reaction mass of sodium acetate, monoethylene glycol and diethylene glycol

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Evaluation of the Developmental Toxicity of Ethylene Glycol Aerosol in the CD Rat and CD-1 Mouse by Whole-Body Exposure.

Author:

Tyl, R.W., Ballantyne, B., Fisher, L.C., Fait, D.L., Savine, T.A., Dodd, D.E., Klonne, D.R., Pritts, I.M.

Year:

1995

Bibliographic source:

Fundamental and Applied Toxicology 24, 57 - 75

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

1985

Report date:

1985

Materials and methods

Principles of method if other than guideline:

Evaluation of the developmental toxicity potential, including embryotoxic, fetotoxic and teratogenic effects of maternally-inhaled ethylene glycol aerosol in the rat and the mouse.

GLP compliance:

yes (incl. QA statement)

Remarks:

testing lab.

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Ethane-1,2-diol

EC Number:

203-473-3

EC Name:

Ethane-1,2-diol

Cas Number:

107-21-1

Molecular formula:

C2H6O2

IUPAC Name:

1,2-ethanediol

Constituent 2

Reference substance name:

ethan-1,2-diol

IUPAC Name:

ethan-1,2-diol

Details on test material:

nearly 100% pure throughout the study

Test animals

Species:

rat

Strain:

other: COBS CD (SD)BR

Details on test animals or test system and environmental conditions:

The rats were housed in one room. They were quarantined for two weeks during which time representative animals were subjected to fecal sampling, histologic examination of selected organs and to serum viral antibody examination. The results of the quality control tests indicated that these animals were suitable for use in this study. All rats received a unique number and a stainless steel ear tag. Rats were housed in stainless steel wire-mesh cages with food and water available ad libitum except during exposures. Deotized Animal Cage Board was placed beneath the cages and changed daily. Animals were kept on a 12-hour photoperiod (06.00 to 18.00 hours for light phase) and room temperatures and humidity were recorded continuously.

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure (if applicable):

whole body

Vehicle:

unchanged (no vehicle)

Details on exposure:

The test material was placed in one-gallon glass bottles and blanketed with nitrogen. The purity and stability were verified by analysis both before and after the study. No significant compositional changes occurred while the study progressed, except for a slight increase (0.2%) in water content . The test material remained nearly 100% pure throughout the study. The nitrogen and hydrogen were Ultra High Purity grade and the air was breathing quality. The inhalation chambers were constructed of stainless steel with glass windows for animal observation, with the dimensions 60 cm (height) x 101 cm (depth) x 101 cm (width). Chamber volume was approximately 900 liters and airflow ranged from 120 to 200 L/min, depending on desired exposure concentration . Chamber temperature and relative humidity were recorded at least five times per exposure with a minimum-maximum thermometer. Target concentrations were 0, 150, 1000, and 2500 mg/m3. These exposure concentrations were based on a range-finding study in pregnant rats and mice which employed concentrations of 0, 50, 250, 1250 and 2500 mg/m3. For the 2500 mg/m3 group, two generation systems, each with a 4-tube Laskin nebulizer, were used. For the 1000 mg/m3 group, a single generation system with a 4-tube Laskin nebulizer was used for Exposure Days 1 through 8. Beginning on Exposure Day 9 and going through Exposure Day 13, a 2-tube Laskin nebulizer was added to the generation system of the 1000 mg/m3 group in order to help achieve the desired concentration. For the 150 mg/m3 group, a 1-tube Laskin nebulizer was used for generation of the aerosol atmosphere. Compressed air supplied to each nebulizer created a negative pressure such that the ethylene glycol was aspirated into the tube where it was then dispersed as a fine liquid aerosol. The liquid aerosol was then introduced into the top of the exposure chamber where it was diluted to the desired concentration and dispersed through the chamber by filtered chamber supply air. The operating pressure of the nebulizers used to generate the 2500, 1000, and 150 mg/m3 target concentrations of ethylene glycol was approximately 30 psig. Chamber concentrations of ethylene glycol were analyzed by both gravimetric determinations and impinger samples in order to determine the aerosol and total ethylene glycol concentrations, respectively. Each chamber atmosphere was analyzed for ethylene glycol three times during each 6-hour exposure. Daily nominal concentrations (an estimated concentration calculated from the amount of test material delivered,and the chamber airflow during the exposure period) were also calculated for each chamber. A Perkin-Elmer Model 3920B gas chromatograph (GC) equipped with a flame ionization detector was,used to monitor the ethylene glycol concentrations in the impinger solutions. Calibration of the gas chromatograph was done with liquid injections of standard solutions of ethylene glycol in distilled water prepared volumetrically. The mass median aerodynamic diameter (MMAD) and geometric standard deviation (ag) were determined from log probability plots of the cumulative percent mass collected on each impactor stage. The flowrate for all samples was 14.2 L/min. The sample collection times for the 2500, 100, and 150 mg/m3 groups were generally 4, 10, and 120 minutes, respectively.

