Reaction mass of 2-methylbutyl acetate and pentyl acetate

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

7 May - 7 Sept 1984

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1985

Report date:

1985

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

Principles of method if other than guideline:

Study was conducted prior to actual guidelines but followed intent of the guidelines

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 2-methylbutyl acetate and pentyl acetate

EC Number:

908-918-1

Molecular formula:

Unspecified

IUPAC Name:

Reaction mass of 2-methylbutyl acetate and pentyl acetate

Details on test material:

The test material is 98.97% (pre-study) and 98.7% (post-study) pure (all isomers totaled).
Approximate isomeric composition Primary Amyl Acetate: 60%, 2-Methyl Butyl Acetate: 35% and 3-Methyl Butyl Acetate: 5%

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animal Species, Source, and Quality Control
One-hundred four (104) male and one-hundred five (105) female rats [COBS CDF@ (F-344/Cr1BR)], 34 days of age, were received on April 16, 1984 from Charles River Breeding Laboratories, Inc. (Kingston, NY). Shortly after delivery, random fecal samples were collected and examined for intestinal parasites by zinc sulfate flotation. Results of the fecal tests were negative. Blood samples were collected from three male and three female rats for serology prior to sacrifice. A gross examination was performed on the same rats used for serology evaluations and the liver, heart, kidneys, salivary glands, nasal turbinates, lungs, trachea, spleen and cervical lymph nodes were fixed and examined microscopically. Physical examination, parasitology,
serology, and histology on the quality control animals were within expected limits for this strain and species. Body weight determinations and clinical
and ophthalmologic examinations of all animals were performed prior to the initiation of exposures.

Animal Husbandry
The rats were housed two per cage in stainless steel, wire-mesh cages (23.5 cm x 40 cm x 18 cm high). All animals were housed separately by test group and sex. A layer of Deotized Animal Cage Board* (Shepherd Specialty Papers, Inc., Kalamazoo, MI) was placed under each cage. The room temperature and relative humidity were recorded continuously (Hygrothermograph Seven-Day Continuous Recorder, Model #8368-00, Cole-Parmer Instrument Company, Chicago, IL). The animals were kept on a 12-hour photoperiod throughout the study.

During non-exposure periods, water (Municipal Authority of Westmoreland County, Greensburg, PA) supplied by an automatic watering system, and powdered food (Purina Certified Rodent Chow #5002, Ralston Purina Company, St. Louis, MO) were available ad libitum. Analyses of the food and water showed no contaminants at concentrations high enough to interfere with the outcome of the study.

During the exposures, the animals were housed two per cage, separated by sex and test group, in stainless steel wire-mesh cages, 12.5 cm x 20 cm x 17 cm high. Food and water were withheld during exposure.

Group Assignment
The body weight and physical condition of all animals were monitored for approximately two weeks prior to placement into exposure groups. Animals were assigned to three test groups and an air-exposed control group (20 per sex per group) using a computer-based (Wil Laboratories Toxicology Data Management System) randomization program. At the time of group assignment, only animals with body weights within two standard deviations of the group mean for each sex were used in the study. Any animal in poor health was rejected from group assignment.

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

air

Remarks on MMAD:

MMAD / GSD: Not applicable.

Details on inhalation exposure:

Inhalation Chamber Description and Operation
The inhalation chambers used in the study were constructed from stainless steel with glass windows for animal observation. The volume was approximately 1330 liters and the airflow was approximately 300 L/min (13-14 air changes per hour). Chamber temperature and relative humidity were recorded using an Ashcroft dial-type thermometer and an Abbeon Certified Hygrometer Model AB167B (Abbeon Cal, Inc., Santa Barbara, CA), respectively. Temperature and relative humidity measurements were recorded at least four times per exposure.

