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Developmental toxicity / teratogenicity

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developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline with acceptable restrictions (reliability adopted from OECD SIDS)

Data source

Referenceopen allclose all

Reference Type:
study report
Report Date:
Reference Type:
secondary source
Report Date:

Materials and methods

Principles of method if other than guideline:
Female rats were exposed to the test substance using four different dosing regimes (control, test substance from day 7-16 of gestation, test substance from day 1-16 of gestation and test substance three weeks pre-mating and from day 1-16 of gestation). The effects of the test substance on developmental toxicity / teratogenicity were examined.
GLP compliance:
Limit test:

Test material

Details on test material:
- Name of test material (as cited in study report): n-Butyl Acetate
- Analytical purity: 99%

Test animals

Details on test animals and environmental conditions:
- Source: Charles River , Portage Facility
- Age at study initiation: 7-8 wks
- Weight at study initiation: 170 - 175 g
- Acclimation period: 3-4 wks

not reported

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
unchanged (no vehicle)
Details on exposure:
Vapor atmospheres of n-butyl acetate were generated using a heated, stainless steel vaporizer with vapor concentrations controlled by a pump metering the amount of liquid available. The chambers were 2.3 m3 in volume and exposure concentrations were within 3% of target.
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
The n-butyl acetate concentrations in the exposure chambers were monitored using the GC system with pneumatically actuated valves
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:2
- Length of cohabitation: maximum 8 nights
- After 8 days of unsuccessful pairing necropsy
- Proof of pregnancy: sperm in vaginal smear referred to as day 1 of pregnancy
Duration of treatment / exposure:
7 h
Frequency of treatment:
Duration of test:
Group 1-Control
Group 2-Day 7-16 of gestation (developmental study)
Group 3- Day 1-16 of gestation [also described in chapter 7.8.1]
Group 4- 3 weeks prior to mating and from Day 1-16 of gestation [also
described in chapter 7.8.1]
Doses / concentrationsopen allclose all
Doses / Concentrations:
7.05 mg/L
analytical conc.
Doses / Concentrations:
1500 ppm
analytical conc.
No. of animals per sex per dose:
37-43 females
Control animals:
yes, concurrent vehicle
Details on study design:
Four groups, each containing 37-43 female Sprague-Dawley rats were exposed to n-butyl acetate air concentrations of either 0 or 1500 ppm (7,050 mg/L) for 7 hours/day. The rats were maintained in the exposure chambers throughout the study period, 3 weeks pre-gestation until gestation day 21. Group 1 was not exposed to test material throughout the study and served as the control. Group 2 was exposed to 1,500 ppm (7,050 mg/L) n-butyl acetate from day 7 to 16 of gestation. Group 3 received n-butyl acetate from Day 1 to 16 of gestation and Group 4 was exposed from 3 weeks pre-gestation through day 16 of gestation. All test material exposures were discontinued from gestation day 17 through study termination. Premating exposures were for 5 days/week while gestational exposures were continuous. On gestation Day 21 (sperm positive = Day 1), the fetuses were collected and examined for both skeletal and visceral malformations.


Maternal examinations:
Data from adult animals, such as food con sumption, body weight, and organ weights, are from pregnant animals only. Although formal randomization of body weights was used to select animals for the experimental groups, removal of data from nonpregnant animals from the group means tends to produce apparent deviations ininitial body-weight values for some groups of animals. Results from histopathology studies are from a random sample of tissues from all females sacrificed at the termination of each study.

