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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:
1984
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study methodology followed was equivalent or similar to OECD TG 414 and the report contains suffcient information to permit a meaningful evalaution of study results
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1983
Report Date:
1983
Reference Type:
study report
Title:
Unnamed
Year:
1984
Report Date:
1984

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
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): Ethylene glycol monopropyl ether
- Physical state: liquid
- Analytical purity: 99.5% (analysed by gas chromatography and mass spectroscopy)
- Impurities (identity and concentrations): The main impurity was ethylene glycol monobutyl ether (0.13%) and the other impurities identified were triethylene glycol n-butyl ether (0.04%) and ethylene glycol isopropyl ether (0.02%)
- Purity test date: not specified
- Lot/batch No.: 907124/82-0234
- Expiration date of the lot/batch: not specified
- Stability under test conditions: not specified
- Storage condition of test material: not specified
- Other: Ethylene glycol monopropyl ether was obtained from the Tennessee Eastman Company, Kingsport, TN

Test animals

Species:
rat
Strain:
other: (COBS-CD-(SD) BR)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories
- Age at study initiation: sexually mature
- Weight at study initiation: sexually mature
- Housing: females individually housed in suspended steel wire bottom cages
- Diet (e.g. ad libitum): Purina Rodent Laboratory Chow 5001 (ground) provided ad libitum
- Water (e.g. ad libitum): tap water, provided by an automatic watering system provided ad libitum
- Acclimation period: 3 weeks prior to mating

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C
- Humidity (%): 50 ± 15%
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12 hours light:dark cycle

Administration / exposure

Route of administration:
inhalation
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 and glass inhalation chambers
- Method of holding animals in test chamber: the animals were housed in suspended wire mesh cages and exposed to vapors of Ethylene glycol propyl ether (EGPE) in 420 liters stainless steel and glass inhalation chambers
- Source and rate of air: ambient
- Method of conditioning air: not specified
- Temperature, humidity, pressure in air chamber: mean temperature 22/23 ± 1 °C, 52-58 ± 6%
- Air flow rate: not specified
- Air change rate: not specified
- Treatment of exhaust air: not specified
- Vapors of EGPE were generated by passing metered air over the surface of the liquid in a 3-neck round bottom flask. Temperature in the flask was slightly elevated (up to 37 °C) to aid vaporization. The atmospheres within the chambers were sampled at least once per hour for concentration, temperature and relative humidity.

TEST ATMOSPHERE
- Brief description of analytical method used: Concentrations were quantitatively analysed using an infrared analyzer (Miran 1A)
- Samples taken from breathing zone: not specified

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The overall mean analytical concentrations (of all individual determinations) of 101, 203, 305 and 405 ppm were all within 2% of the target concentrations. The individual daily means of each exposure level were within ± 6% of their respective target concentrations
Details on mating procedure:
- Impregnation procedure: [cohoused]
- If cohoused:
- M/F ratio per cage: 1:2
- Length of cohabitation: 4 day mating period
- Proof of pregnancy: [sperm in vaginal smear] referred to as [day 0] of pregnancy
Duration of treatment / exposure:
6 hours/day from days 6-15 of gestation
Frequency of treatment:
daily 6 hours/day from days 6-15 of gestation
Duration of test:
days 6-15 of gestation
No. of animals per sex per dose:
30 female rats/dose
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: based on the results of a range finding study (refer to data source, study number 152978R - Exposure to 1600 ppm of 2-propoxyethanol by inhalation for 6/hrs per day on days 6 and 7 of gestation produced maternal lethality (8/10) in rats and was discontinued. Concentrations of 400 and 800 ppm produced maternal toxicity and embryo/fetolethality and/or fetotoxicity. Mean maternal body weight and feed intake was reduced in the 800 pprn animals during exposure. Mean body weight of the 400 ppm dams was comparable to the controls during this time, while the feed intake was slightly reduced from days 6 to 9 and 12 to 15 of gestation. The body weight response seen in the 800 ppm dams was the result of a significant body weight loss in these animals during the first three days of exposure. Red blood cell counts and hematocrit were decreased and the mean corpuscular volume, mean corpuscular hemoglobin, reticulocytes, and nucleated red blood cells were increased in the 400 and 800 ppm groups. The severity of the effect was concentration-dependent. A significant decrease in total white blood cell counts was observed at both exposure levels and reflected the histologic changes seen in the bone marrow at 800 ppm. Terminal body weight and the absolute and relative weights of the thymus glands were lower in the 400 and 800 ppm animals compared to the controls, while 400 and 800 ppm increased the absolute and relative spleen weights. Increased resorptions were seen in the dams exposed to 800 ppm and fetal body weights were reduced in the litters of dams exposed to 400 and 800 ppm of 2-propoxyethanol. Compound related gross and histopathology were seen in both exposed groups, but the incidence and/or severity were greater in the 800 ppm dams. Based on the data presented here, concentrations of 0, 100, 200 and 400 ppm of 2-propoxyethanol appear to be appropriate exposure levels for a teratology study)