Target Concentrations and Exposure Regimen
The animals were acclimated to the exposure chambers (air-only exposure) for two days prior to the initiation of the PAA exposure regimen. Target concentrations of 500, 300, 100, and 0 (control) ppm primary amyl acetate were selected for this study by the Sponsor after results of a preliminary 9-day inhalation study had been reviewed. The rats (55 days of age at initiation of exposures) were exposed for six hours per day, five days a week for thirteen weeks, except for the fourth week when there were 4 exposure days (holiday). During the fourteenth week there were 3 days of exposure for males sacrificed the following day and four days of exposure for all females and male recovery rats. Control (air-exposed) animals were handled in an identical manner as the PAA-exposed animals.

The position of cages was rotated weekly in a predetermined pattern within each chamber to compensate for any possible, but undetected, variations in chamber conditions.

Vapor Generation
The primary amyl acetate liquid was metered from a piston pump (Fluid Metering, Inc., Oyster Bay, NY) into a heated glass evaporator similar in
design to that described by Carpenter -et -a l . (1975). Temperatures in the evaporator were maintained near the minimum temperature required to vaporize the liquid. The resultant vapor was carried into the chamber by a Countercurrent air stream that entered the bottom of the evaporator.

Reference
Carpenter, C. P., Kinkead, E. R., Geary, D. L., Sullivan, L. J., and King, J. M, (1975). Petroleum Hydrocarbon Toxicity Studies. I. Methodology.
Toxicol . Appl . Pharmacol. 32, 246-262.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chamber concentrations of primary amyl acetate were analyzed approximately once each hour by gas chromatography.

Duration of treatment / exposure:

6 hrs/day

Frequency of treatment:

5 days/week except for holidays

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0, 100, 300 and 500 ppm
Basis:
nominal conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0, 101±3.8, 303±8.8, and 509±16.4 ppm (corresponding to approx. 0.54, 1.61 and 2.71 mg/l, calculated assuming test substance molecular weight of 130.19 g/mol)
Basis:
analytical conc.

No. of animals per sex per dose:

20 rat/sex/dose level

Control animals:

yes, concurrent no treatment

Details on study design:

Four groups, each consisting of twenty (20) Fischer 344 rats per sex were exposed for 6 hours per day, 5 days per week for 14 weeks to vapors of primary amyl acetate (PAA) at target concentrations of 0, 100, 300 or 500 ppm. Actual mean concentrations obtained for this study were 101, 303, and 509 ppm PAA. The following parameters served as monitors for toxic effects: clinical observations, body weight, food and water consumption, ophthalmology, hematology, clinical chemistry, urinalysis, organ weights, and macroscopic and microscopic tissue evaluations.

Positive control:

No data.

Examinations

Observations and examinations performed and frequency:

Animal Observation
All animals were observed prior to, during, and following each exposure for signs of toxic effects. Animals were observed once a day on weekends and holidays during the fourteen week exposure regimen. The recovery animals were observed once a day.

Body Weights
All animals were weighed just prior to the initiation of the first exposure. These values were used as the pre-exposure reference weights (labeled study day "zero") and were substracted from each subsequent weight to determine the change in body weight. The animals were weighed weekly during
the exposure period and preceding sacrifice. Body weights for animals held during the epostexposure period were determined weekly and just prior to sacrifice.

Food and Water Consumption
Ten rats per sex per exposure group were individually housed in round polycarbonate metabolism cages with stainless steel, wire-mesh bottoms,
approximately 20 cm diameter x 11 cm high (Nalge Company, Rochester, NY). Food and water consumption were measured for approximately 15 1/2 or 16 hours for male and female rats, respectively, following sixty-seven exposures for the male rats and sixty-eight exposures for the female rats. Food and water consumption evaluations were not conducted on the recovery animals.

Ophthalmologic Evaluation
Prior to the first exposure and at sacrifice, the anterior chambers of the eyes of each animal were examined by a veterinarian using an external light
source and a magnifying lens. At the recovery sacrifice, the rats were examined by a veterinarian using a direct light ophthalmoscope to observe the
anterior chamber of the eye.

Sacrifice and pathology:

Blood Analysis
Serum chemistry and hematologic evaluations were performed on blood samples collected from eighty rats (10 per sex per exposure group) just prior to sacrifice on the day following the last exposure and from eighty recovery rats (10 per sex per group) just prior to sacrifice. Blood was obtained from the orbital sinuses of methoxyflurane- anesthetized animals. Water was available to all animals ad libitum during blood collection and necropsy.