Details on mating procedure:

Rats were mated 1 : 1 (one male at least 250 g : one female 200-300 g) in stainless steel wire-mesh cages and the paperboard beneath the cages was checked twice daily for dropped copulation plugs. The new males and males from the probe study were used as breeders. Successfully mated (plug-positive) females were housed singly in stainless steel wire-mesh cages for the duration of the study except during daily exposures. The day a copulation plug was found was designated gestational day (gd) 0. Twenty-five (25) plug-positive females were randomly assigned to each experimental group using a stratified randomization system on gd 0. The mating period was February 17 through 19, 1985 (gd 0 was February 18 through 20, 1985). For exposures, plug-positive females were transferred, one per cage, to stainless steel wire-mesh exposure cages and the cage carriers, with stainless steel shelves beneath each row of cages, were moved into the chambers. Food and water were withheld-during exposures . Exposures were for six hours per day, gd 6 through 15, with exposures beginning on February 24 for females with gd 0 on February 18 and ending March 7, 1985 for females with gd 0 on February 20. After each exposure, animals were returned to their original cages. The animals were observed daily for clinical signs throughout the study (gd 0 through 21). Food and water consumption was measured for the intervals gd 0-3, 3-6, 6-9, 9-12, 12-15, 15-18, and 18 -21. Maternal body weights were taken on gd 0, 6, 9, 12, 15, 18, and 21. At scheduled sacrifice on gd 21, rats were sacrificed by carbon dioxide asphyxiation. Maternal sacrifice period was March 11 through 13, 1985 .

Duration of treatment / exposure:

g.d. 6 - 15

Frequency of treatment:

6 h/day

Duration of test:

until g.d. 21

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

150 mg/m³ air

Remarks:

aerosol

Doses / concentrations 2

Dose / conc.:

1 000 mg/m³ air

Remarks:

aerosol

Doses / concentrations 3

Dose / conc.:

2 500 mg/m³ air

Remarks:

aerosol

No. of animals per sex per dose:

total: 62 virgin male and 172 female rats

Control animals:

yes

Details on study design:

The exposure concentrations were based on a range-finding study in pregnant rats and mice which employed concentrations of 0, 50, 250, 1250 and 2500 mg/m3.

Examinations

Maternal examinations:

Maternal animals were evaluated for body weight, liver weight, kidney weight, gravid uterine weight, number of ovarian corpora lutea and status of implantation sites, i .e., resorptions, dead fetuses, live fetuses.

Fetal examinations:

Fetuses were dissected from the uterus, counted, weighed, sexed and examined for external, visceral and skeletal malformations.

Statistics:

The unit of comparison was the pregnant female or the litter. Results of quantitative continuous variables were intercompared for the 3 exposure groups and the control group of each species by use of Levene's test for equal variances, analysis of variance and t-tests with Bonferroni probabilities. The t-tests were used when the F value from the ANOVA was significant. When Levene's test indicated homogeneous variances, and the ANOVA was significant, the pooled t-test was used. When Levene's test indicated heterogeneous variances, all groups were compared by an ANOVA for unequal variances followed, when necessary, by the separate vairance t-test.
Non-parametric data obtained following laparohysterectomy were statistically treated using the Kruskal-Wallis test follwed by the Mann-Whitney U test, when appropriate. Incidence data were compared using Fisher's Exact test. For all statistical tests, the fiducial limit of 0.05 (two-tailed) was used as the criterion for significance.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical signs, which increased in incidence with increasing exposure concentrations, included only red fur discoloration on the head and neck, a nonspecific indication of stress, observed in all groups.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The weight gain for dams at 2500 mg/m3 was significantly higher than in controls for the interval gd 6-18.