- Rationale for animal assignment (if not random): randomly assigned to treatment and control groups using a computer generated stratified randomization scheme

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: Maternal body weights were recorded on days 0, 6, 9, 12, 16 and 19 of gestation

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Individual feed consumption was measured on days 6, 9, 12, 16 and 19 of gestation

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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20

OTHER: On the 20th day of gestation, the females were anesthetized by CO2 inhalation and the abdominal viscera were exposed by a mid-line incision through the abdominal wall. All pregnant females were bled from the inferior vena cava for hematologic determinations including total red blood cell, total and relative white blood cell, reticulocyte, nucleated red cell, platelet and Heinz body counts, hemoglobin concentration, and hematocrit. Mean corpuscular volume, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration were calculated. Red cell morphology was examined. All animals were fasted for 10-12 hours before bleeding.

The thoracic and abdominal viscera of the dams were examined in situ for gross abnormalities at the time of cesarean section. The liver, kidneys, spleen and thymus were weighed for organ/body weight comparisons and along with a femur (for bone marrow) and mesenteric lymph node, sections of these organs were collected for histologic examination
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: After exsanguination via the inferior vena cava, the dams were weighed and the uterine horns were opened. The implantation sites were counted and categorized as viable or dead fetuses, early resorptions or late resorptions. A resorption was classified as "early" when no placenta or conceptus was distinguishable, and as "late" when a placenta containing an embryo in the process of being resorbed (partially to fully formed) was present. The ovaries were removed, identified as left or right, and the corpora lutea of pregnancy were counted
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: No data
- The viable fetuses were removed from the placenta, blotted dry on absorbent paper, sexed, examined for gross external abnormalities, and weighed individually. Approximately one half of the fetuses from each litter was fixed in Bouin's solution and examined for internal soft tissue anomalies using Wilson's free-hand razor blade technique.The other half of the fetuses was fixed in 95% ethanol, macerated with potassium hydroxide and stained with Alizarin Red S and examined for skeletal defects
Statistics:
Test groups were compared to the control group at a level of significance of p <0.05. Continuous data (e.g., maternal body weight, feed consumption, hematology) were analyzed using a one way analysis of variance with significant "F" values further analyzed using Duncan's Multiple Range test. Homogeneity of variances was tested by Bartlett's test. Incidence data were compared using Chi square contingency tables (2 X 5). Each test group was compared to the control group using Fisher's Exact Test when Chi square was significant. Both the proportion of fetuses affected and the number of litters involved were analyzed
Indices:
number of corpora lutea or implantation sites/dam, number of resorptions and viable fetuses/litter, Pre- and post-implantation loss, sex ratio and mean fetal weights
Historical control data:
not applicable

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
None of the exposure concentrations had an effect on the maternal body weight gain . Feed consumption was slightly reduced during the first three days of exposure (days 6-9 of gestation). This reduction of feed intake was statistically different from the controls at the 200, 300 and 400 ppm concentrations.

