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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 Oct 1990 - 16 Nov 1990
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report date:
1994

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity 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

Constituent 1
Chemical structure
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 substance was a colorless liquid with a mild and characteristic odor (MSDS) and was a mixture of two isomers, primary amyl acetate (CAS Number 628-63-7) and 2-methyl butyl acetate. These two components comprised > 99% of the primary amyl acetate test substance. The purity of the test substance as determined by the Union Carbide Technical Center (South Charleston, WV) was > 99%. No compositional changes in the test substance occurred during the course of the study.

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
Seventy (70) virgin female New Zealand White rabbits (3.0-3.5 kg, as designated by the supplier, and approximately 5 months old) were received on
October 1, 1990 from Hazleton Dutchland Laboratories, Inc. (Denver, PA). Male New Zealand White rabbits from the BRRC buck colony (originally from the same supplier) were used for breeding.

A pretest health screen, utilizing three females randomly selected from the population of rabbits received for the study, was performed within 1 day of receipt of the animals. Examination for intestinal parasites was performed by the zinc sulfate flotation method. Evaluation for fecal parasites indicated that the animals were free of parasites and considered to be acceptable for use on the study.

The females were individually housed in stainless steel, wire-mesh cages, 61 cm x 61 cm x 41 cm, during the acclimation and study periods. A layer of Deotized Animal Cage Boarde (Shepherd Specialty Papers, Inc., Kalamazoo, MI) was placed under each row of cages to collect solid and liquid excrement. The paper board was changed at least three times per week. Other animal care was performed regularly according to BRRC standard operating procedure. The room temperature and relative humidity were monitored continuously by a Cole-Parmer Hygrothermographo Seven-Day Continuous Recorder, Model No. 8368-00 (Cole-Parmer Instrument Co., Chicago, IL). Temperature and relative humidity were routinely maintained within the protocol-specified range of 61-70°F and 40-70%, respectively. Any minor exceptions to these specified ranges were noted in the raw data. The animals were kept on a 12-hour photoperiod using an automatic timer throughout the study.

During nonexposure periods, water and food were available to the animals ad libitum. Tap water (Municipal Authority of Westmoreland County, Greensburg, PA) was delivered by an automatic watering system with demand control valves mounted on each rack. Water analyses were provided by the supplier, the Halliburton NUS Environmental Laboratories, Materials Engineering & Testing Company, and Lancaster Laboratories, Inc. at regular intervals. EPA standards for maximum levels of contaminants were not exceeded. The animals were supplied with Agway Prolab Certified Rabbit Foodo, Batch No. AUGUST 15 90 W1 (Agway, Inc., St. Marys, OH). Analyses for chemical composition and possible contaminants of each feed lot were performed by Agway, Inc. (Ithaca, NY), and the results were included in the raw data.

During the exposures, the animals were individually housed, separated by exposure group, in stainless steel, wire-mesh cages, 35 cm x 37 cm x 41 cm. Food and water were withheld during the exposures.

The body weight and physical condition of all animals were monitored for approximately 2 weeks prior to mating.

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
unchanged (no vehicle)
Details on exposure:
The inhalation chambers used for this study were constructed from stainless steel with glass windows (Wahmann Manufacturing Company, Timonium, MD) for animal observation. The volume of each chamber was approximately 4320 liters, and the air flow was 1000 liters/minute (14 air changes per hour). Chamber temperature and relative humidity were recorded using Industrial thermometers (Control Specialties, Inc., Houston, TX) and an Airguide humidity indicator (Airguide Instrument Co., Chicago, IL). Temperature and relative humidity measurements were recorded twelve times per exposure. Prior to the start of the study, the distribution of primary amyl acetate vapor in the chamber was evaluated at five different locations in two of the three exposure chambers. In addition, prior to the start of exposures, and on Exposure Days 2 and 16, the oxygen content of all chambers was measured with an 02 Indicator Model 250 (Mine Safety Appliances, Pittsburgh, PA).

Primary Amy1 Acetate Vapor Generation
Liquid primary amyl acetate 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 al. (1975) and, more recently, by Snellings and Dodd (1990). The temperature in the evaporator(s) was maintained at the lowest level sufficient to vaporize the test substance. The resultant vapor was carried into the chamber by a countercurrent air stream that entered the bottom of the evaporator. Prior to and after the exposure regimen, temperature was measured from the inside surface of the evaporators using a Model 400A Doric Trendicator (Doric Scientific Division, San Diego, CA) and a Type K thermocouple.

