Temperature: 67-75°F
- Humidity: 46-60%
- Photoperiod (hrs dark / hrs light): 12/12


Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 4200 L stainless-steel and glass inhalation chambers
- Method of holding animals in test chamber: cages
- System of generating vapour: test substance was metered into glass distillation columns packed with glass beads; filtered, compressed air was passed through the glass bead-packed columns to evaporate the test substance; distillation columns were heated to about 50°C to enhance vaporization; the resultant vapour was directed via glass tubing to a tee just upstream of the inhalation chamber where it was mixed with filtered, conditioned outside air
- Temperature, humidity in air chamber: 21.1-24.7°C; 36.7-68.7%
- Air flow rate: 836 to 965 Lpm
- Air change rate: 12 to 14 air changes per hour
- Method of particle size determination: Micro Laser Particle counter (µLPC-301, Particle Measuring Systems, Inc, Coulder, USA); indicating that an aerosol fo the test subsance was not present



TEST ATMOSPHERE
- Brief description of analytical method used: MIRAN IA infrared gas analyzer (Wilks Foxboro Analytical, South Norwalk, CT) set at a wavelength of 3.38 µM
- Samples taken from breathing zone: no; collection of chamber vapour samples

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- MIRAN IA infrared gas analyzer (Wilks Foxboro Analytical, South Norwalk, CT) set at a wavelength of 3.38 µM
- chamber vapour samples were continuously collected from each chamber throught TEFLON tubing (3/16" i.d.)
- valve position was pepriodically changed to sample from each chamber at least once each hour
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 hours per day, 5 days per week
Remarks:
Doses / Concentrations:
500, 1500, 3000 ppm
Basis:
nominal conc.
No. of animals per sex per dose:
15
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: Range finding study: 2-Weeks repeated exposure in which animals were exposed to 0, 750, 1500 or 3000 ppm n-butyl acetate. The test substance produced concentration-related reductions in general activity levels during exposure periods. Animals appeared to acclimate to the 750 and 1500 ppm concentrations but not to 3000 ppm. Mean body weights for the female 1500 ppm animals and for the 3000 ppm male and female animals were lower than the control group on Days 7 and 14, but no statistically significant differences were noted. 3000 ppm was selected as an exposure concentration that would produce overt signs of toxicity, and 500 ppm was selected as an exposure concentration that was expected to have no effect. An exposure concentration of 1500 ppm was selected as the intermediate exposure concentration.
Positive control:
none
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: during exposure and once per day on weekends
- Cage side observations checked were included.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before and after exposure


BODY WEIGHT: Yes
- Time schedule for examinations: weekly


FOOD CONSUMPTION: YES
- Time schedule for examinations: weekly

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: prior to the start of the study and during the last week of exposure
- Dose groups that were examined: all animals prior to the start of the study; animals from the control and high-concentration group during the last week of exposure


HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 30 and 90 of the study
- Anaesthetic used for blood collection: Yes (Metofane)
- Animals fasted: Yes
- How many animals: 5 per sex per dose group
- Parameters checked:
Whole blood: hemoglobin concentration, red blood cell count, white blood cell count, hematocrit, red blood cell indices, prothrombin time
Blood smears: cellular morphology and differential white blood cell count


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day 30 and 90 of the study
- Anaesthetic used for blood collection: Yes (Metofane)
- Animals fasted: Yes
- How many animals: 5 per sex per dose group
- Parameters checked:
Blood serum: aspartate aminotransferase, sorbitol dehydrogenase, alkaline phosphatase, creatinine, albumin, total protein, total bilirubin, alanine aminotransferase, gamma glutamyltranspeptidase, urea nitrogen, glucose, calcium, phosphorus


URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: Organ weights (liver, kidneys, testes or ovaries, spleen adrenal glands, lungs, brain)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes (embedded in paraffin, sectioned at 5 µm and stained with hematoxylin and eosin; nasal passages were decalcified prior to being embedded and sectioned); all tissues (see below) were examined microscopically from the control and high-concentration groups, in addition, the lungs, nasal passages, thymus (males only), stomach (females only), and gross lesions were examined from the mid- and low concentration group
- tissues examined: nasal passages, trachea, larynx, lungs, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, heart, aorta, adrenal glands, pituitary gland, thymus, pancreas, urinary bladder, thyroid gland, parathyroid gland, spleen, liver, kidneys, mesenteric lymph nodes, sternum (with bone marrow), right testis, right epididymis, male accessory sex glands, ovaries, vagina, uterus, fallopian tubes, salivary glands, gross lesions
Other examinations:
- assessment of sperm morphology and development (reported in section 7.8)
Statistics:
Bartlett's test, one-way analysis of variance, duncan's multiple range test, Kruskal-Wallis H-test and Mann-Whitney U-test
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 specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
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 examined
Details on results:
CLINICAL SIGNS AND MORTALITY
- no spontaneous mortality occurred during the study

