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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
year of publication: 1982
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1982
Report date:
1982

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
: e.g. additional exposure during pregestation period
GLP compliance:
yes
Limit test:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
N-butyl acetate
EC Number:
204-658-1
EC Name:
N-butyl acetate
Cas Number:
123-86-4
Molecular formula:
C6H12O2
IUPAC Name:
butyl acetate
Details on test material:
- Name of test material (as cited in study report): n-butyl acetate
- Physical state: liquid
- Analytical purity: 99.1%
- Supplier: McKesson Chemical Company

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, Portage facility
- Age at study initiation: 7-8 weeks
- Weight at study initiation: males: 200-225 g; females: 170-175 g
- Fasting period before study: none
- Housing: individually in stainless steel cages
- Diet: Wayne Lab-Blox, ad libitum except during the exposure periods
- Water: ad libitum except during the exposure periods
- Acclimation period: no data
- rats were exposed and housed within the chamber


Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
unchanged (no vehicle)
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: stainless steel chamber (U.S. patent # 4,216,741)
- Method of holding animals in test chamber: in stainless steel cages
- Source and rate of air: HEPA-filtered air
- Method of conditioning air: test item vapour introduced into the filtered air system
- System of generating vapours: Liquid was pumped from a liquid reservoir to the vaporizer. Nitrogen was used to replace the depleted liquid in the reservoir, to prevent formation of an explosive mixture. The vaporizer consisted of a stainless stell cylinder covered with a glass-fiber wick from which the liquid was vaporized. An 80-watt heater and a temperature-sensi element were incorporated within the cylinder and connected to a remote temperature controller. Vaporizer surface temperatures were set at about 80°C. Each cylindrical vaporizer was positioned in the fresh-air duct leading directly into ghe exposure chamber to minimize mateila loss due to condensation on duct walls. The vapour generation system was capabel of vapourizing up to 7 ml/min of liquid into 280 L/min fresh air to produce vapour concentrations as high as 9000 ppm. The system maintained the required chamber concnetrations of 1500 ppm n-butyl acetate within +/- 3% of target concentration.
- Temperature, humidity, pressure in air chamber: 23-27°C, 40-60% RH, -0.3 to -2.0 cm H2O (relative to room)
- Air flow rate: 280 L/min
- Treatment of exhaust air: Exhaust was pumped from the chamber through a flow monitor and into the building exhaust system. The exhaust from exposure rooms was diluted with the building exhaust prior to release form the building stack to produce environmenally acceptable stack concentrations.
- at the end of the exposure period the exposure chambers were flushed with fresh air for at least 2 h

TEST ATMOSPHERE
- Brief description of analytical method used: GC/FID analysis with n-nonane as internal standard
- Samples taken from breathing zone: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- chamber monitoring system for constant collection of fresh samples
- GC/FID analysis
- target mean daily concentration of n-butyl acetate in the exposure chambers was 1500 +/- 150 ppm
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/2
- Length of cohabitation: for up to 8 nights
- After 8 days of unsuccessful pairing the population of females in which sperm was not detected were held without further mating or exposure until pregnancy could be readily deteced, at which time they were necropsied (9 to 10 days).
- Proof of pregnancy: sperm in vaginal smear referred to as day 1 of pregnancy
Duration of treatment / exposure:
7 h/d, 5 d/w during the pregestation period
7 h/d on the designated gestation days
Frequency of treatment:
Exposure schedule

Days of gestation
Group pregestation 1 to 6 7 to 16 17 to 20 21
1 Filtered air Filtered air Filtered air No exposure Sacrifice
2 Filtered air Filtered air Test chemical No exposure Sacrifice
3 Filtered air Test chemical Test chemical No exposure Sacrifice
4 Test chemical Test chemical Test chemical No exposure Sacrifice

pregestation exposure period: 3 weeks
Duration of test:
6 weeks
Doses / concentrations
Dose / conc.:
1 500 ppm (nominal)
No. of animals per sex per dose:
group 1: 37 females
group 2: 42 females
group 3: 38 females
group 4: 43 females
Control animals:
yes, sham-exposed
Details on study design:
positive control group:
- pregnant rats, which received filtered air during the premating period, were randomly assigned to the positive control group and housed in a separate room instead of further filtered air exposure
- they received a single intraperitoneal injection of the known teratogenic substance 6-aminonicotinamide on gestation day 12 (6.5 mg/kg)

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: no data, probably daily


DETAILED CLINICAL OBSERVATIONS: No


BODY WEIGHT: Yes
- Time schedule for examinations: twice per week during pregestation; on day 1, 6, 11, 16 and 21 of gestation


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule for examinations: on a weekly basis druing the pregestational exposure and over 5-day intervals during gestation