Food consumption and compound intake (if feeding study):

no effects observed

Water consumption and compound intake (if drinking water study):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Maternal liver weights (absolute and relative to corrected body weight) were significantly higher in dams at the highest concentration of ethylene glycol versus those in controls. Body weight, maternal body weight corrected for gravid uterine weight, gravid uterine weight, and absolute and relative kidney weights were unaffected by exposure to ethylene glycol aerosol.

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

Reproductive parameters were also unaffected by treatment. These parameters included number of corpora lutes, total non-viable and viable implantations per litter, percent live fetuses, sex ratio ( X males) and fetal body weight (males, females or total) per litter.

Effect levels (maternal animals)

Dose descriptor:

NOAEC

Effect level:

1 000 mg/m³ air

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

Remarks on result:

other:

Remarks:

Exposure to ethylene glycol aerosol during organogenesis resulted in minimal maternal toxicity in rats at 2500 mg/m3 and minimal fetotoxicity in rats at 1000 and 2500 mg/m3.

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

There was some evidence of treatment-related reductions in ossification of the fetal skeleton. These included an increase in the incidence of poorly ossified humerus (upper arm) and zygomatic arch (face) at 2500 mg/m3 and an increase in the incidence of poorly ossified metatarsals and proximal phalanges of the hindlimb at 1000 mg/m3 (but not at 2500 mg/m3). One additional incidental finding, that of bilobed sternebra number 1, exhibited a decreased incidence at 2500 mg/m3 relative to that of controls.

Visceral malformations:

no effects observed

Description (incidence and severity):

There were no significant increases in any fetal external or visceral variations in any ethylene glycol-exposed groups relative to controls.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

There was a significant decrease in the incidence of red foci on thymus at 2500 mg/m3 relative to controls.

Details on embryotoxic / teratogenic effects:

There was no statistically significant increase in the number of fetuses or of litters with one or more affected fetuses with individual malformations or pooled external, visceral or skeletal malformations or with total malformations. The incidence of total visceral malformations was significantly lower at 150 and 2500 mg/m3 relative to controls because of the finding of hydroureter and hydronephrosis in control litters, a malformation to which the CD rat is predisposed.

Effect levels (fetuses)

1

Dose descriptor:

NOAEC

Effect level:

150 mg/m³ air

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: no adverse effects seen at this dose

2

Dose descriptor:

LOAEC

Effect level:

1 000 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: fetotoxicity.

Remarks on result:

not determinable

Remarks:

True NOAEC from inhalation exposure alone cannot be determined due to confounding oral exposure during whole-body exposure. There was no maternal or embryofetal toxicity at 150 mg/m3 (the no observable effect level) and no teratogenicity at any aerosol concentration employed.

Fetal abnormalities

Abnormalities:

effects observed, treatment-related

Localisation:

other: ossification delays

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Generation of Atmospheres and Chamber Parameters: The total ethylene glycol concentrations were 79.3, 88 .8 and 83 .6% of target for the 150, 1000 and 2500 mg/m3 concentrations. The mass median aerodynamic diameter for the aerosol particles was 2.3 M, within the respirable range. Temperature was maintained at 69-72°F and relative humidity was maintained at 32-37%.

Applicant's summary and conclusion

Conclusions:

Exposure to rats and mice to EG glycol aerosol during organogenesis resulted in minimal maternal toxicity in rats at 2500 mg/m3 and minimal fetotoxicity in rats at 1000 and 2500 mg/m3. There was no maternal or embryofetal toxicity at 150 mg/m3 (the no observable effect level) and no teratogenicity at any aerosol concentration employed. In mice, evidence of maternal and embryofetal toxicity, including teratogenicity, was observed at 1000 and 2500 mg/m3. There were no observable effects to the mouse dams or conceptuses at 150 mg/m3. The amount of EG on the fur, groomed and ingested by the mice, may have been sufficient, per se, to produce the terata observed in mice in the present study. The role of inhaled ethylene glycol on teratogenesis cannot be determined from the present study.