Urinalysis
Urine was collected while the rats were in the metabolism cages. Food and water were available ad libitum.

Necropsy and Pathology
Ten male rats per exposure group were sacrificed on August 9; ten female rats per exposure group were sacrificed on August 10, 1984. The recovery rats, ten males and ten females per exposure group were sacrificed on September 6 and 7, 1984, respectively. The rats were killed by exsanguination via the brachial blood vessels following anesthesia with methoxyflurane. Gross examinations were performed and selected tissues were saved in neutral buffered 10% formalin for possible future histologic evaluation. Histologic evaluation was performed on selected tissues from animals in both the 500 ppm and control groups.

Tissues collected at necropsy included:
adipose tissues
adrenals*
aorta
bone and bone marrow - femoral, sternal*, vertebral
bone marrow smear
brain* - brain stem*, cerebellum*, cerebrum*
cervix
ears
epididymides*
esophagus
eyes
fallopian tubes
heart*
intestine - large (3 levels) small (3 levels)
kidneys*
lacrimal glands
larynx*
liver*
lungs
lymph nodes - cervical*, mesenteric, thoracic (mediastinal)
mammary tissue
muscle - gastrocnemius*
nasal turbinates*
nerve - plantar, sciatic*, tibial
ovaries
pancreas
parathyroids*
pituitary*
prostate (and associated sex glands)
salivary glands
skin (flank)
spinal cord (lumbosacral section)
spleen*
stomach
testes*
thymus*
thyroids*
trachea*
urinary bladder*
uterus
vagina
Zymbal ' s gland
any lesions
* tissues examined histopathologically

Organ Weights
The brain, liver, kidneys, spleen, lungs, and testes (males only) from all animals were weighed at sacrifice. Organ weights were recorded as absolute
weights and as relative (as a percentage of body weight) weights.

Other examinations:

No additional information available.

Statistics:

Results of quantitative continuous variables were intercompared among the three exposure groups and one control group by use of Bartlett's homogeneity of variance (Sokal and Rohlf, 1969), analysis of variance (ANOVA) (Sokal and Rohlf, 19691, and Duncan's multiple range tests (Snedecor and Cochran, 1967). The latter was used to delineate which exposure groups differed from the control, when F from the analysis of variance was significant. If Bartlett's test indicated heterogeneous variances, all groups were compared by an ANOVA for unequal variances (Welch or Brown and Forsythe, 1974) followed if necessary by t-tests. Corrected Bonferroni probabilities were used for t-test comparisons. Statistical procedures for hematologic continuous variables were similar. The fiducial limit of 0.05 (two-tailed) was used as the critical level of significance for all comparisons.

References
Sokal, R. R. and Rholf, F.J. (1969). Biometry, W. H. Freeman and Company, San Francisco, pp. 299-340 and 369-371.

Snedecor, G. W. and Cochran, W. G. (1967). Statistical Methods, Iowa State University Press, Ames, 10, 274-275.

Brown, M. B. and Forsythe, A. B. (1974). The Small Sample Behavior of Some Statistics Which Test the Equality of Several Means. Technometrics 16,
129-132.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Control group appeared to have faster growth rate than other control animals of same age which resulted in an apparent effect in rats exposed to PAA.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not specified

Details on results:

Chamber Concentration, Temperature and Relative Humidity
The target concentrations were 100, 300 and 500 ppm of PAA vapor. Control animals were exposed to filtered air only. Gas chromatographic analyses of the chamber atmospheres resulted in mean (+/- standard deviation) concentrations of 101 (+/- 3.8), 303 (+/- 8.8) and 509 (+/- 16.4) ppm, respectively. The mean analytical to nominal concentration ratios were 0.97, 0.99 and 0.98, respectively. No amyl acetate was detected in the control chamber.

During the exposures, the daily mean chamber temperature and relative humidity ranged between 73-79F and 42-58%, respectively.

Housing condition
The temperature and relative humidity in the animal housing room ranged between 68-79F and 35-70%, respectively, throughout the study period.

