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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:
1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report Date:
1990

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EPA OTS 798.4900 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Triethylene Glycol Monomethyl Ether (TGME)
- Molecular formula (if other than submission substance):
- Molecular weight (if other than submission substance):
- Physical state: Clear liquid
- Analytical purity: Details on file with the sponsor
- Storage condition of test material: Room temperature.

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
Females were assigned temporary numbers used for identification.
Males were individually identified with monel self-piercing ear tag inscribed with unique permanent number.

TEST ANIMALS
- Source: Hazelton Research Products, Denver, PA 17517
- Age at study initiation: 5 months old
- Weight at study initiation: 2.54 – 3.89 kg
- Fasting period before study:
- Housing: Individually housed in units of eight stainless-steel cages. Nesting material was not provided since rabbits were terminated before day of natural delivery.
- Diet: 150g Certified Rabbit Chow® #5322 (Ralston Purina) daily in an individual stainless-steel “J-type” feeder attached to each cage.
180g Rabbit Chow® #5322 (Ralston Purina) was given beginning on the day insemination occurred.
- Water: Ad libitum via individual glass bottles with attached stainless-steel sipper tubes.
- Acclimation period: 3 weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 64 – 72°F
- Humidity: 43 – 66%
- Photoperiod: 12 hours light/12 hours dark

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
unchanged (no vehicle)
Details on exposure:
Test material was administered as received. Dosages were adjusted daily on the basis of individual body weights recorded immediately prior to intubation. All dosages were followed by a 3 to 5 mL flush with R.O. deionized water. The control group rabbits were given reverse osmosis membrane processed deionized water (R.O. deionized water) at a dosage volume (1.44 mL/kg) that was equivalent to that given to the high dosage group.
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
Test material was administered as received.
Details on mating procedure:
- Impregnation procedure: artificial insemination, untreated proven male rabbits used specifically for breeding. The female rabbits were intravenously administered 20 USP Units/kg of HCG approximately three hours prior to insemination of October 16 through October 19, 1989. An estimated 0.25 mL of semen that had been diluted with normal saline to a concentration of 6.0x 10^6 spermatozoa/0.25 mL saline was used to artificially inseminate each rabbit.
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: The day artificial insemination was performed was designated as day 0 of presumed gestation.
Duration of treatment / exposure:
13 exposures: once daily on days 6 - 18 of gestation.
Frequency of treatment:
Once daily. Each daily dosage given to the rabbits was administered at approximately the same time each day [between 0759 and 1125 hours EDT (0659 and 1025 EST)].
Duration of test:
Animals were sacrificed on gestation day 29.
Doses / concentrationsopen allclose all
Dose / conc.:
250 mg/kg bw/day
Dose / conc.:
500 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
Dose / conc.:
1 500 mg/kg bw/day
No. of animals per sex per dose:
20
Control animals:
yes

Examinations

Maternal examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: female rabbits were observed for clinical signs and/or general appearance several times during the acclimation period and on day 0 of presumed gestation. Viability in the rabbits was noted twice daily throughout the study. Observations for clinical signs of test substance effects, abortions, premature deliveries and/or viability were made daily for each rabbit immediately prior to dosage (beginning on day 6 of presumed gestation and continuing through day 18 of presumed gestation) and approximately onehalf hour and one hour postdosage. These observations were also made once daily during the postdosage period (days 19 through 29).

BODY WEIGHT: Yes
- Time schedule for examinations: weights of the rabbits were recorded at least once weekly prior to insemination and on day 0 of presumed gestation and days 6 through 29 of presumed gestation.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Feed consumption values were recorded daily during the acclimation and study periods.

