The following abstract presents results of a study conducted by a contract laboratory for the National Toxicology Program. The findings were not evaluated in accordance with the levels of evidence for reproductive or developmental criteria established by NTP in March 2009. The findings and conclusions for this study should not be construed to represent the views of NTP or the U.S. Government.
Ethylene glycol monomethyl ether (EGMME, Methyl Cellosolve®) is a commonly used solvent for low-viscosity cellulose acetate, natural resins, some alcohol-soluble dyes, leather dyeing, moisture-proof cellophane sealing, nail polishes, quick-drying varnishes and enamels, and wood stains1. Based on the instances reported in the literature of human intoxication from exposure to EGMME, Cornish (1981)2 suggested that it can cause neurologic symptoms, macrocytic anemia, and abnormal leukocyte counts.
The reproductive toxicity of EGMME was evaluated according to the Fertility Assessment by Continuous Breeding (FACB) protocol.
In Task 1, the dose-range finding study, EGMME was tested at the following concentrations: 0 (control), 0.25, 0.5, 1.0, 2.5, and 5.0% (w/v). During the 2 weeks of Task 1, animals in the control, 0.25, 0.5, and 1.0% dose groups, on the average, gained 8 to 11% of their original body weight. The male mice exposed to 2.5% and 5.0% EGMME lost 19 and 40% of their initial body weight, respectively. The female CD-1 mice in the 2.5 and 5.0% dose groups lost 11 and 37% of their initial body weight, respectively. Out of the 16 animals in the 5.0% EGMME group, 7 females and 5 males died. There were no deaths in the control or any dose groups. A dose-dependent decrease in the average daily consumption of water containing 1.0 to 5.0% EGMME levels was noted during two weeks of Task 1.
Task 2 was the continuous breeding phase of the protocol. This phase employed a vehicle control group (40 males and 40 females) and three dose groups (20 males and 20 females per group). Since it was required that the highest dose should not suppress body weight gain more than 10 percent compared to controls and allow 90 percent or greater survival in male and female CD-1 mice, 2.0% EGMME was chosen as the high dose. The mid-dose was 1.0% and the low dose, which was expected to be a no effect dose level, was 0.5% administered in drinking water. Eleven-week old male and female CD-1 mice were exposed to the chemical during a 7-day premating period, after which they were randomly paired (one male: one female) within each dose group and cohabited for 14 weeks. Newborn litters were evaluated and immediately sacrificed.
Continued EGMME treatment was extremely toxic to CD-1 mice. All 40 animals in the 2.0% dose group died within 11 weeks of treatment. Twenty-four out of the 40 animals in the 1.0% EGMME group died during the 14 weeks of cohabitation; 5 more animals died during the 3 weeks of the holding period. In the 0.5% dose group, 5 animals died during the Task 2 cohabitation and 1 during the holding period. There were no deaths in the control group during the Task 2 phase. Since all breeding pairs in all 3 dosed groups were infertile, data from breeding pairs in which one or both partners died during cohabitation were not excluded.
Daily consumption of control/dosed water by animals in the three dose groups was significantly decreased; the observed response was dose dependent. The average daily water consumption and mean body weight data during the first 14 weeks of Task 2 indicated that the male mice in the 0.5, 1.0, and 2.0% dose groups received approximately 0.779, 1.270, and 1.909 g/kg bw of EGMME, respectively.
All breeding pairs in the 0% (control) group delivered at least one litter. None of the breeding pairs in the three dose groups delivered any litters. The reproductive performance data of fertile pairs in the control group were parallel to similar control data from the other FACB studies conducted in our laboratory.
The continuous breeding portion of the protocol, Task 2, indicated that EGMME treatment was extremely toxic at the 1.0 and 2.0% dose levels and significantly affected fertility at all three dose levels. Since Task 2 does not discriminate which sex (or sexes) is susceptible to the chemical exposure, it was followed by a crossover mating study, Task 3.
In this particular study, during Task 3, animals from the 0.5% dose group were tested in a crossover mating trial to determine whether the males or females or both sexes had compromised reproductive performance when matched with control animals.