Exposure Regimen
The mated females assigned to the study were exposed to primary amyl acetate vapor or filtered air for six hours per day during the period of major
organogenesis (gd 6 through 18, October 21 through November 5, 1990). The 6-hour exposure interval was defined as the time when the vapor generation system was turned on and subsequently turned off. Within each chamber, the cage placement pattern was changed daily to compensate for any possible, but undetected, variations in chamber exposure conditions.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chamber concentrations of primary amyl acetate vapor were analyzed by flame ionization gas chromatography. Approximately nine samples were obtained from each exposure chamber including the control chamber during the 6-hour exposure period. The nominal concentration was calculated from the total amount of test substance delivered to the chamber and chamber air flow rate.
Details on mating procedure:
New Zealand White rabbit females were mated to "provenn males from the BRRC breeding colony. Following successful copulation, the animals were returned to their respective cages; the date of the copulation was designated gestation day (gd) 0. The mating period was October 15-18, 1990. On each mating day successfully mated females were assigned to one of three exposure groups or one air-exposed control group using a computer-based randomization program. At completion of mating, each exposure group consisted of 15 mated female rabbits.
Duration of treatment / exposure:
6-hour exposure on gestation days 6-18
Frequency of treatment:
Daily on gestation days 6-18
Duration of test:
gestation days 6-18
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 500, 1000 and 1500 ppm
Basis:
other: target concentration
Remarks:
Doses / Concentrations:
0, 509±4.6, 995±9.2, and 1522±20.1 ppm (corresponding to approx. 2.7, 5.4 and 8.1 mg/L air)
Basis:
analytical conc.
No. of animals per sex per dose:
15 timed-pregnant New Zealand White rabbits
Control animals:
yes, concurrent no treatment
Details on study design:
Target Concentration Selection
The developmental toxicity range-finding study was conducted at target concentrations of 0, 500, 1000, and 1500 ppm primary amyl acetate vapor. Selection of target vapor concentrations for the range-finding study was based on a previous 9-day vapor inhalation study in rats and other available acute toxicity data (see BRRC Report 47-103). Following acute dynamic exposure of Wistar rats to concentrations up to 5.2 mg/L, decreases in body weight gain were observed on postexposure day 1, but weight gains returned to normal limits by postexposure day 3. No deaths occurred following 6-hour static exposures of male and female Wistar rats to concentrations up to 19.3 and 19.7 mg/L primary amyl acetate vapor, respectively. In the 9-day study, repeated exposure of Wistar rats to concentrations up to 1200 ppm produced no clear evidence of toxicity. Although above the limit test exposure level of 5 mg/L (940 ppm), 1500 ppm was selected as the high vapor concentration for the developmental toxicity exposure range-finding study in order to maximize the likelihood of producing sustained maternal effects. While no concentration-related maternal effects were observed in the range-finding study, target concentrations of 0 (control), 500, 1000, and 1500 ppm primary amyl acetate vapor (the same as those in the range-finding study) were selected for the definitive study by the Sponsor. A high vapor concentration of 1500 ppm was selected in order to maximize the likelihood of producing sustained maternal effects using a larger number of animals in the dose group.

Examinations

Maternal examinations:
Maternal In-Life Observations and Evaluations
All does were observed twice daily for morbidity and mortality. Does were observed for clinical signs of toxicity once daily. In addition, during the
exposures, animals were observed from outside their respective exposure chambers for overt signs. Maternal body weights were taken on gd 0, 6, 12, 18, 24, and 29. Food consumption was measured daily throughout the study (gd 0 through 29).

Ovaries and uterine content:
Maternal Necropsy and Laparotomy
At scheduled sacrifice on gd 29, does were sacrificed by injection of Beuthanasia-D Special solution (Schering Corporation USA, Kenilworth, NJ).
The maternal sacrifice period was November 13-16, 1990.

The maternal body cavities were opened by midline thoracolaparotomy. The uterus, ovaries ( including corpora lutea) , cervix, vagina, and abdominal and thoracic cavities were examined grossly. Ovarian corpora lutea of pregnancy were counted. The maternal liver and kidneys were weighed. Each uterus was externally examined for signs of hemorrhage, removed from the peritoneal cavity, weighed and dissected longitudinally to expose the contents. All live and dead fetuses and resorption sites (early and late) were noted and recorded. Uteri from females that appeared nongravid were placed in a 10% ammonium sulfide solution for detection of early resorptions (Salewski, 1964).

