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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:
Not applicable
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-study according to EPA guideline 560/6-84-002
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1987
Report Date:
1987

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: "New and Revised Health Effects Test Guidelines" EPA 560/6-84-002 (reproduced by National Technical Information Service - PB84-233295)
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Ethyl 3-Ethoxypropionate EEP
- Physical state: Colorless liquid; mild ester odor
- Analytical purity: 99%
- Impurities (identity and concentrations):
- Purity test date: 26 March 1986; 3 September 1986
- Lot/batch No.: X 18806-142
- Expiration date of the lot/batch: One year from date of receipt
- Storage condition of test material: Store at room temperature away from heat and flame.

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Hazl eton Research Products, Inc.
- Age at study initiation: Males (1-2 years); Females (8 months)
- Weight at study initiation:
- Fasting period before study:
- Housing: Individually, except during mating in suspended, stainless steel cages with wire mesh floors. Each cage was fitted with a stainless steel, sub-cage floor pan to retain urine and feces. During exposure, females were individually housed. Stainless steel shelf pans were placed under each row of cages to prevent urinary and fecal contamination of animals on lower levels .
- Diet: Feed was withheld on the day of arrival. The following day, each rabbit received one leaf of cabbage; thereafter, Purina High-Fiber Rabbit Chow (Certified diet No. 5325) was fed ad libitum during the non-exposure. During the exposure intervals, animals did not have access to food.
- Water: Tap water delivered by automatic watering system, ad li bitum, during the non-exposure intervals. During the exposure intervals, animals did not have access to water.
- Acclimation period: 68 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16 - 24
- Humidity (%): 37-77
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light):12 hour light/dark cycle (7 AM to 7 PM) via automatic timer.


IN-LIFE DATES: Dates of Treatment: Day 6 of gestation: 6-9 and 13-16 June 1985.
Day 18 o f gestation: 18-21 and 25-28 June 1985.

Administration / exposure

Route of administration:
inhalation
Type of inhalation exposure (if applicable):
not specified
Vehicle:
unchanged (no vehicle)
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 10m3 (10,000 liters) exposure chambers
- Method of holding animals in test chamber: housed individually
- System of generating particulates/aerosols:
- Temperature, humidity, pressure in air chamber: 17.2°C to 22.8°C, 40% to 70%
- Air flow rate: 2800-3060 ppm
- Air change rate: 3.3-3.6 min
- Method of particle size determination: TSI Aerodynamic Particle Sizer (Model #3300) equipped with a TSI Diluter (Model #3302).



TEST ATMOSPHERE
- Brief description of analytical method used: Atmospheric samples were drawn directly from the exposure chamber into a MIRAN@ 1A Gas Anlayzer Spectrophotometer by a Thomas pump (Model #107CA183). Absorbance was read directly from the MIRANQ absorbance scale. For reference, the response was traced on a Fisher Recordall series 5000 strip chart recorder.
- Samples taken from breathing zone: no - back middle sampling point

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical data indicated good agreement of the analytical concentration with the target concentration. Analytical concentrations were about 86% of the nominal for the two lower concentrations and 106% of the nominal for the two higher concentrations. Analytical concentrations lower than nominal are expected due to chamber absorption losses; variability in measurement of chamber flow or delivery of test material used for calculation of nominal concentration may have been responsible for the reverse results noted for the two higher concentration levels.
Details on mating procedure:
Males: Proven breeders used solely for mating purposes (Bio/dynamics in-house breeding colony)

Natural mating was used. Each female selected for mating was placed into the male's cage. When coitus was observed, the female was removed and returned to her original cage. Following an interval of one to two hours, the female was placed into the cage of a second, different male. When coitus was observed with the second male, the female was considered mated and returned to her cage. The day on which coitus was observed with both males was designated Day 0 of gestation.
Duration of treatment / exposure:
Animals exposed 6 hourslday during the Day 6-18 gestation interval.
Frequency of treatment:
Day 6 o f gestation: 6-9 and 13-16 June 1985.
Day 18 o f gestation: 18-21 and 25-28 June 1985.
Duration of test:
from Days 6-18 of gestation
Doses / concentrations
Remarks:
Doses / Concentrations:
125, 250, 500 and 1000 ppm
Basis:
nominal conc.
No. of animals per sex per dose:
Four groups of mated female rabbits (18 females/group)
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: 1000 ppm EEP is the highest vapor concentration that can be practicably generated before an aerosol is formed.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily (morning; afternoon)
During the exposure intervals animals in the chamber and visible through the chamber windows were observed for clinical signs of toxicity.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice daily (morning; afternoon). In addition, each female was given a detailed physical exam on Days 0, 6-18, 24 and 30 of gestation. These evaluations were performed daily after one, three and six hours of exposure.


