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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:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: R&R Rabbitry (Stanwood, WA)
- Age at study initiation: females: 5-6 month; males: 6-7 month
- Weight at study initiation: females: about 3 kg; males: about 3-4 kg
- Fasting period before study: none
- Housing: individually in stainless steel cages
- Diet: Wayne Rabbit Diet, ad libitum except during the exposure periods
- Water: ad libitum except during the exposure periods
- Acclimation period: no data
- Does were isolated for 24 daysbefore the initiation of exposure to obviate the possibility of pseudopregnancy.
- Rabbits were removed from the chamber and housed in other caging between exposure periods.





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 ag 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 liqquid 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 withing +/- 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: 560 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: artificial insemination;
One third of each group was artificially inseminated in the afternoon of each day during a 3 day period. On the day of artificial insemination semen samples were collected from at least 3 bucks. To induce ovulation the does were administered chorionic gonadotropin at a dose level of 100 USP units/doe. The morning following insemination was designated as day 1 of gestation.
Duration of treatment / exposure:
7 h/d on the designated gestation days
Frequency of treatment:
Exposure schedule

Days of gestation
Group 1 to 6 7 to 19 20 to 29 30
1 Filtered air Filtered air No exposure Sacrifice
2 Filtered air Test chemical No exposure Sacrifice
3 Test chemical Test chemical No exposure Sacrifice

pregestation exposure period: 3 weeks
Duration of test:
30 days
Doses / concentrations
Dose / conc.:
1 500 ppm (nominal)
No. of animals per sex per dose:
group 1: 30 inseminated; 22 pregnant at sacrifice
group 2: 30 inseminated; 21 pregnant at sacrifice
group 3: 30 inseminated; 25 pregnant at sacrifice
Control animals:
yes, sham-exposed
Details on study design:
positive control group:
- Six does were assigned to the positive control group. They received a single intraperitoneal injection of the known teratogenic substance 6-aminonicotinamide on gestation day 9 (3 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: on day 1, 5, 10, 15, 20 and 25 of gestation

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule for examinations: for 2 weeks prior to the initiation of 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 30
- 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)
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)

Mortality:
no mortality observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
FOOD CONSUMPTION AND BODY WEIGHT:
The lowest values for food consumption were consistently observed in animals from the group inhaling filtered air. When the two groups of n-butyl-acetate-exposed rabbits were compared, there were significant differences in food consumption after the onset of exposure. From 1 to 5 dg, values for Group 2 (exposed to filtered air at this time) were higher than those of Group 3. Once the n-butyl-acetate exposure was initiated in Group 2, food consumption was lower than that of Group 3 until the exposure was terminated on 19 dg. Body weights of the rabbits were similar in both n-butyl-acetate-exposed groups, but the filtered-air-exposed rabbits tended to have the lowest body weights throughout the experimental period, particularly from 15 through 30 dg.

ORGAN WEIGHTS AND HISTOPATHOLOGY
Weights of the lungs, kidneys, and spleens of the pregnant, filtered-air exposed rabbits were significantly lower than those of the n-butyl-acetate exposed groups. When relative organ weights (which correct for differences in body weight) were considered, these differences were not significant, although spleen weights tended to be lower in the filtered-air-exposed animals (P < 0.06).
Histopathologic studies performed an the rabbits necropsied at 30 dg did not relate any observed lesions to the n-butyl acetate exposures. A variety of lung lesions were seen, some of which are probably within the range of normal; others may be related to Pasteurella infections. Small foci of mononuclear inflammatory cells were common around alveoli, small airways and small blood vessels. A few bronchi and bronchiales contained small accumulations of heterophils and minimal to mild increases in bronchus-associated lymphoid tissue (BALT). Epithelial hyperplasia of bronchi was mild in the Group 1 rabbits and minimal in the two n-butyl-acetate-exposed groups. The changes were evenly distributed in all groups.
Renal changes, consisting of minimal to mild tubular mineralization and subacute to chronic interstitial nephritis, were evenly distributed throughout all exposure groups. Incidental findings of extramedullary hematopoiesis in the spieen appeared minimally to mildly excessive in only four animals, one of which had peritonitis.
Regressing corpora lutea corresponded well with the status of uterine sections from nonpregnant rabbits or from animals in which pregnancy was detected by uterine staining (two rabbits in Group 1). One Group 3 rabbit had such severe suppurative metritis and peritonitis that ovarian tissue could not be identified.