Animal Observations
There were no exposure-related clinical signs observed for either sex of any of the exposure groups.

On June 27, 1984, Exposure Day 38, there was an error made in placement of the 100 and 300 ppm group males in the chambers. The 100 ppm group males were placed in the 300 ppm exposure chamber; the 300 ppm group males were placed in the 100 ppm exposure chamber. The mean exposure concentrations were 99.4 and 306 ppm in the 100 and 300 ppm exposure chamhers, respectively. The error occurred only on Exposure Day 38. On June 29, 1984, all animals were weighed and individually observed for clinical signs. There was neither a perturbation in body weight gain for the 100 ppm exposure group males nor were any clinical signs present in those rats. Thus, this error did not alter the interpretation of the results of this study.

Mortality
There were no unscheduled deaths during the study.

Body Weights
All PAA exposure groups of males had body weight gains that were statistically significantly lower than the controls by the second week of the exposure, with the 300 and 500 ppm groups also exhibiting significantly lower weight gains during the first week of exposures. The lower weight gains persisted throughout the entire exposure period for all three PAA exposure groups, and the absolute body weights for all PAA-exposed groups of males were also significantly lower than controls at the end of the exposure regimen. A significant decrease in body weight gain was observed during the first week of the recovery period (days 95-102) for the 100 and 300 ppm group males. Although the body weight gains were still below those of controls for the last three weeks of the recovery period, the difference was not statistically significant. There were also no statistically significant differences for the absolute body weights between groups of males at the end of the recovery period. All PAA-exposed groups of males had similar body weight gains throughout the study, i. e., there were no concentration-response decreases in body weight gain.

For females, there were no exposure-related effects observed on body weight gain or absolute body weight during the study. The 100 ppm females had significantly higher body weight gains and/or absolute body weights at various intervals during the exposure period, but not during the recovery period.

Food and Water Consumption
There were no significant differences in food or water consumption observed in either sex of the study groups.

Ophthalmologic Evaluations
There were no exposure-related ocular effects observed during the study.

Blood Analysis and Urinalysis
There were no statistically significant differences between control and PAA-exposed groups of animals for any of the clinical chemistry,
hematology or urinalysis parameters evaluated for the study.

Necropsy
There were no exposure-related macroscopic lesions observed during the exposure or recovery periods.

Organ Weights
There were signficantly decreased absolute liver and testes weights in all groups of PAA-exposed males at the end of 14 weeks of exposure. Significantly increased absolute brain and spleen weights were observed in all groups of PAA-exposed females, compared to controls, at the end of the 14-week exposure period. There were significantly increased relative brain weights in the 100 and 500 ppm exposure group males at the end of the exposure period. There were no statistically significant differences observed for any organ weights for the study groups at the end of the recovery period.

Anatomic Pathology
There were no exposure-related differences between the control and 500 ppm exposure groups.

Effect levels

Dose descriptor:

NOAEC

Effect level:

2.66 mg/L air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no treatment related effects observed at any dose level during this study (original value: 500 ppm)

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

The exposure of rats to primary amyl acetate at air concentrations up to 509 ppm did not result in adverse effects on weight gain or food and water consumption. Toxicologically significant changes were not noted in clinical examination of blood or urine. Clinical signs of toxicity were not observed. Finally, there was no evidence of organ or tissue injury in animals exposed for 90 days to air concentrations of primary amyl acetate up to 509 ppm.

Executive summary:

Four groups, each consisting of twenty (20) Fischer 344 rats per sex were exposed for 6 hours per day, 5 days per week for 14 weeks to vapors of primary amyl acetate (PAA) at target concentrations of 0, 100, 300 or 500 ppm. Actual mean concentrations obtained for this study were 101, 303, and 509 ppm PAA. The following parameters served as monitors for toxic effects: clinical observations, body weight, food and water consumption, ophthalmology, hematology, clinical chemistry, urinalysis, organ weights, and macroscopic and microscopic tissue evaluations. There were no mortalities during the study and no exposure-related effects were observed in any of the parameters evaluated for this study.