GROSS PATHOLOGY
- Necropsy observations and uterine contents were recorded for the rabbits that were found dead and/or sacrificed due to abortion. Maternal tissues that appeared normal were discarded following evaluation. Maternal tissues with gross lesions (with the exception of parovarian cysts, which are considered to be common spontaneous lesions in rabbits) were fixed in neutral buffered 10% formalin for possible 'further evaluation.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 29 of presumed gestation with T-61® Euthanasia Solution.
- Organs examined: Thoracic and abdominal viscera were examined for gross lesions.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes
- Soft tissue examinations: Yes
- Skeletal examinations: Yes
Each fetus was removed from the uterus and placed in an individual container. Each Caesarean-delivered fetus was weighed, examined for gross external alterations and individually identified with a tag noting litter, uterine placement, and study number. Live fetuses were sacrificed by an intraperitoneal injection of pentobarbital sodium. All fetuses were examined to identify sex and for visceral alterations; the brain was free-hand crosssectioned (a single cross-section made between the parietals and the frontals) and examined. Abnormal fetal tissues considered appropriate for retention were preserved in neutral buffered 10% formalin for possible future evaluation. The fetuses were eviscerated, stained with alizarin red 5(5) and evaluated for skeletal alterations. All skeletal preparations were stored in 80% glycerin with thymol crystals added to retard fungal growth.
Statistics:
Maternal and fetal incidence data were analyzed using the Variance Test for Homogeneity of the Binomial Distribution. Maternal body weight, gravid uterine weight and feed consumption means (g/day and g/kg/day), as well as litter means for percent male fetuses, fetal body weight, fetal ossification sites and percent fetal alterations were analyzed using Bartlett's Test of Homogeneity of Variances and the Analysis of Variance, when appropriate [i.e., when Bartlett's Test was not significant (P>0.05)]. If the Analysis of Variance was significant (P<0.05), Dunnett's Test was used to identify the statistical significance of the individual groups. If the Analysis of Variance was not appropriate [i.e., when Bartlett's Test was significant (P<0.05)], the
Kruskal-Wallis Test was used when less than or equal to 75% of the values were equivalent ("ties"); when more than 75% of the values were "ties",
the Fisher's Exact Test was used. In cases in which the Kruskal-Wallis Test was statistically significant (P~0.05), Dunn's Method of Multiple Comparisons(15) was used to identify the statistical significance of the individual groups. All other Caesarean-sectioning data were evaluated using the procedures described for the Kruskal-Wallis Test.
Indices:
gross external or internal soft tissue variations in the fetuses.
Historical control data:
No further data available.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The animals that subsequently went on to die in the 1500mg/kg group showed decreased motor activity, lethargy, laboured breathing, dehydration, no faeces, ataxia and red substance in the cage pan. No significant observations in other dose groups.
Mortality:
mortality observed, treatment-related
Description (incidence):
At the 1500 mg/kg/day dosage, TEGME caused the deaths of eight (one of these does aborted prior to death) (P≤0.01) does. One 1000 mg/kg/day dosage group doe died. It was unclear whether this death was related to TEGME since no clinical signs noted in the 1500 mg/kg/day dosage group does which died were observed in this animal, but this doe had weight loss and decreased feed consumption for several days prior to death.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The animals that died had clinical signs of treatment, including weight loss. Dosages of 500 mg/kg/day and higher caused increased body weight gain during the postdosage period, a common phenomenon seen in developmental toxicity studies after the termination of dosing.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The animals that died had clinical signs of treatment, including decreased feed consumption. Dosages of 500 mg/kg/day and higher caused increased feed consumption during the postdosage period, a common phenomenon seen in developmental toxicity studies after the termination of dosing.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
The animals that died, four of these does had gastric ulcerations.