FERTILITY AND REPRODUCTIVE STATUS
Intrauterine mortality rate and reproductive performance were unaffected by exposure of rabbits to n-butyl acetate:

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEC
Remarks:
maternal toxicity
Effect level:
1 500 ppm (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Changes in postnatal survival:
not examined
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
FETAL MEASURES AND MORPHOLOGY
Fetal growth measures, such as body weight and crown-rump length, were similar for n-butyl-acetate and filtered-air-exposed rabbits. Placental weights and sex ratios were also similar.
No major malformations were observed in fetuses from any exposure group. There was a significantly higher incidence of misaligned sternebrae and of retinal folds among fetuses from rabbits inhaling n-butyl acetate from 1 to 19 dg (Group 3) than in those exposed to filtered air (Group 1). The presence of a clear liquid in the gallbladder, rather than bile, was a more frequent variation in Group 3 fetuses than in those of Group 1.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEC
Remarks:
developmental toxicity
Effect level:
1 500 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
rabbit fetuses were unaffected by chemical exposure; 1500 ppm = 7230 mg/m3

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

Table 1: Food consumptionaof pregnant rabbits exposed to 1500 ppm n-butylacetate (BA) or filtered air (FA).

Group 1       Group 2       Group 3      

EXPOSURE INTERVAL

1 to 6 dg                FA                     FA          BA                      Contrast b

7 to 19 dg                    FA                       BA          BA                     I              II

NO.   RABBITS:

22

21

25

 

 

TIME OF MEASUREMENT

 

 

 

 

 

Pregestation

 

 

 

 

 

Week 1

220 ± 47

228 ± 40

239 ± 31

0.19

0.33

Week 2

221 ± 47

233 ± 42

245 ± 37

0.10

0.36

Gestation Days

 

 

 

 

 

1 to     5

189 ± 52

215 ± 29

190 ± 35

0.22

0.04*

6 to 10

175 ± 36

173 ± 40

201 ± 35

0.22

0.01*

11     to 15

148 ± 48

154± 52

185 ± 51

0.10

0.04*

16 to 20

169 ± 54

173 ± 55

207 ± 74

0.21

0.07

21 to 25

171 ± 49

190 ± 57

197 ± 75

0.16

0.70

26 to 30

142 ± 56

166± 82

156 ± 70

0.29

0.61

 

a Grams/rabbit/day (mean ± SD)

b Probability for Contrast: I = Group 1 versus 2,3; II = Group 2 versus 3.

*indicates Contrast.is significant (P </= 0.05).

Table 2: Body weight (kg, mean±SD) of pregnant rabbits exposed to 1500 ppm n-butyl acetate (BA) or filtered air (FA); no significant differences between exposure groups

Group 1

Group 2

Group 3

EXPOSURE INTERVAL

1 to 6 dg

FA

FA

BA

7 to 19 dg

FA

BA

BA

No. Rabbits

22

21

25

TIME OF MEASUREMENT

Randomization

4.00± 0.22

4.05± 0.35

3.96± 0.62

Gestation day

1

4.09± 0.25

4.20± 0.35

4.20± 0.33

5

4.05± 0.26

4.20± 0.34

4.14± 0.32

10

4.05± 0.25

4.14± 0.36

4.17± 0.30

15

4.05± 0.23

4.18± 0.37

4.22± 0.34

20

4.06± 0.24

4.20± 0.42

4.27± 0.40

25

4.16± 0.29

4.34± 0.45

4.39± 0.47

30

4.18± 0.30

4.34± 0.38

4.41± 0.49

Applicant's summary and conclusion

Conclusions:
Although rabbits exposed to n-butyl acetate showed an increased food consumption in comparison to control rabbits this effect was not judged to be adverse, because body weights were similar in both exposed groups, and was lowest in the control group. n-Butyl acetate exposure did not influence reproductive performance. Rabbit fetuses were unaffected by chemical exposure. Therefore it is concluded that n-butyl acetate is not a developmental toxicant in rabbits under these test conditions.
Executive summary:

Maternal toxicity, reproductive performance, and developmental toxicology were evaluated in New Zealand rabbits following 7 h/d inhalation exposure to 1500 ppm (7230 mg/m3) n-butyl acetate (30 animals/group artificially inseminated; 21 -25 animals per group pregnant at sacrifice). Three different exposure regimens were used: Group 1(control): filtered air; Group 2: test item exposure from gestation day 7 through 19; Group 3: test item exposure from gestation day 1 to 19. Unexposed males were used in artificial insemination procedure (Hackett et al., 1982).

Necropsies were performed on rabbits at gestation day 30. 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.

Exposure of rabbits to 1500 ppm n-butyl acetate affected food consumption: food consumption was generally higher in rabbits exposed to n-butyl acetate than in rabbits exposed to filtered air; onset of n-butyl acetate exposure resulted in a decrease of food consumption. No related changes in body weights were observed. Reproductive performance was unaltered by n-butyl acetate exposure. Fetal effects of n-butyl acetate exposure included increased incidences of retinal folds, misaligned sternebrae, and morphologic variations of the gallbladder in litters or rabbits exposed from gestation day 1 trough 19. No major malformations were observed. The effects on food consumption were not regarded to be adverse. The NOAEC for maternal and developmental toxicity in this study was 1500 ppm (7.2 mg/L).

This study is reliable without restrictions (RL1).

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