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Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1993

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2-methoxyethanol
EC Number:
203-713-7
EC Name:
2-methoxyethanol
Cas Number:
109-86-4
Molecular formula:
C3H8O2
IUPAC Name:
2-methoxyethanol
Details on test material:
- Lot Nr: Lot E16

Test animals

Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Taconic Farms (Germantown, NY)
- Age at study initiation: approximately 4 to 5 weeks of age
- Housing: individually
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 1 to 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 60° to 77° F
- Humidity: 20% to 70%
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: drinking water
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
13 wk
Doses / concentrations
Remarks:
0, 2000, 4000, 6000, 8000, or 10000 ppm corresponding to 0, 295, 529, 765, 992, or 1367 mg/kg bw/d in males and 492, 902, 1194, 1489, or 1839 mg/kg bw/d in females
No. of animals per sex per dose:
10
Control animals:
yes
Details on study design:
- Dose selection rationale:
Dose selections for each 13-week study were based on the results of the respective 2-week studies. Due to a dose-related decrease in water consumption in the 2-week studies, the test articles were administered at a constant concentration (ppm) in the 13-week studies rather than on a mg/kg body weight basis.

Examinations

Observations and examinations performed and frequency:
In the 13-week studies of ethylene glycol ethers, hematology and clinical chemistry evaluations were performed on supplemental rats at Weeks 1 and 3 (10 males and 10 females per dose group per time point for each chemical) and on base-study rats at study termination (Week 13). Urine samples were collected from base-study rats for evaluation at the end of the study.

At all time points, rats were anesthetized with 70% CO2:30% O2, and blood samples were collected from the retroorbital sinus using capillary tubes. Blood samples were placed in EDTA tubes for hematologic analyses and in plain tubes devoid of an anticoagulant for clinical chemistry analyses. After blood samples were collected, bone marrow cells were collected from the right femur of rats for determination of total nucleated cell counts (Thompson et aL, 1991). On Day 90, rats were placed individually in metabolism cages for the collection of 16-hour urine samples. During this period, animals had access to feed but not water.
Reticulocyte counts were determined by microscopic examination of blood smears that had been incubated with new methylene blue. Leukocyte differentials were calculated from percentages of cell types determined from microscopic examination of Wright's-stained blood smears. Methemoglobin concentrations were measured using a spectrophotometric method (Evelyn and Malloy, 1938). Clinical chemistry variables were measured with a Cobas Fara chemistry analyzer (Roche Diagnostic Systems, Inc., Montclair, NJ).

Vaginal cytology and sperm morphology evaluations were performed on rats (10 animals per sex per dose level) and mice (10 animals per sex per dose level) from the 13-week studies. Male rats receiving 2-methoxyethanol at dose levels of 0, 750, 1500, or 3000 ppm and female rats receiving 2-methoxyethanol at dose levels of 0, 1500, 3000, or 4500 ppm were evaluated. Male mice receiving 0, 2000, 4000, or 6000 ppm 2-methoxyethanol and female mice receiving 0, 6000, 8000, or 10,000 ppm 2-methoxyethanol were evaluated.
Rats administered 2-ethoxyethanol at dose levels of 0, 2500, 5000, or 10,000 ppm were evaluated. Methods were those described by Morrissey et al. (1988). Briefly, for the 7 days prior to sacrifice, the vaginal vaults of 10 females of each species per dose group were lavaged and the aspirated lavage fluid and cells were stained with Toluidine Blue. Relative numbers of leukocytes, nucleated epithelial cells, and large squamous epithelial cells were determined and used to ascertain estrous cycle stage (ie., diestrus, proestrus, estrus, and metestrus).

Sperm morphology was evaluated at necropsy in the following manner. The left epididymis was isolated and weighed. The tail of the epididymis (cauda epididymis) was then removed from the epididymal body (corpus epididymis) and weighed. Test yolk (rats) or Tyrode's buffer (mice) was applied to slides and a small incision was made at the distal border of the epididymal tail. The sperm effluxing from the incision were dispersed in the buffer on the slides and the numbers of motile and nonmotile spermatozoa were counted for five fields per slide.
Following completion of sperm motility estimates, each cauda epididymis was placed in buffered saline solution (0.9%). Cauda were gently minced and the tissue was incubated in the saline solution and then heat fixed at 65° C. Sperm density was then determined microscopically with the aid of a hemacytometer. To quantify spermatogenesis, testicular Spermatid head count was determined by removing the tunica albuginea and homogenizing the left testis in phosphate buffered saline containing 10% dimethyl sulfoxide. Homogenization-resistant Spermatid nuclei were enumerated using a hemacytometer.

Dose selections for the stop-exposure studies were based on the results of the 2-week studies. In each stop-exposure study, 30 male rats per dose group were administered 2-ethoxyethanol in drinking water. Dose levels were 0, 5000, 10,000, or 20,000 ppm. Test article was administered daily for 60 days in drinking water that was available ad libitum. At the end of the treatment period, 10 rats per dose group were killed, except
in the case of early deaths. If lesions were found at the 60-day necropsy, half of the remaining animals were killed after a 30-day recovery period, and the other half were killed after a 56-day recovery period. Animals were housed five per cage in the same room as the animals in the 13-week studies. At necropsy, the testes and epididymides were removed. The right testis and epididymis were weighed, and the testes and the caput and cauda of the left epididymis were examined microscopically. Organs for rats in the 30- and 56-day recovery groups in the 2-butoxyethanol stop-exposure study were not processed for histology because no microscopic lesions attributable to chemical exposure were found after the 60-day exposure period.