Significant clinical signs of maternal toxicity were red colored urine seen on the dropping trays of 16/30, 30/30 and 28/30 animals after the first exposure to 200, 300 and 400 ppm of EGPE respectively. It was also seen on the trays of one animal each in the 300 and 400 ppm groups after the second exposure. Red colored urine was not seen at the 200, 300 and 400 ppm levels after subsequent exposures (3-10). One female exposed to 100 ppm EGPE had red colored urine only after the 6th and 7th exposures. One dam exposed to 100 ppm EGPE died during the 7th exposure. Daily clinical signs previous to this time were normal. The animal was autopsied and selected tissues were collected for histologic examination. Grossly, the spleen, liver and renal lymph nodes were enlarged and petechial hemorrhage was seen in the lungs. Histologic examination indicated that the animal died from diffuse malignant lymphoma, which was not considered to be compound related Death occurred on eay 12 of gestation and the uterus contained 11 implantations. Except for one embryo, which appeared dead, the other 10 were alive and appeared to be normal when examined externally

A dam in the 300 ppm group was moribund after the 9th exposure. The animal had an unkempt hair coat, was lethargic and red colored urine was found on the dropping tray. The abdomen was wet and stained brownish. The only abnormal clinical sign seen previously was red colored urine after the first exposure. The dam was euthanized and autopsied. The spleen and adrenals were enlarged, the kidneys were congested and the urinary bladder was acutely inflamed. The bladder and the ureters were dilated. Histologic examination revealed acute suppurative nephritis, cystitis and tracheitis accompanied by adrenal cortical hemorrhage. These lesions were not considered to be compound related. The female was pregnant (gestation day 14) and the uterus contained 14 implantations. Two implants were resorbed and the others were live embryos, which, when eNamined under a dissecting microscope, appeared to be developing normally.

Exposure to 200, 300 or 400 ppm of EGPE produced a statistically significant reduction in the number of red blood cells (Table 4). Mean corpuscular volume and mean corpuscular hemoglobin were increased at these concentrations. Reticulocyte counts were increased by all exposure levels. Minimal polychromasia of the red cells was increased by all concentrations of EGPE; while 300 and 400 ppm increased the number of cells with anisocytosis and 200, 300 and 400 ppm increased macrocytosis. Differential white blood cell counts were comparable to the controls, except in the 300 ppm animals where the lymphocytes were increased and the polymorphonuclear leukocytes were decreased.
Since this effect was not seen at the other exposure concentrations, it is not considered to be compound-related. Total white blood cell, platelet, nucleated red cell and Heinz body counts, hemoglobin concentration and hematocrit were comparable to the controls.

Terminal body weights (day 20 of gestation) of all exposed groups were comparable to the controls. The absolute and relative spleen weights of the animals exposed to 200, 300 or 400 ppm were significantly increased compared to controls. None of the exposure concentrations affected the absolute or relative weight of the liver, kidneys or thymus.

No compound related gross pathology was found in the dams at the time of cesarean sections. Histopathologic examination revealed compound related effects in the spleen, thymus and liver. The splenic changes, which reflected the red blood cell effects and the increased absolute and relative spleen weights, included increased congestion, hemosiderosis and extremedullary hematopoiesis. These changes were seen at all exposure concentrations as well as in the controls and were minor in severity. The incidence differed statistically from the controls only at 300 and 400 ppm. Hepatic effects included minor eosinophilic cytoplasmic changes at all exposure concentrations which were statistically increased at 300 and 400 ppm EGPE. Minor to moderate medullary necrosis of the thymus was present in all EGPE groups as well as the controls with the incidence increased at 300 and 400 ppm. No compound related effects were seen in the kidneys, bone marrow or mesenteric lynph nodes.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEC
Effect level:
200 ppm
Based on:
test mat.
Basis for effect level:
haematology

Results (fetuses)

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

Details on embryotoxic / teratogenic effects:
None of the exposure concentrations of EGPE affected the pregnancy rate, number of corpora lutea or implantation sites per dam or the number of resorptions and viable fetuses per litter. The pre- and post- implantation losses of the treated groups were comparable to the controls. The sex ratio was not affected by exposure to EGPE and the mean fetal body weights of all exposure groups were comparable to the control weights.

Externally, major defects were observed in 2 of 1,386 fetuses examined. One fetus from a litter of 12 exposed to 300 ppm EGPE had a severely atrophied tail and one fetus from a litter of 12 exposed to 400 ppm EGPE had thoraco gastroschisis. The fetus with thoracogastroschisis was examined immediately upon removal from the uterus and placenta and all of the thoracic and abdominal structures appeared normal e.g. the heart was beating and the major blood vessels were properly located. Both of these defects were considered to be spontaneous events and not compound related.

