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Toxicological information

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:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals 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, 2500, 5000, 10,000, 20,000, or 40,000 ppm, coresponding to 587, 971, 2003, 5123, or 7584 mg/kg bw/d in males and 722, 1304, 2725, 7255, or 11172 mg/kg bw/d in females
No. of animals per sex per dose:
10
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:
effects observed, treatment-related
Description (incidence and severity):
The only treatment-related clinical sign of toxicity noted for mice treated with 2-ethoxyethanol was emaciation, which was observed in males and females in the 20,000 and 40,000 ppm groups.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
For male and female mice in the 20,000 and 40,000 ppm 2-ethoxyethanol groups, body weight gains were lower than those of the control groups.
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. Average compound consumption increased with dose for male and female mice treated with the glycol ethers.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Most changes in absolute and relative organ weights in the 13-week study of 2-ethoxyethanol in mice could be attributed to low final mean body weights, excluding decreases in testis weights. Absolute testis weights were significantly decreased for males in the two highest dose groups (20,000 and 40,000 ppm).
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
In the 13-week study of 2-ethoxyethanol, chemical-related gross lesions consisted of small testes and epididymides in mice from the 40,000 ppm group. Histopathologic changes were present in the spleen and testis of male mice and the spleen and adrenal gland of female mice. In male mice, degeneration of the testis was characterized as a marked, diffuse loss of germinal epithelium in the seminiferous tubules. Histopathologic changes were not seen in the testis of mice in the lower dose groups. In the spleen of female mice in the 20,000 ppm group and males and females from the 40,000 ppm groups, there was a minimal to mild increase in hematopoiesis; there was also a minimal increase in splenic hematopoiesis in one female mouse in the 10,000 ppm group. Splenic hematopoiesis was characterized by an increase in the number of erythroid elements and megakaryocytes and was similar to that seen in mice from the 2-methoxyethanol study.
Based upon histologic sections, there was no apparent effect in the bone marrow. In the adrenal gland, hypertrophy of the X-zone was present in all dose groups and was morphologically identical to that described for mice in the 2-methoxyethanol study.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Sperm morphology and vaginal cytology evaluations were performed on mice treated with 0, 5000, 10,000, or 20,000 ppm 2-ethoxyethanol. Epididymal and testicular weights were significantly lower than control values for males in the high-dose group (20,000 ppm). Values for sperm motility, Spermatid heads per testis, and Spermatid count were significantly lower than control values for males receiving 20,000 ppm 2-ethoxyethanol. All treated females had significantly longer estrous cycles than did controls.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Sperm morphology and vaginal cytology evaluations were performed on mice treated with 0, 5000, 10,000, or 20,000 ppm 2-ethoxyethanol. Epididymal and testicular weights were significantly lower than control values for males in the high-dose group (20,000 ppm). Values for sperm motility, Spermatid heads per testis, and Spermatid count were significantly lower than control values for males receiving 20,000 ppm 2-ethoxyethanol. All treated females had significantly longer estrous cycles than did controls.

Effect levels

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Dose descriptor:
NOAEL
Effect level:
20 000 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. 5123 mg/kg bw/d
Dose descriptor:
NOAEL
Effect level:
5 000 mg/L drinking water
Based on:
act. ingr.
Sex:
female
Basis for effect level:
other: adrenal gland hypertrophy and increased hematopoiesis in the spleen
Remarks on result:
other: corresponds to ca. 1300 mg/kg bw/d

Target system / organ toxicity

open allclose all
Critical effects observed:
yes
Lowest effective dose / conc.:
10 000 mg/L drinking water
System:
immune system
Organ:
thymus
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Critical effects observed:
yes
Lowest effective dose / conc.:
40 000 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-ethoxyethanol for 13 weeks, the NOAEL for testicular degeneration and increased hematopoiesis in the spleen was 20,000 ppm. For female mice in the 13-week study of 2-ethoxyethanol, the NOAEL for adrenal gland hypertrophy and increased hematopoiesis in the spleen was 5000 ppm.