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Effects on fertility

Additional information

In a special study with the aim to investigate the testicular and epididymal toxicity of some short-chain ethers, including dibutyl ether, in comparison to the known testicular toxin 1,6-dimethoxy hexane, male Sprague-Dawley rats (seven weeks old, 7 per group) were treated by gavage with 2, 20, and 200 mg dibutyl ether/kg bw in corn oil, 5 days per week for 4 weeks. Negative control and positive control groups were treated with 10 ml corn oil and 200 mg 1,6-dimethoxy hexane/kg bw, respectively.A post-exposure recovery group was not included in this study.Body weights and food consumption were measured weekly. Cage-side observations were made weekly. At study termination, urine was analyzed and blood was withdrawn from the abdominal aorta for hematological and blood chemistry analyses including plasma and urine concentrations of 2-methoxy acetic acid (MAA), the toxic metabolite of the positive control substance 1,6-dimethoxy hexane. In liver homogenates reduced glutathione, thiobarbituric acid-reactive substances (TBARS), protein carbonyl content, and activity of UDP-glucuronosyltransferase (UDPGT), and in liver S9 fraction the activities of ethoxyresorufin-O-deethylase (EROD), pentoxyresorufin-O-dealkylase (PROD), benzyloxyresorufin-O-dealkylase (BROD), and glutathione-S-transferase (GST) were measured. Brain, heart, thymus, liver, lung, kidneys, spleen, ventral prostate, epididymides and testes were weighed and prepared for histopathological examinations.

All animals survived the 4-week treatment with no abnormal clinical signs. Food intake, final body weight and the relative organ weights including ventral prostate, epididymides and testes were not significantly different from the negative control group. There were no treatment-related changes in the measured hematological parameters and serum chemistry parameters including serum thyroxin (T4) and triiodothyronine (T4). In the liver BROD activity was significantly elevated in the high dose group receiving 200 mg dibutyl ether/kg bw. In animals treated with dibutyl ether, no significant changes were found in urinary MAA levels and MAA was not detected in plasma. The histopathological examination of the testicles and the epididymides of animals treated with dibutyl ether, where special focus was put on seminiferous tubule degeneration and interstitial vacuolation in the testicles, reduction in sperm density and the existence of spermatid giant cells in the caput (head), corpus (body) and cauda (tail) sections of the epididymides, revealed no treatment related changes.Minimal histologicalchanges were found in the thyroid (reduced follicle size, nuclear vesiculation) of all dibutyl ether treated animals, but also in some negative control animals and, more prominent, in all positive control animals. In the absence of significant modulation in serum thyroxin (T4) and triiodothyronine (T3), the thyroid effects were discussed by the study authors as probably adaptive and reversible. Some bone marrow changes (increased granulocytes and myeoloid/erythroid ratio), minimal in severity, were seen in animals treated with 200 mg dibutyl ether/kg bw. In the liver, the high dose of dibutyl ether produced minimal to mild histopathological changes such as vesiculation of nuclei and increase in cytoplasmatic homogeneity. Urinary ascorbic acid, a biomarker of hepatic response to xenobiotics, was also elevated at the high dose of 200 mg dibutyl ether/kg bw, suggesting that the hepatic glucuronic acid pathway, which is associated with the glucuronide pathway of detoxification, was stimulated. A hepatic response was further indicated by the increase in hepatic xenobiotic enzyme activity (benzyloxyresorufin-o-dealkylase (BROD)) in high dose animals. However, as the study authors discussed, in the absence of signs of necrosis or elevated level of aspartate aminotransferase in serum, the urinary ascorbic acid and hepatic enzyme changes and histopathological changes can be considered as mild metabolic responses. All other organs were free of any histopathological changes.

In conclusion, the animals treated with dibutyl ether did not show any significant testicular or epididymal effect. The reproductive organs were of normal weight and free of histopathological changes. The urinary creatine/creatinine ratio (as sensitive biomarker of testicular injury) was unchanged, and the amount of 2-methoxy acetic acid (the toxic metabolite of the positive control substance 1,6-dimethoxy hexane) in urine was within the control levels and 2-methoxy acetic acid was not detected in plasma. At the high dose of 200 mg dibutyl ether/kg bw changes to the thyroid, liver and bone marrow that were mild and adaptive in nature were caused. In contrast, the positive control substance 1,6-dimethoxy hexane caused decreased testis and thymus weights, degeneration of the seminiferous tubules and reduction of sperm density in the epididymides, an elevated creatine/creatinine ratio, an increase in plasma and urinary 2-methoxy acetic acid levels, histopathological thymus changes, mild dyserythropoiesis and dysthrombopoiesis in the bone marrow, thyroid changes of moderate severity and more pronounced adaptive liver changes (Poon et al., 2004, 2005).