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined:
- organ weight: liver, lung, spleen, kidneys, ovaries, uteri
- organs for histopathologic investigations: ovaries, uteri, liver, lungs with trachea, spleen, kidneys and any grossly abnormal tissue; tissues from 25% of the females (a maximum of 8 per group) and any grossly abnormal tissues were processed by routine techniques (paraffin embedding, hematoxylind and eosin staining) and subjected to histopathologcal examination
- observations internal abnormalities in pregnant and nonpregnant animals were recorded (e.g. adhesions, tumors, evidence of infection)

OTHER:
- examination for the evidence of infections in at least 5 pregnant rats from group 1 and 4: screened for Mycoplasma, Corynebacterium, Sendai virus, PVM, KRV
- female rats not inseminated after 8 nights were also investigated macroscopically and histopathologically
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other:
- uteri of all apparent nonpregnant females werer stained and examined for implantation sites
Fetal examinations:
- External examinations: Yes [all per litter]
- Soft tissue examinations: Yes [all per litter]
- Skeletal examinations: Yes [all per litter]
- Head examinations: Yes [half per litter]
Statistics:
Binary response variables were compared among groups by chi-square tests for independence (Siegel, 1956). Pairwise comparisons for significant findings used either a two-tailed chi-square test or a Fisher's Exact Test (Siegel, 1956).
Analysis of variance (ANOVA) method was used 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 comparisons 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 extragestational 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 account the effects of treatment, litter, and sex an the body weight and crown-rump length measurements.
Repeated-measures data, such as maternal body weight, were analyzed by a multivariate repeated-measures analysis. Orthogonal polynomials were fit for each animal for which there were complete data, and a multivariate analysis of variance was performed an the coefficients to identify differences in growth patterns among exposure groups (Bock, 1975).
Indices:
No Formulas for calculation were presented

- percent sperm-positive females pregnant at 21 dg
- no. corpora lutea/dam
- no. implantation sites/dam
- no. resorptions/litter
- resorptions/implantation sites
- resorptions/litter
- no. dead fetuses/litter
- no. live fetuses/litter


Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
FOOD CONSUMPTION AND BODY WEIGHT:
Food consumption was decreased in the group of rats exposed to n-butyl acetate during the first week of their pregestational exposure. These rats may have become conditioned to exposure during the second week since their food consumption for this period was higher than that of the three filtered-air exposed groups. Food consumption during the gestational exposure (1 to 16 dg) was higher in the control animals (Group 1) than in the n-butyl-acetate-exposed rats. Food consumption declined significantly in the period immediately following the initiation of chemical exposure (1 dg for Group 3 and 7 dg for Group 2). Values for Group 2 recovered and were higher than those of Group 3, even during the period following termination of exposure.
There was a loss of body weight in the Group 4 rats during the acclimatization period (between randomization and the initiation of pregestational exposure). This may have been a random variation since no apparent cause for this weight loss was found: food and water were readily available and there were no obvious signs of disease. By the end of the first week of exposure, body weights of these rats had recovered to the level of the other three exposure groups. By day 6 of the gestational exposure, body weights for Groups 3 and 4, which were inhaling n-butyl acetate, were lower than those of the filtered-air-exposed groups. Body weights for the rats exposed to this chemical remained lower than those of control rats until sacrifice.

ORGAN WEIGHTS AND HISTOPATHOLOGY
At sacrifice, extragestational weight depression in n-butyl-acetate-exposed. rats reflected patterns observed in body-weight comparisons. Liver weights were also lower in rats inhaling n-butyl acetate. The relative weights of lungs and kidneys of n-butyl-acetate exposed rats were higher than those of control animals, and these relative weights were highest in the rats exposed for 31 days.
Tissue lesions observed during histopathologic examinations could not be related to n-butyl acetate exposure. The only lung changes noted were small foci of mononuclear inflammatory cells in alveolar areas and around small blood vessels, and small foci of histiocytosis. These changes, observed in all groups, were considered minimal, nonspecific, and unimportant.
Liver changes, observed in all exposure groups, were apparent only as minimal mononuclear inflammatory cell populations in portal areas and minimal foci of mixed inflammatory cells scattered in the hepatic parenchyma. No changes were apparent in the spleens. Renal lesions were infrequently observed in any group; when observed, lesions were minimal to mild, except for one rat in Group 3 that had moderate hydronephrosis. Ovarien changes were limited to apparently regressing corpora lutea that correlated well with nonpregnant uteri.