3000 ppm group:
- reduced activity levels of generally minor severity during exposure (less movement, decreased alertness, slower response to tapping on the chamber wall)
- Sialorrhea was observed in three animals for no more than one or two exposure days
- red discoloration of the chin hair occurred in several females for no more than one or two exposure days

1500 ppm group:
- reduced activity of generally minimal severity during exposure periods (except the first 5 hours of Day 0 and the first hour or two of Days 1 and 2)

Animals in all groups had prophyrin nasal discharges and dried porphyrin stains around the nose, which occasionally persisted till the next morning. Animals from all groups exhibited discolored-red facial hair, usually the chin hair (slightly higher in the 3000 ppm group than in other groups)

BODY WEIGHT AND WEIGHT GAIN

3000 ppm group:
- significantly reduced mean body weights and body weight gains in males and females; overall weight gains were 62% and 78% of weight gains for the control group (males and females, respectively)

1500 ppm group:
- significantly reduced mean body weights and body weight gains in males and females; overall weight gains were 77% and 70% of weight gains for the control group (males and females, respectively)

500 ppm group:
- body weights and body weight gains were not affected; overall weight gains were 90% and 107% of weight gains for the control group (males and females, respectively)

FOOD CONSUMPTION

3000 ppm group:
- significantly lower than for the control group
- mean weekly feed consumption values were 14-25% or 6-16% lower than the control group in male and female rats, respectively

1500 ppm group:
- significantly lower than for the control group
- mean weekly feed consumption values were 4-17% or 10-15% lower than the control group in male and female rats, respectively
500 ppm group:
- significantly (p - mean weekly feed consumption values were 3-12% lower than the control group in male rats and from 2% higher to 7% lower than the control group for female rats
FOOD EFFICIENCY
- no data

WATER CONSUMPTION
- no data

OPHTHALMOSCOPIC EXAMINATION
- no effects

HAEMATOLOGY
No significant differences in hematologic parameters were seen after 30 days on test. Significantly higher (p Evaluation of blood cell morphology did not suggest any compound-related effects. After 30 days on test, spherocytosis and poikilocytosis were seen in blood smears of animals from most groups. Increased polychromasia was observed in one male control rat (# 613), while decreased polychromasia was see in two female 3000 ppm rats (# 719 and 720). Howell-Jolly bodies in the blood and anisocytosis were also noted for Rat # 720. After 90 days on test, poikilocytosis was seen in animals from all groups. Anisocytosis was noted in animals from the control, 500, and 1500 ppm groups. Microcytosis was seen in one male 1500 ppm rat (# 632) and in one male 500 ppm rat (#624) and spherocytosis was seen in two male 1500 ppm rats (# 631 and 638).

CLINICAL CHEMISTRY
After 30 days on test, mean sodium concentrations for the male and female 3000 ppm groups were significantly lower (p After 90 days on test, mean albumin and total protein concentrations for the 3000 ppm female group were significantly lower (p
URINALYSIS
- not examined

NEUROBEHAVIOUR
- not examined

ORGAN WEIGHTS
Mean terminal body weights measured after exsanguination were significantly lower (p kidney weights for the 1500 female and 3000 ppm male and female groups were also significantly lower (p

GROSS PATHOLOGY
- no exposure related effects were observe in male rats.
- Two females of the 3000 ppm group showed hemorrhage involving the glandular stomach (minimal severity). White discoloration in the non-glandular stomach was also observe for these animals.
- No changes were seen in female rats from the 1500 and 500 ppm groups.

HISTOPATHOLOGY: NON-NEOPLASTIC
- exposure related changes were observed in the nasal passages and stomach of 1500 and 3000 ppm rats
- all male and female 3000 ppm rats and 4/10 male and 6/10 female 1500 ppm rats had necrosis of the olfactory epithelium (mild to moderate for the 3000 ppm group, mild for the 1500 ppm group).
- no lesions were observed in the nasal passages of the 500 ppm group
- 3/10 female rats of the 3000 ppm group had inflammation of the stomach mucosa (glandular or forestomach) of mild to minimal severity; the pathologist concluded that this effect was probably due to stress
- no effects were seen in the low- and mid concentration group
- thymus atrophy was observed in one male of the 3000 ppm group, this lesion was attributed to stress by the pathologist