BODY WEIGHT: Yes
- Time schedule for examinations: Recorded on days 0, 6, 9 , 12, 15, 18, 25 and 30 of gestation.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Calculated for the following intervals: days 0-1, 3-4, 6-7, 7-8, 8-9, 9-10, 12-13, .15-16, 18-19, 25-26 and 29-30 of gestation.


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 30
- Organs weighed: liver, kidneys, spleen, and thymus for all females sacrificed on Day 30 of gestation.
- Tissues Preserved and examined microscopically: Yes. The following tissues/organs were dissected free, cleaned of extraneous adhering tissue and preserved (10% neutral buffered formalin) for all females: liver, bone and bone marrow, kidneys (femur), spleen, mesenteric lymph nodes, thymus gross lesions. Additionally, the liver, kidneys, spleen and thymus were weighed (paired organs weighed together) for all females sacrificed on Day 30 of gestation:
- Reproductive System: Yes




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
When no uterine implants were grossly apparent, the uterus was stained w ith ammonium sulfide. Foci visualized following this staining procedure were counted and females w ith such foci were included as pregnant in the calculation of pregnancy rate. The number of foci was not included in the calculation of uterine implantation data.
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: No data

All fetuses were given a gross examination for malformations of the external form to include observation for palatal defects. Subsequently, each fetus was weighed and tagged individually for identification.

Fetal evaluations: Fetuses/delivered pups recovered during the Day 26-30 gestation interval were evaluated for external malformations, weighed and preserved intact in 10% neutral buffered formalin.

Soft Tissue Evaluations: All fetuses were sacrificed (intraperitoneal injection of T-61 euthanasia solution) and evaluated for soft tissue malformations and variations using the microdissection procedure similar to that described by Staples and internal sex noted. Following this procedure, the viscera were removed from the thoracic and abdominal cavities and discarded. The eviscerated fetus was then skinned. As the skin of the head was removed, the eyes were evaluated grossly for obvious malformations. The brain was evaluated by making a transverse cut with a razor blade parallel and just posterior to the frontal- parietal suture and through the cerebral hemispheres. The microdissection procedure was performed under a dissecting microscope (10-20X) and magnification lens (2.0X). Eyes and brain observations were performed grossly.

Fetal Skeletal Evaluations : The eviscerated/skinned fetal specimens were then processed for staining of the ossified skeletal structures using the Alizarin Red S staining procedure of Crary as modified by Bio/dynamics. Fetal skeletal specimens were evaluated under a 2.0X magnification lens for malformations and/or ossification variations.

Late Resporations: Late resorptions were weighed and examined grossly for external malformations. Only late resorptions with external malformations were saved (10% neutral buffered formalin) .
Statistics:
Data were analyzed between control and treated Groups. Statistically significant differences between the control and treated group wre noted. Statistical evaluation of equality of means was made by the appropriate one way analysis of variance technique, followed by a multiple comparison procedure
if needed. First, Bartlett's test was performed to determine if grouphad equal variance. If the variances were equal, parametric procedures were used; i f not, nonparametric procedures were used. The parametric procedures were the standard one way ANOVA using the F distribution to assess signifiace. If significant differences among the means were indicated, Dunnett's test was used to determine which means were significantly different from the control. If a nonparametric procedure for testing equality of means was needed, the Kruskal- Wallis test was used, and if differences were indicated a summed rank test (Dunn) was used to determine which treatments differed from control.
A statistical test for trend in the dose levels was also performed. In the parametric case (i.e., equal variance) standard regression techniques with a test for trend and lack of fit were used. In the nonparametric case, Jonckheere's test for monotonic trend was used.
The test for equal variance (Bartlett's ) was conducted at the 1%, two-sided risk level. All other statistical tests were conducted at the 5% and 1% , two-sided risk level.
All ratios were transformed via the arc sine transformation prior to analysis. Data is presented untransformed.
References for these techniques are Snedecor, G.W., and Cochran, W.G., Statistical Methods, 6th edition, Iowa State University Press (1967) ; Hollander and Wolfe, Nonparametric Statistical Methods, John Wiley and Sons, New York (1973); Dunnett, C.W., J. Am. Sta. Assn., Vol. 50 (1955) and Biometries, Vol. 20 (1964).
Statistical analysis of incidence data was performed using contingency tables. A standard chi-square analysis; 2x2 Fisher Exact test; Armitage
Indices:
Mean pre- implantation loss (PL) indices. PL indices were not considered to be adversely affected by treatment. Indices were calculated from cesarean section records of animals in the study as follows: Corpora Lutea; Implantation sites; Early and Late resorptions; Live/Dead Fetuses; Sex M/F; Fetal body weight
Historical control data:
Yes. Historical Control Data - New Zealand White Rabbit (Bioldynamics Inc. 1977- 1984).