FERTILITY AND REPRODUCTIVE STATUS
Mating performance, intrauterine mortality rate, and reproductive performance were unaffected by exposure of rats to n-butyl acetate:

Effect levels (maternal animals)

Key result
Dose descriptor:
LOAEC
Effect level:
1 500 ppm (nominal)
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
FETAL MEASURES AND MORPHOLOGY
Body weights and crown-rump lengths of both male and female fetuses were lower in all n-butyl-acetate-exposed groups than in the filtered-air-exposed rats. Neither the duration of exposure to the chemical for the period of gestation at the initiation of exposure had a significant effect an fetal growth indices. Placental weight reductions also occurred in n-butyl-acetate-exposed rats. Sex ratios were unaffected.
Two fetuses in Group 2, one in Group 3, and three in Group 4 had major malformations which included: multlple facial defects, eye defects, diaphragmatic hernias, and generalized brain dysmorphology. The lesions described a "generalized brain dysmorphology", included massive distortion of the external and internal architecture of the brain; inequalities in size of the olfactory lobes, and abnormalities in shape and size of the cerebral hemispheres. Hemorrhage was apparent around exterior brain surfaces.
The incidence of rib dysmorphology was increased in fetuses of rats exposed to n-butyl acetate during gestation. The incidence of wavy, fuse 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 Groups 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)

open allclose all
Key result
Dose descriptor:
LOAEC
Remarks:
developmental toxicity
Effect level:
1 500 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: developmental toxicity
Key result
Dose descriptor:
NOAEC
Remarks:
teratogenicity
Effect level:
1 500 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: teratogenicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
yes
Lowest effective dose / conc.:
1 500 ppm (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects

Any other information on results incl. tables

Table: Morphologic alterations a in fetal rats exposed in utero to 1500 ppmn-butyl acetate (BA) or filtered air (FA).

Group 1                 Group 2                Group 3                  Group 4

EXPOSURE INTERVAL

Pregestation

1 to    6 dg

7 to 16 dg

OBSERVATION

FA
FA
FA

 

FA
FA
BA

 

FA
BA
BA

 

BA
BA
BA

 

No.  litters with live fetuses

37

 

42

 

38

 

43

 

No.      fetuses examined

455

 

538

 

458

 

539

 

No.    heads examined

223

 

267

 

229

 

272

 

MAJOR MALFORMATIONS

 

 

 

 

 

 

 

 

Hydrocephaly (internal)

0

 

0

 

(1/1)

2.6

8

 

Generalized brain dysmorphology

0

 

0

 

0

 

(1/1)

2.3

Cleft lip/palate

Aglossia/agnathia

0
0

 

(1/1)
(1/1)

2.4b

2.4 b

0
0

 

0
0

 

Eye defects

0

 

0

 

0

 

(2/2)

4.6

Microphthalmia

0

 

0

 

0

 

(2/2)

4.6c

Aphakia

0

 

0

 

0

 

(1/1)

2.3c

Retinal disorganization

0

 

0

 

0

 

(1/1)

2.3c

Diaphragmatic hernia

0

 

(1/1)

2.4

0

 

0

 

MINOR ANMALIES

 

 

 

 

 

 

 

 

Visceral anemalies

 

 

 

 

 

 

 

 

Lung lobe agenesis

(1/1)

2.7

0

0

 

0

 

Organ agenesis (unilateral)

0

 

(1/1)

2.4d

0

 

0

 

Asplenia

0

 

(1/1)

2.4

0

 

0

 

Situs inversus totalis

0

 

(2/1)

2.4

0

 

0

 

Cardiovascular anomalies

0

 

(1/1)

2.4

(2/2)

5.3

(1/1)

2.3

Retroesophageal great vessels

0

 

(1/1)

2.4e

(1/1)

e

2.6

0

 

Missing innominate

0

 

0

 

(1/1)

2.6

(1/1)

2.3

Skeletal anomalies

 

 

 

 

 

 

 

 

Fused vertebra

0

 

(1/1)

2.4f

(171)

2.6f

0

 

Rib dysmorphology

0

 

(6/6)

14.3

(8/5)

13.1

(2/2)

4.7

Wavy

0

 

(4/4)

9.5

(4/3)

7.9

(2/2)

4.7

Fused

0

 

(1/1)

2.4

(4/2)

5.3

0

 

Bifid

0

 

(1/1)

2.4

(1/1)

2.6

0

 

Sternebral anomalies

(9/7)

18.9

(1/1)

2.4

(3/2)

5,3

(9/8)

18.6

Misaligned

(6/5)

13.5

(1/1)

2.4

(2/2)

5.3

(7/7)

16.2

Scrambled

(2/1)

2.7

0

 

0

 

0

 

Bipartite

(2/2)

5.4

(1/1)

2.4

0

 

(3/3)

7.0

Extra ossification site

8

 

0

 

(1/1)