The females treated with 0.5% EGMME and cohabited with control males (Group 1) had 0% fertile matings as compared to a control male X control female (Group 2) value of 61%. The corresponding value for 0.5% male X control female group (Group 3) was 6%. The mating index (No. with copulatory plugs/No. cohabited X 100) values for groups 1, 2, and 3 were 93, 61, and 59%, respectively. These data suggest that a significant number of females exposed to 0.5% EGMME successfully mated with control males but were unable to deliver any litters. It is possible that EGMME treatment alters ovulation, fertilization, implantation, and/or embryonic development and thereby causes infertility in female mice.
The group mean whole body, liver, and kidney(s) weights in the female mice exposed to EGMME were significantly decreased relative to the controls (p<0.01). When organ weights were adjusted for body weight at necropsy, the mean adjusted liver and kidney(s) weights in treated animals were no longer significantly different (p<0.05).
For male mice, a significant decrease (p<0.01) was noted in the EGMME treated animals with respect to the right epididymis, right testis, and seminal vesicle(s) weights at necropsy . The average liver weight in the treated animals was increased (p<0.01). The group mean weights for whole body, kidneys, right cauda epididymis, and prostate were essentially the same (p<0.05) as the control values. Male organ weights were adjusted for body weight at necropsy; once again the right epididymis, right testis, and seminal vesicles weights for treated mice were significantly lower (p<0.01) than the corresponding control values and liver weight was significantly higher (p<0.01).
The incidence of abnormal sperm in the treated mice was 73% vs. a control value of 4%. The average sperm motility in EGMME treated mice was 13% vs. 91% in the control animals. The sperm density was also significantly reduced (p<0.01). EGMME treatment did not appear to interfere with the relative frequency of various estrous stages. The average estrous cycle length in the treated animals increased to 5.3 days vs. a control value of 4.6 days. The difference was statistically significant (p<0.05).
Testes were atrophic in 16 out of the 20 treated males and 0 out of the 40 control males. Distinct treatment related histopathologic changes were noted in 20 out of the 20 male CD-1 mice. The lesions were mainly seen in the testes and epididymis and included degeneration of seminiferous tubules, interstitial cell hyperplasia in the testes, reduction of sperm content, and accumulation of fluid and degeneration of duct epithelial cells in the epididymis. The degeneration of tubules consisted of loss of spermatozoa, spermatids, spermatogonia, spermatocytes, and vacuolization of epithelial cells. In the control group, 23 out of the 40 males showed degenerated epithelial cells; 22 were rated minimal and the other 1 mild. Among 20 animals in the 0.5% EGMME group, 1 was rated minimal, 2 mild, 7 moderate, and 10 severe. Interstitial cell hyperplasia was present in 95% of the testes of the treated animals and only 2.5% of the control mice.
EGMME treatment did not produce any significant gross or histopathologic changes in the reproductive organs of the treated female mice except an increased incidence of amyloidosis in the ovaries. Histologic changes of phases of estrous were observed in the vagina of the control and treated animals indicating that exposure to EGMME did not affect the normal estrous cycle.
In essence, EGMME administered in drinking water was extremely toxic at the 1.0 and 2.0% dose levels. Breeding pairs exposed to this chemical at all three dose levels (0.5 to 2.0%) were infertile during Task 2. When male/female partners from these pairs were mated with control male/female partners (Task 3), the fertility index was once again 0 and 6% vs. a control value of 61%. SMVCE studies showed that the testes, epididymides, and seminal vesicles were atrophic in the treated animals. Other male reproductive parameters adversely affected were sperm density, sperm motility, and sperm morphology. These observations were parallel to the histopathological findings. For example, the high rate of degeneration of seminiferous tubules, interstitial cell hyperplasia, and reduction in sperm count was apparent in treated male mice.
Based on the present study, EGMME can be designated as a potent reproductive toxicant in mice at the 0.5% and higher dose levels.
1The MERCK Index (1983), 10th Edition, p. 866, #5915.
2Cornish, H.H.: Solvents and Vapors in Casarett and Doull's Toxicology. Doull, J., Klaassen, C.D., and Amdur, M.O. (Eds.), MacMillan Publishing Co., Inc. (1981), p. 468496.