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Body Weight: In the high dose group (1000 ppm), the mean weight loss encountered for the Day 6-9 gestation period was statistically significantly higher than control. Over the entire Day 6-18 treatment period, the mean weight loss seen for the high-dose group (1000 ppm) during the treatment period differed statistically from control (p <0.05) and this change was considered indicative of a treatment- related effect. During the Day 18-30 post-treatment interval, in dose groups 250, 500 and 1000 ppm mean weight gain for the Day 18-30 post-treatment interval of gestation was higher than control and these differences were statistically significant .

Food Consumption: The only adverse effect of treatment on food consumption occurred early in the treatment period (Day 6 and 7) at exposure levels of 250, 500 and 1000 ppm.

Clinical Observations: In dose groups 500 and 1000 ppm, several animals were noted with excessive lacrimation on Day 6 following the first exposure. Several high dose 1000 ppm animals continued to show excessive lacrimation at various interval s during the treatment period. Excessive salivation was also noted i n several 1000 ppm females on Day 6. Clinical signs noted during exposure intervals were primarily restricted to reduced activity and closing of the eyes. The incidence of these observations seemed to be directly related to concentration. Labored breathing and salivation were noted with increased frequency among the animals at the higher concentrations ( i .e., 500 and 1000 ppm).

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOEC
Effect level:
1 000 ppm
Basis for effect level:
other: developmental toxicity
Dose descriptor:
NOEC
Effect level:
500 ppm (nominal)
Basis for effect level:
other: maternal toxicity

Results (fetuses)

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

Details on embryotoxic / teratogenic effects:
Not applicable

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

- Pregnancy rates were comparable between the control and treated groups.

Gestation Period

- Mean body weights were comparable between the control and test Groups II, III and IV throughout the gestation period. In Group V (1000 pprn) , a slight (-3% to -4.6%) decrease in mean weights was seen during the treatment period; these differences from control were not statistically significant.

Mean weight change for the Day 0-6 pre-exposure interval of gestation was considered comparable between the control and treated groups.

During the treatment period, control and all treated groups experienced a mean weight loss for the Day 6 -9 interval with initiation of exposures. These changes were considered comparable between the control and treated Groups II, III and IV. In Group V (1000 ppm), the mean weight loss encountered for the Day 6-9 gestation period was statistically significantly higher than control. Considerable variability in mean weight change data was seen for the Day 9 -12, 12 -15 and 15 -18 intervals of the treatment period; however, these data did not differ statistically between the control and treated groups.

Over the entire Day 6 -18 treatment period, the control group experienced a slight mean weight gain and Groups II, III and IV experienced a slight mean weight loss; however, these data did not differ statistically from control and in the absence of a dose relationship were not considered indicative of a treatment- related response. The mean weight loss seen for the high-dose group (Group V) during the treatment period differed statistically from control (p <0.05) and this change was considered indicative of a treatment-related effect.

During the Day 18-30 post-treatment interval , a comparable mean weight gain was seen for the control and Group II. In Groups III, IV and V, mean weight gain for the Day 18-30 post-t reatment interval of gestation was higher than control and these differences were statistically significant.

Food Consumption: The only adverse effect of treatment on food consumption occurred early in the treatment period (Day 6 and 7) at exposure levels of 250, 500 and 1000 ppm.

Physical Examinations: No adverse effect of treatment at the 125 or 250 ppm exposure levels was evident from physical observation data. In Groups IV and V (500 and 1000 ppm), several animals were noted with excessive lacrimation on Day 6 following the first exposure. Several Group V animals continued to show excessive lacrimation at various intervals during the treatment period.

Excessive salivation was also noted in several Group V females on Day 6.

Clinical signs noted during exposure intervals were primarily restricted to reduced activity and closing of the eyes. The incidence of these observations seemed to be directly related to concentration. Labored breathing and salivation were noted with increased

frequency among the animals at the higher concentrations ( i .e., 500 and 1000 ppm).

Applicant's summary and conclusion

Conclusions:
Slight maternal toxicity was seen in the form of slightly reduced feed consumption at the 250, 500, and 1000 ppm concentrations of EEP and slighly reduced body weight gain at the 1000 ppm level. Excessive lacrimation was seen in the 500 and 1000 ppm animals on the first day of exposure. No effects were seen on the reproductive indices and no treatment- related external, internals of tissue, or skeletal anomalies were seen at concentrations as high as 1000 ppm EEP (the highest practicably generated vapor concentration). Thus, 1000 ppm EEP was a no-observed-effect-level (NOEL) for developmental toxicity in this study.
Executive summary:

This inhalation developmental toxicity study was conducted for Eastman Kodak Company to evaluate the maternal toxic , embryotoxic, fetotoxic and teratogenic potentia1 of ethyl 3-ethoxypropionate in the New Zeal and White rabbit. Animals were exposed to test material for 6 hours/day during the Day 6 -18 gestation period in stainless steel and glass 10 cubic meter chambers. Target exposure levels were 125, 250, 500 and 1000 ppm; included in the study was a chamber-housed, sham air control group. Each test group contained 18 mated females (natural mating was used and the day of mating was Day 0 of gestation).