Reference
Salewski, E. (1964). Farbemethode Zum Makroskopischen Nachweis Von Implantations-Stellen am Uterus Der Ratte. Naunyn-Schmiedebergs, Arch.
Exp. Pathol. Pharmakol. 247, 367.
Fetal examinations:
Immediately upon exteriorization, fetuses were euthanized by intraperitoneal injection of sodium pentobarbital. All live and dead fetuses were weighed and examined externally for variations and malformations including cleft palate. All live fetuses in each litter were examined for thoracic and abdominal visceral abnormalities by methods described by Staples (1974). Fetal sexes were determined during the visceral examination. One-half of the live fetuses (even-numbered fetuses from litters with an even number of live fetuses, odd numbered fetuses from litters with an odd number of live fetuses) in each litter were decapitated and their heads were fixed in Bouin's solution for examination of craniofacial structures by sectioning methods modified from van Julsingha and Bennett (1977). All fetuses (50% intact, 50% decapitated) in each litter were eviscerated, air-dried, and processed for skeletal staining with alizarin red S (Crary, 1962; Peltzer and Schardein, 1966), and examined for skeletal malformations and variations.

References
Crary, D. D. (1962). Modified Benzyl Alcohol Clearing of Alizarin-Stained Specimens without Loss of Flexibility. Staining Technology 37, 124-125.

Peltzer, M. A. and Schardein, J. L. (1966). A convenient method for processing fetuses for skeletal staining. Stain Technology 4l, 300-302.

Staples, R. E. (1974). Detection of visceral alterations in mammalian fetuses. Teratology, 9, A-37.

van Julsingha, E. B. and C. G. Bennett (1977). A dissecting Procedure for the Detection of Anomalies in the Rabbit Foetal Head. In: Methods in Prenatal Toxicology (D. Neubert, H. J. Merker, and T. E. Kwasigroch, Editors). PsG Publishing Company, Inc., Littleton, Massachusetts, pp. 126-144.
Statistics:
The unit of comparison was the pregnant dam or the litter. The data for quantitative continuous variables were intercompared for the exposure groups and the control group by use of Levene's test for equality of variances, analysis of variance (ANOVA), and t-tests. The t-tests were used when the F value from the ANOVA was significant. When Levene's test indicated similar variances, and the ANOVA was significant, a pooled t-test was used for pairwise comparisons. When Levene's test indicated heterogeneous variances, all groups were compared by an ANOVA for unequal variances followed, when necessary, by a separate variance t-test for pairwise comparisons.

Nonparametric data obtained following laparohysterectomy were statistically evaluated using the Kruskal-Wallis test followed by the Mann-Whitney U-test when appropriate. Incidence data were compared using Fisher's Exact Test. With the exception of the data analysis for fetal malformations and variations, all statistical analyses were performed using BMDP Statistical Software (Dixon, 1990). Incidence data for fetal malformations and variations were analyzed using a program developed at BRRC. For all statistical tests, the probability value of < 0.05 (two-tailed) was used as the critical level of significance.

Indices:
No data
Historical control data:
No additional information available.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: Effects at 1500 ppm included body weight losses during the first week of the exposure period and reduced food consumption during the entire exposure period.

Details on maternal toxic effects:
Chamber Concentration, Temperature, and Relative Humidity
The means of daily mean analytical concentrations (+/- SD) were 509 (+/- 4.6), 995 (+/-9.2), and 1522 (+/- 20.1) ppm for target concentrations of 500, 1000, and 1500 ppm, respectively. The overall mean A/N ratios were 1.03, 1.02, and 1.05 for the 500, 1000, and 1500 ppm concentration, respectively. Among exposure groups, the daily mean chamber temperature and relative humidity ranged from 21-23C and 45-50%, respectively. For all measurements, the chamber oxygen content was 20.8%.

Animal Fate and Observations
No mortality was observed in any exposure group prior to scheduled sacrifice. One doe in the 500 ppm group aborted on gd 24. Two females each from the control and 1500 ppm exposure groups were not pregnant. All pregnant females contained viable fetuses. The overall pregnancy rate was equivalent for all exposure groups, ranging from 87 to 100%. Exposure-related clinical signs observed during the study included full food hoppers for several does in the 1500 ppm group.

Maternal Food Consumption
Food consumption was generally decreased in the 1500 ppm group during the exposure period with differences attaining statistical significance on gd 7-8, 8-9, 9-10, and 11-12. The statistically significant decrease observed in the 1000 ppm group for gd 7-8 was not considered to be biologically significant due to the transient nature of the decrease and the fact that the decrease was observed on the day after the first exposure.

Maternal Body Weights and Weight Changes
No statistically significant differences in body weights were observed in any exposure group. Statistically significant body weight losses were observed in the 1000 and 1500 ppm groups during the gd 6 to 12 interval. In addition, body weight loss was observed in the 500 ppm group. The body weight losses observed in the 1500 ppm group were considered to be related to the exposure since during this period, all does in this group except one lost weight. This body weight loss early in the exposure period contributed to the slightly reduced body weight gain observed during the entire exposure period. Losses observed in the 500 and 1000 ppm groups were not considered to be biologically significant since the magnitude of the losses was small, were within the range of losses commonly seen in rabbit studies, and were unaccompanied by consistent reductions in food consumption.