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
- not examined


OTHER FINDINGS
- No exposure-related effect on epidydimidal or testicular sperm count was observed. Effects on testicular staging or spermatogenic were not evaluated due to the unacceptable condition of tissue.
Dose descriptor:
NOAEC
Effect level:
500 ppm
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Critical effects observed:
not specified

- overall time-weighted average analytical concentrations:

548.4; 1487.5, 3009.6 ppm for males

547.9, 1487.6, 3008.8 ppm for females

- target analytical concentration for the 500 ppm group was increased to 550 ppm to ensure that exposure concentration was at least 500 ppm at all locations of the eposure chamber

Conclusions:
Subchronic exposure of rats to n-butyl acetate vapour resulted in acute, transient signs of reduced activity levels during exposure to 1500 and 3000 ppm. Decreased body weight and feed consumption were noted for the 1500 and 3000 ppm groups, but there was no systemic or organ-specific toxicity. Signs of upper respiratory tract irritation were seen in the nasal passages of 1500 and 3000 ppm animals, but there was no evidence of pulmonary toxicity. The no-observed-adverse-effect concentration (NOAEC) for this study is considered to be 500 ppm (2.4 mg/L).
Executive summary:

Male and female Sprague-Dawley rats (15 animals/sex/dose group) were exposed to nominal concentrations of 0, 500, 1500 or 3000 ppm of n-butyl acetate for 6 hours per day, 5 days per week for 13 consecutive weeks. The time-weighted average analytical concentrations were within 10% of the target concentrations. Transient signs of sedation were observed during exposure to the 1500 and 3000 ppm concentrations. Body weights were significantly reduced in the mid and high concentration groups. Feed consumption was significantly lower in the 1500 and 3000 ppm group in comparison to the control group. Organ weights affected: weights of liver, kidneys and spleen were significantly lower for the males of the highest concentration goup. Testes and adrenal gland weights for the mid and high concentration groups and the lung weights for the 3000 ppm males were significantly higher than for the control group. Additionally, effects on the stomach (probably stress related) and pulmonary system were observed: Females of the highest concentration group showed signs of irritation of the glandular stomach and necrosis in the non-glandular stomach. Some rats of the 1500 and 3000 ppm group showed degeneration of the olfactory epithelium along the dorsal medial meatus and ethomtubinates of the nasal passages. The severity was mild to moderate for the 3000 ppm group and minimal to mild for the 1500 ppm group. There was no systemic, organ specific toxicity. The no-observed-adverse-effect concentration (NOAEC) for this study is 500 ppm (2.4 mg/L) (Bernard and David, 1996; David et al., 2001).

The study is reliable without restriction (RL1).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
2 400 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
Results from reliable, GLP conform subchronic toxicity study with rats are of high reliability (Klimisch score 1). Supported by results from reliable, GLP conform Two-Generation toxicity study with rats of high reliability (Klimisch score 1). Other studies with repeated exposure support the findings and thus the quality of the database is high.

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:

In the key study male and female Sprague-Dawley rats (15 animals/sex/dose group) were exposed to nominal concentrations of 0, 500, 1500 or 3000 ppm of n-butyl acetate (6 h/d, 5 d/w, 13 weeks). Transient signs of sedation were observed during exposure to the 1500 and 3000 ppm concentrations. Body weights were significantly reduced in the mid and high concentration groups. Feed consumption was significantly lower in the 1500 and 3000 ppm group in comparison to the control group. Organ weights affected: weights of liver, kidneys and spleen were significantly lower for the males of the highest concentration group. Testes and adrenal gland weights for the mid and high concentration groups and the lung weights for the 3000 ppm males were significantly higher than for the control group. Additionally, effects on the stomach (probably stress related) and pulmonary system were observed: Females of the highest concentration group showed signs of irritation of the glandular stomach and necrosis in the non-glandular stomach. Some rats of the 1500 and 3000 ppm group showed degeneration of the olfactory epithelium along the dorsal medial meatus and ethomtubinates of the nasal passages. The severity was mild to moderate for the 3000 ppm group and minimal to mild for the 1500 ppm group. The no-observed-adverse-effect concentration (NOAEC) for this study is 500 ppm (2.4 mg/L) (Bernard and David, 1996; David et al., 2001; corresponds to IUCLID study record OPP/CMA, 1996).