Maternal developmental toxicity

Number of abortions:
effects observed, treatment-related
Description (incidence and severity):
Two does given the 1500 mg/kg/day dosage of TEGME aborted; the does that aborted also had treatment-related clinical signs: weight loss and reduced feed consumption. One low dosage group doe aborted but this was not considered related to the test substance because the incidence was not dosage-dependent.  No other does aborted, and no does prematurely delivered litters. Note that these were late abortions and there was no apparent abnormalities of the conceptuses.
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
effects observed, treatment-related
Description (incidence and severity):
Seen in animals that aborted in 1500mg/kg dose group.
Changes in number of pregnant:
no effects observed

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOEL
Effect level:
250 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
histopathology: non-neoplastic

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
Description (incidence and severity):
Pups sacrificed for examination
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
The 1500 mg/kg/day dosage significantly increased the fetal and/or litter incidences of two common skeletal variations. This maternally toxic dosage significantly increased only (P<0.01) the litter and/or fetal incidences of variations in fetal ossification (angulated hyoid alae- a frequent spontaneous finding - and reversible delays in ossification of the xiphoid). Thus,

Effect levels (fetuses)

Dose descriptor:
NOAEC
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Commonly observed skeletal variations at a dose well in excess of maternally toxic dose

Fetal abnormalities

Abnormalities:
effects observed, treatment-related
Localisation:
skeletal: sternum
other: skeletal: hyoid
Description (incidence and severity):
Increase in two common variations at maternally toxic dose, one a reversible ossification delay.

Overall developmental toxicity

Developmental effects observed:
no

Any other information on results incl. tables

TEGME should not be classified as a developmental toxicant since it does not present a unique hazard to the development of rabbit conceptuses.

Applicant's summary and conclusion

Conclusions:
Based on the data in this study, the maternal no-observed-effect-level (NOEL) for TEGME is 250 mg/kg/day. Dosages of 500 mg/kg/day and higher caused increased body weight gain and feed consumption during the postdosage period, a common phenomenon seen in developmental toxicity studies after the termination of dosing. The 1500 mg/kg/day dosage of TGME (and possibly the 1000 mg/kg/day dosage) caused death. The 1500 mg/kg/day dosage also produced abortions, clinical and necropsy observations, reduced maternal body weight, and feed consumption, compared with controls. Thus, 500 mg/kg/day was a NOAEL for maternal toxicity. The NOEL for developmental toxicity was 1000 mg/kg/day. The NOAEL for developmental toxicity was 1500 mg/kg/day. This maternally toxic dosage significantly increased only the litter and/or fetal incidences of variations in fetal ossification (angulated hyoid alae and reversible delays in ossification of the xiphoid). Thus, TGME should not be classified as a developmental toxicant since it does not present a unique hazard to the development of rabbit conceptuses.
Executive summary:

In a guideline (TSCA) and GLP study, the developmental toxicity of 2 -(2 -(2 -methoxyethoxy)ethoxy) ethanol or TGME was evaluated in rabbits following oral (gavage) administration at dosages up to 1,500 mg/kg bw/day on Days 6-18 of gestation. Dosages of 500 mg/kg/day and higher caused increased body weight gain and feed consumption during the postdosage period, a common phenomenon seen in developmental toxicity studies after the termination of dosing. The 1500 mg/kg/day dosage of TGME (and possibly the 1000 mg/kg/day dosage) caused death. The 1500 mg/kg/day dosage also produced abortions, clinical and necropsy observations, reduced maternal body weight, and feed consumption, compared to the study controls. Based on the data in this study, the maternal NOEL for TGME is considered to be 250 mg/kg/day, and 500 mg/kg/day is considered the NOAEL for maternal toxicity.

The 1500 mg/kg/day dosage significantly increased the fetal and/or litter incidences of two common skeletal variations. This maternally toxic dosage significantly increased (P<0.01) only the litter and/or fetal incidences of variations in fetal ossification (angulated hyoid alae and reversible delays in ossification of the xiphoid). Thus, TGME should not be classified as a developmental toxicant since it does not present a unique hazard to the development of rabbit conceptuses. Based on these findings the NOEL for developmental toxicity is considered to be 1000 mg/kg/day, and the NOAEL for developmental toxicity is considered to be 1500 mg/kg/day.