Liver, lungs , spleen , kidneys , ovaries and the gravid or nongravid uterus were weighed and the weights recorded. Uteri of all apparently nonpregnant females were stained and examined for implantation sites (Kopf et al ., 1964). Observations of intern abnormalities of the pregnant and nonpregnant animals were recorded (e. g., adhesions, tumors, or evidence of infection). Samples (of appropriate size for proper fixation) were taken of ovaries, uterus, liver, lungs with trachea, snleen, and kidneys of each actual or potential parental female. A randomized sampling of tissues from 25% of the females (a maximum of eight per group) and any grossly abnormal tissues were processed by routine techniques (paraffin embedding, hematoxylin and eosin staining) and subjected to histopathological examination. The residual tissues, and the tissues from the remaining 75% of the females, were preserved for possible future examination. The uterus, with ovaries attached, was removea from each animal. The ovaries were excised, identified as to right and left, and the number of corpora lutea estimated by counting. The excised uterus was opened, the membranes and amniotic fluid were observed for abnormalities, and living and dead fetuses. and resorptions, were counted. Mortality in utero was classified and recorded as "early" (E, placenta and conceptus indistinguishable, or metrial gland), "mid" (M, placenta distinct, embryo partially to fully formed), and "late " (L, fully formed but not viable fetus) . Beginning at the right ovary, numbers were assigned, in order, to each implantation site down the right horn, to the cervix. Consecutive numbers for implantation sites in the left horn proceeded from ovary to cervix.
Ovaries and uterine content:
Live and recently dead fetuses were removed in serial order , blotted on a moist surface , freed of adherent material , and we i ghed . A fetus was designated as stunted when its size was below the norma l range of variation of its littermates, as determined by a statistical tes t to reject extreme obs ervations in one direction (McLaren and Michie , 1960). The crown-rump length of each fetus was measured and recorded. Concurrently , the placentas were removed , weighed and examined; abnormal placentas, if observed, were fixed for histological preparation and examination. Each fetus was examined for gross external abnormalities under an illuminated magnifier. The fetuses were randomly divided into two equal groups for more detailed teratologic examination. In one group, the heads were removed and placed in Bouin's fixative for subsequent examination of serial razor-blade-cut sections by the methods of Wilson (1965) and van Julsingha and Bennett (1977) for rats and rabbits, respectively.
Fetal examinations:
All fetuses were examined for internal abnormalities using Staples' ( 1974) technique. The sex of each fetus was determined by external genitalia and visceral examination of the gonads . All fetuses were eviscerated ; rat fetuses were immediately fixed in alcohol, and rabbit fetuses were skinned and air-dried prior to fixation. Following staining with alizarin red S, maceration with KOH, and clearing in glycerol (Staples and Schnell, 1964 ; Dawson, 1926) , each skeleton was examined for abnormalities in size, shape, relative position, and degree of ossification . Results from fetal morphologic examinations were grouped into three categories (major malformations, minor anomalies, or morphologic variations) according to degree of severity, locus of fetal structural change, and incidence of these changes (Palmer, 1968, 1969, 1972, 1974, 1977, 1978 ; Perraud, 1976).
Binary response variables were compared among groups by chi-square tests for independence (Siegel, 1956). Pairwise comparisons for signi ficant findings used either a two-tailed chi-square test or a Fisher's Exact Test (Siegel, 1956). Analysis of variance (ANOVA) method was us ed to analyze continuous variable data. Response proportions were analyzed by ANOVA with an arcsin transformation of the response proportion. Orthogonal a priori comparisons were made among treatment group means for rabbits and rats. All orthogonal comparis ons were two-tailed tests.
Absolute maternal organ weights were analyzed by analysis of covariance using the terminal body weight minus the weight of the gravid uterus (extragestational body weight) as the covariate. Relative organ weights were also analyzed as a percentage of the extragestati o nal body weight by analysis of variance.
Body weights and crown-rump lengths for live male and female fetuses were analyzed by nested analysis of variance. The analysis takes into accunt the effects of treatment, litter, and sex on the body weight and crown-rump length measurements.
Repeated-measures data, such as maternal body weight, were analyzed by a multivariate repea ted measures analysis. Orthogonal polynomials were fit for each animal for which there were complete data, and a multivariate analysis of variance was performed on the coefficients to identify differences in growth patterns among exposure groups (Bock, 1975).

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Details on maternal toxic effects:
Feed consumption was decreased in each test group in the week following initiation on n-butyl acetate exposure. The decrease in feed consumption was accompanied by decreases in body weight in Groups 3 and 4. Relative kidney and lung weights were increased in animals exposed to n-butyl acetate, with the greatest increase occurring in the animals receiving the longest exposure. There were no changes in histopathology that could be related to n-butyl acetate exposure. Mating and reproductive performance and intrauterine mortality was unaffected.

Effect levels (maternal animals)

Dose descriptor:
Effect level:
7.05 mg/L air
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:
Fetal growth measures (fetal body weights and crown-rump growth) and placental weights were lower in Groups 2, 3, and 4. However, the duration of exposure and period of gestation during which exposure occurred did not affect fetal growth indices. Sex ratios were unaffected.
The incidence of rib dysmorphology was increased in fetuses of rats expised to n-butyl acetate during gestation. The incidence of wavy, fused and bifid ribs increased in rats exposed from 7 to 16 dg (P = 0.05), or from 1 to 16 dg (P= 0.07). Reduced pelvic ossification was also observed in fetuses of group 2 and 3 (P=0.08 and P= 0.002, respectively. A larger number of fetuses with dilated ureters was noted in rats that were exposed to
n-butyl acetate for 31 days than in the filtered-air-exposed animals.

Effect levels (fetuses)

Dose descriptor:
Effect level:
7.05 mg/L air
Basis for effect level:
other: teratogenicity

Fetal abnormalities

not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Authors stated: "The significantly increased incidence of rib dysmorphology in rat fetuses of Group 2 and the suggestive increase in Group 3 might be considered indicators of an effect on development. We hesitate to define this as a teratogenic effect of n-butyl acetate, since a similar increase was not seen in the group of rats exposed during this period of gestation subsequent to a pregestational exposure."

Applicant's summary and conclusion