Maternal Necropsy and Laparotomy
There were no exposure-related necropsy findings in the does at scheduled sacrifice on gd 29 or in the doe which aborted prior to scheduled sacrifice. There were no statistically significant differences in terminal body weight, liver weight or kidney weight. While not statistically significant, corrected weight changes appeared to be decreased in the 1500 ppm group. There were no effects of exposure on the number of ovarian corpora lutea, on the number of total, viable or nonviable (early and late resorptions and dead fetuses) implantations per litter or on sex ratio (percent males). Percent preimplantation and postimplantation losses were equivalent across groups.

Effect levels (maternal animals)

Dose descriptor:
NOEC
Effect level:
5.4 mg/L air (analytical)
Based on:
test mat.
Basis for effect level:
body weight and weight gain

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There were no effects of exposure on the number of ovarian corpora lutea, on the number of total, viable or nonviable (early and late resorptions and dead fetuses) implantations per litter or on sex ratio (percent males). Percent preimplantation and postimplantation losses were equivalent across groups.

No exposure-related effects on fetal body weights (all fetuses, male or female) were observed in any group. Statistically significant decreases
observed in mean fetal weight of male or female fetuses in the 1000 ppm group were not considered to be exposure-related due to the lack of concentration response relationship.

There were no statistically significant differences in individual external, visceral or skeletal malformations by category, or of total malformations
among all groups. There were no statistically significant differences among groups in the incidence of individual fetal external or visceral variations. Of the 73 different types of individual fetal skeletal variations observed, none were statistically significantly increased due to exposure. There were no exposure-related increases in the incidence of variations by category (external, visceral or skeletal) or of total variations.

Effect levels (fetuses)

Dose descriptor:
NOAEC
Effect level:
>= 8.1 mg/L air (analytical)
Based on:
test mat.
Basis for effect level:
other: teratogenicity

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
Exposure of pregnant New Zealand White rabbits to primary amyl acetate vapor during organogenesis at 0, 500, 1000, and 1500 ppm resulted in minimal maternal toxicity at 1500 ppm and no embryolethality or developmental toxicity (including teratogenicity). The no-observed-effect level" (NOEL) for maternal toxicity was 1000 ppm. The NOEL for developmental toxicity was greater than 1500 ppm.
Executive summary:

Four groups, each consisting of 15 timed-pregnant New Zealand White rabbits were exposed to primary amyl acetate vapor for six hours/day on gestational days (gd) 6 through 18. Target concentrations of primary amyl acetate vapor were 0 (control), 500, 1000, and 1500 ppm. Clinical observations were made daily, and body weights were measured on gd 0, 6, 12, 18, 24, and 29. Maternal food consumption was measured daily throughout gestation, gd 0-29. At scheduled sacrifice on gd 29, the does were subjected to a necropsy and were evaluated for body weight, liver and kidney weights, and gravid uterine weight. Ovarian corpora lutea of pregnancy were counted and status of implantation sites (i.e. resorptions, dead fetuses, live fetuses) were identified and recorded. All fetuses were dissected from the uterus, counted, weighed, and examined for external abnormalities. All live fetuses in each litter were examined for visceral malformations and variations and sexed. Approximately one-half of the live fetuses in each litter were examined for craniofacial malformations and variations. All fetuses (50% intact, 50% decapitated) in each litter were stained with alizarin red S and examined for skeletal malformations and variations.

No mortality occurred during the study. One female in the 500 ppm group aborted. The pregnancy rate was equivalent for all groups and ranged from 87 to 1001. Maternal toxicity was observed in the 1500 ppm group and included body weight losses during the first six days of the exposure period accompanied by reduced food consumption during the entire exposure period.

Fetal examination indicated no evidence of fetotoxicity or developmental toxicity in any of the exposure groups. The number of total, viable or nonviable implantations per litter, the sex ratio, and the percent preimplantation and postimplantation losses were similar in all groups. No clear exposure-related decreases in mean fetal body weight were observed. External, visceral and skeletal examinations of the fetuses revealed no exposure-related differences in the incidences of variations or malformations. Exposure of pregnant New Zealand White rabbits during organogenesis to primary amyl acetate vapor at 0, 500, 1000, and 1500 ppm resulted in maternal toxicity at 1500 ppm and no developmental toxicity (including teratogenicity). The no-observed-effect level (NOEL) for maternal toxicity was 1000 ppm. The NOEL for developmental toxicity was at least 1500 ppm.