2.6

(1/1)

2.3

Other anomalies

 

 

 

 

 

 

 

 

Edena

0

 

0

,

0

 

(1/1)

2.3

MORPHOLOGIC VARIATIONS

 

 

 

 

 

 

 

 

Renal variations

(3/3)

8.1

(5/5)

11.9

(5/3)

7.9

(22/10)

23.3

Hydroureter

(3/2)

5.4

(5/5)

11.9

(5/3)

7.9

(22/10)

23.39

Renal pelvic cavitation

(1/1)

2.7

(2/2)

4.8

(2/1)

2.6

(5/4)

9.3

Supernumerary ribs

(4/3)

8.1

(9/4)

9.5

(4/3)

7.9

(2/2)

4.7

Extra

0

 

(1/1)

2.4

0

 

0

 

Ossification at luxbar 1

(4/3)

8.1

(8/4)

9.5

(4/3)

7.9

(2/2)

4.7

Reduced ossification

(389/36)

100.0

(535/42)

100.0

(452/38)

100.0

(515/43)

100.0

Skull

(9/6)

16.2

(13/11)

26.2

(16/10)

26.3

(5/5)

11.6

Vertebra

(162/33)

89.2

(253/39)

92.9

(206/36)

94.7

(238/43)

100.0

Sternebra

(381/36)

100.0

(534/42)

100.0

(449/38)

100.0

(511/43)

100.0

Ribs

(18/11)

29.2

(27/12)

28.6,

(18/10)

26.3

(34/15)

34.9

Pelvis

(7/2)

5,4

(18/9)

21.4 h

(33/14)

36.8h

(3/3)

7.0

Limbs

0

 

0

 

0

 

(1/1)

2.3

Phalanges

(4/1)

2.7

(7/3)

7.1

(5/3)

7.9

(3/3)

7.0

aExpressed as: (number of fetuses/nomber of litters) percentage of litters affected

b Fetus 12, litter 3197

cFetus 1, litter 3016

d Unilateral agenesis of kidney, ovary and uterus

e Retroesophageal aortic arch, pulmonary artery or right subclavian

f Chi-square test for rib dysmorphology: P = 0.05 for Group 1 versus 2; P = 0.07 for Group 1 versus 3

g Chi-square test for hydroureter: P = 0.05 for Group 1 versus 4

h Chi-square test for reduced ossification of pelvis: P = 0.08 for Group 1 versus 2; P - 0.002 for Group 1 versus 3

Applicant's summary and conclusion

Conclusions:
Maternal toxicity, indicated by reduced body and liver weight, occurred when rats were exposed to concentrations of 1500 ppm (7230 mg/m3) of n-butyl acetate before and during gestation. n-Butyl acetate exposure did not influence reproductive performance in rats. Additionally, developmental toxicity ( reduced fetal size) was observed in the presence of n-butyl acetate, but the influence of maternal toxicity cannot be ruled out. 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. But, a similar increase was not seen in the group of rats exposed during this period of gestation subsequent to a pregestational exposure. Therefore it is concluded that the observed effects are probably due to maternal toxicity and that n-butyl acetate is not teratogenic.
Executive summary:

Maternal toxicity, reproductive performance, and developmental toxicology were evaluated in Sprague-Dawley rats following 7 h/d inhalation exposure to 1500 ppm (7230 mg/m3) n-butyl acetate (37 -43 animals/group). Four different exposure regimens were used: Group 1(control): filtered air; Group 2: test item exposure from gestation day 7 through 16; Group 3: test item exposure from gestation day 1 to 16; Group 4: test item exposure for 5 days/week for 3 weeks prior to mating and daily from gestation day 1 through 16. Unexposed males were used in mating (Hackett et al., 1982).

Necropsies were performed on rats at gestation day 21. Pregnant animals were examined for toxic changes, including altered food consumption, body weight, tissue weights and histopathology. Reproductive measures included the determination of numbers of corpora lutea, implantation sites, resorptions, dead fetuses and live fetuses. Live fetuses were weighed, measured, and subjected to external, visceral and skeletal examinations to detect morphologic anomalies.

Food consumption, body weight and liver weight was reduced in maternal rats exposed to n-butyl acetate. Fetal size was reduced in all n-butyl acetate exposed litters. Increased incidences of fetal rib dysmorphology were observed in rats exposed from gestation day 7 through 16, and more numerous hydroureters in fetuses from rats exposed prior to mating and from gestation day 1 through 16. There was no evidence of teratogenic effects following exposure of rats to 1500 ppm of n-butyl acetate. The LOAEC for maternal toxicity and developmental toxicity in this study was 1500 ppm (7.2 mg/L).

This study is reliable without restrictions (RL1).