Similar results were obtained in the range finding study for the subchronic study: Sprague-Dawley rats were exposed to 0, 750, 1500 and 3000 ppm n-butyl acetate for two consecutive weeks (6 h/d, 5 d/w). Groups consisted of 5 males and 5 females, which received food ad libitum, and 5 feed-restricted males. Exposure to n-butyl acetate produced concentration-related reductions in activity levels during exposure but not immediately after exposure. Animals appeared to acclimate to the 750 and 1500 ppm concentrations but not to the 3000 ppm concentration. There were no apparent differences in the clinical conditions of ad libitum-fed and feed­restricted groups during or after exposure. Mean body weights and feed consumption were reduced as a result of exposure to n-butyl acetate, and feed-restricted 3000 ppm animals lost weight while animals in other feed-restricted groups gained weight. Based on acclimation to exposure at 750 ppm, this concentration appears to be a no-observed-adverse-effect concentration (NOAEC). A no-effect concentration (NOEC) was not determined (Bernard and David, 1995; corresponds to IUCLID study record OPP/CMA, 1995).

Similar findings were observed in a study of lower reliability (RL 3) performed with cats: Cats (one male and one female per dose group) were exposed to 14.5 or 20 mg/L n-butyl acetate for 6 hours/day on 6 consecutive days. During exposure mild signs of irritation were observed. Animals showed apathy at the end of exposure and decreased body weight gain (low dose) or body weight loss (high dose).One animal of the high dose group died on exposure day 5. In the low exposure group increased hemoglobin content, erythrocyte and leucocyte were observed (not analysed for the high exposure group). From this study no NOAEC can be derived, the LOAEC was 14.5 mg/L (Flury and Wirth, 1933). In another study it was reported that rats (10 males per group), exposed to 210 mg/m3 n-butyl acetate for 4 month (5 h/d; 5 d/w) had significantly reduced albumin concentration, increased beta- and gamma globulin concentrations and an increased beta lipoprotein concentration in blood serum at the end of the exposure duration (Makshanova, 1977). But no such findings were observed in the key study. The reliability of this study could not be assigned (thus RL4), as the original study report was only availble in Russian and thus was cited from secondary source.

Local effects:

In the second publication by David et al. (2001, cf. IUCLID study record OPP/CMA, 1996) dose dependent degeneration of the olfactory epithelium was observed (). Local irritating effects were of mild to moderate severity in the high dose group and reported as mild in the mid dose group. The NOAEC for local irritating effects is 500 ppm (2.4 mg/L).

In the reliable and GLP conform Two-Generation toxicity study with rats (RL1, Nemec, 2010 - corresponds to IUCLID study record ACC/WIL, 2010) degeneration of the olfactory epithelium in the nasal cavity is observed already at the lowest test concentration of 750 ppm (i.e. 3620 mg/m³) in F0 and F1 males and females (this was also true for the high dose group F0 and F1 males and females, but not for the mid dose group (1500 ppm) as these effects were not investigated there).

Mechanistically most probably these local irritating effects are caused by the metabolite acetic acid, which is a known corrosive substance and is produced when n-butyl acetate is converted via the carboxyl esterase.

Oral:

In a guideline study with the read-across substance n-butanol rats were exposed by gavage to 0, 30, 125 and 500 mg n-butanol/kg bw/d for 13 weeks (US EPA, 1986; RL2). The only adverse effects observed were ataxia and hypoactivity which were observed 2 -3 minutes after dosing (lasting less than 1 h) in both sexes of the high-dose group during the final 6 weeks of dosing. Similar observations during the first weeks of treatment were not described. But, this is possibly due to the high number of animals to be dosed in the beginning which was reduced after the interim sacrifice which enabled the personnel to perform post-dosing observation more quickly as discussed by the authors. The NOAEL is 125 mg n-butanol/kg bw/d, corresponding to 196 mg n-butyl acetate/kg bw/d.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
No study with repeated oral exposure of n-butyl acetate is available, but a reliable subchronic study with the read-across substance n-butanol (metabolite of n-butyl acetate) is available.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
This study with the longest duration (subchronic) and the lowest NOAEC was chosen (KEY study).

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
This study with the longest duration (subchronic) and the lowest NOAEC was chosen (KEY study).

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
Inhalation exposure is the most relevant route of exposure based on the vapour pressure of the substance. Adequate studies using this route of exposure are available.

Repeated dose toxicity: via oral route - systemic effects (target organ) neurologic: central nervous system

Repeated dose toxicity: inhalation - systemic effects (target organ) neurologic: central nervous system

Justification for classification or non-classification

It is concluded that no classification for repeated dose toxicity is necessary because adverse effects after repeated exposure were only observed at doses above the guidance values for classification.