Minor defects seen externally included small hematomas on the head, limbs and interscapular area. These were seen at all exposure concentrations as well as in the control group. The incidence of these hematomas was not concentration-dependent and not considered toxicologically significant. The total external defects found in the exposed groups were not statistically different from the controls.

Another external observation was two fetuses in a litter exposed to 200 ppm EGPE with a common placenta. Both of these fetuses appeared normal, externally, but one was half the size of the other and was wrapped around its littermate.

Internal soft tissue examinations of 693 fetuses revealed four fetuses with major defects. One fetus from a litter exposed to 200 ppm EGPE had internal micrencephaly (the cerebral hemispheres were not fully developed), one fetus from a control litter had unilateral anophthalmia and one fetus from a control litter had incomplete septation of the ascending aorta and pulmonary artery forming a fistula between these two vessels. The latter fetus was much smaller than normal and also had slight hydrocephalus (i.e. dilation of the lateral ventricles of the brain).

The fourth fetus with a major defect internally was the smaller fetus described above that shared a common placenta with a littermate. Internal examination of this fetus revealed that the ribs rather than being perpendicular to the spine, were displaced cephalad and parallel to the spine. The thoracic viscera were also displaced cephalad and dorsally. When the torso was sectioned transversely (Wilson's Technique) the blood vessels cut
obliquely and the ribs cut in cross section. The right fourth aortic arch was persistent and formed a ringed aorta with the left aortic arch. The esophagus and trachea were severely constricted in this area, while the trachea was greatly dilated cephalad. Innominate arteries were present on both right and left arches and 4 (2 pairs?) carotid arteries were seen in the neck region. Subclavian arteries were not seen. The pulmonary artery and the ductus arteriosus were associated with the correct aortic arch (i.e. the left). This fetus also had bilateral hydronephrosis with equivocal hydroureter. It also appeared that a pair of cervical ribs were present at about the level of the 6th or 7th cervical vertebra, but this could not be determined definitely due to the distortion of the rib cage. The abnormalities seen in this fetus are not considered to be compound related, but these defects, as well as its small size, are attributed to the stress caused by sharing the same placenta and the physical contact (conpression) with its littermate.

Minor defects seen internally included slight to moderate hydrocephalus and hydronephrosis and/or hydroureter in controls as well as in the exposed groups. One control fetus had a severely distended urinary bladder. Two fetuses in a control litter, two fetuses in a 100 ppm litter and one fetus in a 300 ppm litter had subcutaneous hemorrhage of the head. The incidence of fetuses or litters with internal soft tissue defects was not statistically different among the controls and the groups exposed to EGPE.

No major skeletal malformations (i.e., any skeletal defects that would have compromised the survival or health of the fetus) were seen in this study.

Minor skeletal defects were seen in all treated groups and the control group. Neither the.specific skeletal defect nor the total minor skeletal defects were statistically different between the EGPE exposed groups and the control group. One Fetus from a control litter had only 12 pairs of ribs with the 12th pair partially ossified. The 9th and 10th right ribs of one fetus from a litter exposed to 300 ppm EGPE were partially ossified. Wavy and/or
knobby ribs were seen at a low incidence in all test groups and the controls. The incidence seen in the EGPE exposed fetuses or litters was not statistically different from the controls. Additionally, bone spurs (small unilateral or bilateral sites of ossification) were seen adjacent to the 7th cervical vertebra in 2, 3, 5 and 1 litter(s) exposed to 0, 200, 300 and 400 ppm of EGPE, respectively. The incidence of bone spurs seen in the treated
groups was not statistically different from the controls. Other skeletal alterations consisted of variations in the pattern of ossification of some skeletal elements and the supernumerary ribs (e.g. rudimentary ribs) frequently seen in teratology studies. The variations included partial ossification of some intramembranous skull bones, partial or non-ossification of the hyoid bone, the first and second thoracic centrae, the sternebrae, and a bilobed appearance of the lower thoracic centrae (8th to 13th). The number of fetuses with at least one variation of the skull, including the hyoid bone, was statistically increased at the 300 ppm EGPE concentration only; while the incidence of litters involved was not significantly different from controls at any exposure concentration.