During gestation, animals were weighed and food consumption was recorded at regular intervals. Additionally, females were observed twice daily for pharmacologic/toxicologic effects and mortality and given a detailed physical examination on Day 0, 6-18, 24 and 30 of gestation. Animals that aborted or delivered prematurely were sacrificed on the day such evidence was observed. Remaining females were sacrificed on Day 30 of gestation. Each female received a gross postmortem examination at which time selected tissues were taken, preserved and evaluated histopathologically. The following organs were weighed at necropsy; intact uterus with ovaries attached; 1iver; kidneys; spleen and thymus. Prior to sacrifice, a blood sample was collected from the ear vein and standard hematological and clinical chemistry parameters evaluated.

Corpora lutea were counted on each ovary and uterine implantation sites identified as either fetuses or resorption sites. All fetuses were removed from the uterus, weighed, evaluated for external malformations, individually identified and processed for visceral (microdisection procedure with internal sexing) and skeletal (Alizarin Red S staining) evaluations.

Mean analytical concentrations were generally in good agreement with targeted concentrations. The mean daily analytical concentrations ( ± standard deviations) for the groups targeted at the 125, 250, 500 and 1000 ppm exposure levels were 124 (± 4.7), 247 ( ± 8.0), 498 ( ±20.2) and 997 ( ± 32.4) ppm, respectively.

No mortality occurred in the control or treated groups. Pregnancy rates and the incidence of pregnancies lost to abortion or premature delivery were comparable between the control and treated groups.

No adverse effect of treatment was evident from maternal hematology or clinical chemistry data, organ weight data, gross postmortem evaluations or histopathological evaluations of selected tissues.

At the 125 ppm exposure level , no adverse effects of treatment were evident from maternal or fetal parameters. Evaluation of fetuses recovered from females treated at this exposure level revealed no increase in malformation rate.

At the 250 ppm exposure level , a statistically significant reduction in mean maternal food consumption was seen on Days 6 and 7 of gestation. No other maternal toxicity was seen at this dose level and no effect on fetal parameters (i.e., fetal weight, sex distribution) was evident. Evaluation o f fetuses recovered at this exposure level revealed no increase in malformation rate.

At the 500 ppm exposure level , mean food consumption was statistically significantly lower than control at Days 6 and 7 of gestation and several females were noted with excessive lacrimation on Day 6 of gestation. No other adverse effects of treatment (maternal, fetal ) were evident at this exposure level. During the fetal visceral evaluation, four fetuses (3.5%), three fetuses from one litter and one fetus from a second litter, had severely distended lateral ventricles of the brain. Since this malformation was not seen among fetuses at the highest dose level (1000 pprn) which had approximately 30% more fetuses available for evaluation, its occurrence in this group was not considered to be related to treatment. Fetal external evaluations did not indicate an adverse effect of treatment.

At the 1000 ppm exposure level, statistically significant changes in body weight gain were seen for the Days 6-9 and Days 6-18 gestation intervals. In both instances this group experienced a mean weight loss in respect to the control which also experienced a slight mean weight loss for the Days 6 -9 interval but a slight mean weight gain for the Day 6 -18 interval. Mean food consumption was lower than control a t Days 6 and 7 of gestation and females in this group were noted with an increased incidence of excessive lacrimation and excessive salivation (Day 6). No adverse effect of treatment at the 1000 ppm level was evident from u t e r i n e implantation data, fetal weight data or fetal sex distribution data. External and visceral examination of fetuses recovered at the 1000 ppm exposure level did not indicate an adverse effect of treatment.

The incidence of fetuses with major skeletal malformations at the 500 and 1000 ppm exposure levels (2.6% and 2.7%, respectively), was slightly higher than control (0.7%). In each of these groups, one fetus with major skeletal malformations also had severe dissimilar external malformations which appeared to be of spontaneous origin and not treatment- related. If these fetuses were excluded, the incidence of fetuses with major skeletal malformations for the 500 and 1000 ppm levels was 1.8% and 2.0%, respectively, both of which were just slightly higher than the control incidence (0.7%). A broad spectrum o f skeletal

malformations was seen during the evaluation of the 134 control fetuses (15 1itters) and 559 treated group fetuses (62 1itters); however, 1ittle consistency was seen in the type or severity of the malformations to indicate an adverse effect of treatment.