Sacrifice and pathology:
Complete necropsies were performed on all base-study animals in the 2-week and 13-week studies. The following organs from rats and mice were weighed: heart, right kidney, liver, lung, thymus, and right testis. Organs and tissues were examined for gross lesions and were fixed in 10% neutral buffered formalin. Tissues to be examined microscopically were trimmed, embedded in paraffin, sectioned, and stained with hematoxylin and eosin.

For animals in the 2-week studies, complete histopathologic examinations were performed only on those organs showing gross evidence of lesions. For animals in the 13-week studies, complete histopathologic examinations of protocol-required tissues were performed on all control animals, all animals in the highest dose group with at least 60% survivors at the time of sacrifice, and all animals in higher dose groups inclusive of
early deaths and survivors. Gross lesions and selected tissues were examined in the lower dose groups to a no-observed-effect level.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
There were no significant clinical observations in male or female mice during the 13-week studies of 2-methoxyethanol.
Mortality:
no mortality observed
Description (incidence):
No male or female mice receiving 2-methoxyethanol died or were killed before the end of the studies.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean body weight gains of male mice receiving 10,000 ppm 2-methoxyethanol and female mice receiving 8000 or 10,000 ppm 2-methoxyethanol were notably lower than those of the control groups. For
Water consumption and compound intake (if drinking water study):
effects observed, non-treatment-related
Description (incidence and severity):
In the 13-week study of ethylene glycol ethers, average water consumption was variable, and no clear treatment-related patterns were evident
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
With the exception of decreases in thymus and testis weights, most changes in absolute and relative organ weights in the 13-week study of 2-methoxyethanol in mice could be attributed to low final mean body weights. Dose-related decreases were noted for the absolute and relative testis weights of male mice and the absolute and relative thymus weights of male and female mice.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
In the 13-week study of 2-methoxyethanol, chemical-related gross lesions were identified in the testis and thymus. Testes from mice in the 6000, 8000, and 10,000 ppm groups were small. The thymuses of males in the 8000 and 10,000 ppm groups and females in the 10,000 ppm (high-dose) group were also smaller than those of the control animals. In male mice, degeneration of the testis was characterized microscopically by a doserelated, minimal to marked degeneration of the germinal epithelium in seminiferous tubules (Table 21); at the higher doses, the lumen of many tubules contained only Sertoli cells (Plate 9). In the thymus of most males from the two highest dose groups and females in the high-dose group, there was minimal to mild lymphoid depletion (atrophy) consisting of a reduction in the thickness of the thymic cortex and in the number of thymocytes.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathologic changes were also present in the spleen of male and female mice and in the adrenal gland of female mice. Increased hematopoiesis was present in the spleen of mice from all dosed groups, excluding male mice in the lowest dose group (2000 ppm), and was characterized by a marked increase in the number of megakaryocytes present in the red pulp. In the adrenal gland of female mice in all dosed groups, there was hypertrophy of the X-zone. In dosed mice, there was a marked increase in the lipid vacuolization normally present in this region of the adrenal gland in young female mice.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Sperm morphology evaluations were performed on male mice treated with 0, 2000, 4000, or 6000 ppm 2-methoxyethanol. Vaginal cytology evaluations were performed on female mice treated with 0, 6000, 8000, or 10,000 ppm 2-methoxyethanol. Results showed significant decreases in epididymal and cauda epididymal weights for males in the 6000 ppm group and in testicular weight for males in the 4000 and 6000 ppm groups. The values for sperm motility were significantly less than controls for the 2000 and 6000 ppm groups, as were sperm concentration measurements for males treated with 2000 to 6000 ppm 2-methoxyethanol. Spermatid measurements were significantly lower than controls for males receiving 4000 or 6000 ppm 2-methoxyethanol.
For females, all dose groups differed significantly from controls in the relative frequency of time spent in estrous stages.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
2 000 mg/L drinking water
Based on:
act. ingr.
Sex:
male
Basis for effect level:
other: testicular degeneration and increased hematopoiesis in the spleen
Remarks on result:
other: corresponds to ca. 100 mg/kg bw/d
Dose descriptor:
LOAEL
Effect level:
2 000 mg/L drinking water
Based on:
act. ingr.
Sex:
female
Basis for effect level:
other: adrenal gland hypertrophy and increased hematopoiesis in the spleen

Target system / organ toxicity

open allclose all
Critical effects observed:
yes
Lowest effective dose / conc.:
2 000 mg/L drinking water
System:
haematopoietic
Organ:
spleen
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Critical effects observed:
yes
Lowest effective dose / conc.:
4 000 mg/L drinking water
System:
male reproductive system
Organ:
testes
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Applicant's summary and conclusion

Conclusions:
For male mice treated with 2-methoxyethanol for 13 weeks, the NOAEL for testicular degeneration and increased hematopoiesis in the spleen was 2000 ppm. A NOAEL was not reached for female mice treated with 2-methoxyethanol, since adrenal gland hypertrophy and increased hematopoiesis in the spleen occurred at the lowest concentration administered (2000 ppm).