Variations in the ossification of the vertebral column consisted primarily of partial or non-ossification of the 1st and 2nd thoracic centrae, a bilobed appearance of the 8th, 9th, 10th, 11th, 12th or 13th thoracic centrae and a few lumbar centrae and partial ossification of the sacral vertebrae. When the incidence of these variations was analyzed collectively (total vertebral variations), the number of fetuses per total fetuses examined with at least one variation was statistically increased by 200, 300 and 400 ppm EGPE compared to the controls. However, the incidence of litters affected (i.e. at least one fetus in the litter had a vertebral variation) was not statistically different between the exposed and control groups.

The number of fetuses per total fetuses examined with less than 4 ossification sites in the sternum and the incidence of litters with at least one affected fetus were comparable between test group and controls while the number of fetuses with partial ossification of the sternum were increased at the 200, 300 and 400 ppm levels; the number of litters involved were not significantly increased. The number of fetuses with non-ossified sternebral sites and the number of litters involved were comparable to the controls. Most importantly, when overall retarded ossification of the sternum was statistically analyzed (i.e. total sternebral variations), the groups exposed to EGPE did not differ from the controls.

A concentration-dependent increase in the incidence of fetuses and litters with a 14th thoracolumbar rudimentary rib (< 1/2 the length of the 13th rib) was statistically different compared to controls in the 200, 300 and 400 ppm groups exposed to EGPE. Extra 14th thoracolumbar ribs (> 1/2 the length of the 13th rib) were found in one fetus each from 4 litters exposed to 300 ppm and in one fetus each in 5 litters exposed to 400 ppm EGPE. Extra ribs were not found in control, 100 or 200 ppm fetuses. The incidence of extra ribs in the 300 and 400 ppm fetuses and litters was not statistically increased compared to controls.

Effect levels (fetuses)

Dose descriptor:
NOEL
Effect level:
400 ppm
Based on:
test mat.
Basis for effect level:
other: teratogenicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

not applicable

Applicant's summary and conclusion

Conclusions:
Based on the results of the study, the no-observed-effect-level (NOEL) for teratogenicity for ethylene glycol monopropyl ether was 400 ppm in rats, when exposed via inhalation
Executive summary:

Pregnant rats were exposed to vapor concentrations of 100, 200, 300 or 400 ppm of ethylene glycol monopropyl ether (EGPE) for 6 hours per day on days 6 through 15 of gestation. Maternal effects included a slight reduction in red blood cell count and increased mean corpuscular volume and mean corpuscular hemoglobin at the 200, 300 and 400 ppm concentrations. Reticulocyte counts and polychromasia of the red blood cells were increased at all exposure levels; while anisocytosis was increased at 300 and 400 ppm and macrocytosis was increased at 200, 300 and 400 ppm.

Hematocrit, hemoglobin concentration, platelet and total and differential white blood cell counts were comparable to the controls. Red colored urine was seen in the females after the first and second exposures to 200, 300 and 400 ppm of EGPE, but not after subsequent exposures. Absolute and relative spleen weights were increased by 300 and 400 ppm EGPE. Histologically, the maternal spleen, liver and thymus were affected; particularly after exposure to 300 and 400 ppm. Kidneys, bone marrow and mesenteric lymph nodes were normal.

 

Pregnancy rate, number of corpora lutea, implantation sites and viable fetuses per dam, the number of resorptions per litter and .the mean fetal body weights were not affected by any of the exposure concentrations. Gross external, internal soft tissue and skeletal examinations of the fetuses revealed that EGPE did not produce teratogenicity or significant embryo/ fetotoxicity in the rat at vapor concentrations as high as 400 ppm EGPE. Variations in the ossification of certain skeletal elements and the incidence of 14th rudimentary ribs were increased by exposure to 200, 300 and 400 ppm EGPE.

 

Ethylene glycol monopropyl ether did not produce teratogenic effects or significant minor emryo/fetotoxicity when pregnant rats were exposed, during the period of major organogenesis, to vapor concentrations as high as 400 ppm. Common variations in the ossification of certain skeletal elements and the incidence of supernumerary ribs (rudimentary 14th) were significantly increased at maternally toxic concentrations of 200, 300 and 400 ppm and may be an indication that exposure of rats to concentrations of EGPE greater than 400 ppm may produce significant embryo/fetotoxicity. However, in this study 400 ppm was a no-observed-effect-level (NOEL) for teratogenicity and significant minor embryo/fetotoxicity.