In a 28-day inhalation guideline study on male and female Wistar rats, exposed towards 0 (control), 150, 500 and 1500 mg dibutyl ether/m³ for 6 hours/day, 5 days/week for four weeks, no concentration-related findings of pathological significance were noted at organ weight evaluation and histopathological examination in testes, epididymides, prostata, seminal vesicles and coagulating glands of males nor in ovaries, uterus, vagina and mammary glands of females in comparison with untreated control animals both immediately after study termination and after a two week recovery period, respectively, thereby revealing no adverse effects on male and female reproductive organs (for detailed study description see chapter 3.1.5; TNO, 2005).

In the prenatal developmental toxicity study which is described below, there were no indications on adverse effects on female reproductive organs at doses up to and including 1000 mg/kg bw/day (Degussa AG, 2005e).


Short description of key information:
No special animal studies were conducted with dibutyl ether to investigate possible substance-related effects on the reproductive performance and reproductive organs of females. The testicular toxicity of dibutyl ether has been assessed in comparison to the known testicular toxicity of 1,6-di¬methoxy hexane in a 4 week oral study on rats. Furthermore there is a 4 week inhalation GLP study, performed according to standard guidelines, which also evaluated the reproductive organs and can therefore be taken into account to assess this endpoint. According to a review by Mangelsdorf and Buschmann (2002), histopathology of the testes and weights of reproductive organs are the most sensitive endpoints to detect toxic effects on male fertility and effects on these endpoints may already be detected after 4 weeks of treatment with high sensitivity. A prenatal developmental toxicity study in rats by oral administration has been performed under GLP conditions and in accordance with directive 88/302/EEC, B.31 (1988) and OECD TG 414 (2001).

Effects on developmental toxicity

Description of key information
A prenatal developmental toxicity study in rats by oral administration has been performed under GLP conditions and in accordance with directive 88/302/EEC, B.31 (1988) and OECD TG 414 (2001).
Effect on developmental toxicity: via oral route
Dose descriptor:
NOAEL
300 mg/kg bw/day
Additional information

Dibutyl ether (purity 99.7 %) was administered in a prenatal developmental toxicity study, performed under GLP conditions and in accordance with directive 88/302/EEC, B.31 (1988) and OECD TG 414 (2001), to pregnant female rats once daily from the 6th to the 19th day of pregnancy in doses of 0 (control, corn oil), 100, 300 and 1000 mg/kg bw. The test item was diluted in the vehicle (corn oil) to the appropriate concentrations and was administered orally at a constant volume of 2 ml/kg bw. The day on which sperm was found in the vaginal lavage was considered as the day of conception (day 0 of pregnancy). Dose selection was based on the results of a dose-range-finding study (Degussa AG, 2005d). Individual animals were observed daily for viability, behavior, external appearance and nature of the faeces. Immediately after administration, any sign of illness or reaction to treatment were recorded. In case of changes, the animals were observed until the symptoms disappeared. In addition, animals were checked regularly throughout the day. Maternal body weights, food and drinking water consumption were recorded daily. Early in the morning of gestation day 20 blood was taken from all animals of the control and the high dose group to determine the plasma activity of alanine aminotransferase (ALAT), alkaline phosphatase (AP), aspartate aminotransferase (ASAT) and lactate dehydrogenase (LDH). The dams were laparotomised under ether narcosis on day 20 of gestation. 20 dams/group were evaluated. In order to obtain the required number of pregnant animals/group, additional animals (5/group) were used at the start of study. The ovaries, the uteri and the liver of the dams were removed; the uteri (in toto) and the liver were weighed. A dissection with macroscopic examination of the internal organs and placentae of the dams was carried out. In case of macroscopical findings, the affected maternal tissues were preserved for possible further histopathological examinations. The fetuses were removed and the following examinations were performed: macroscopic inspection of the placentae, number of fetuses, sex and viability of fetuses, number and size of resorptions, corpora lutea in the ovaries, implantations and location of fetuses in uterus, weight of fetuses and weight of placentae, external inspection of fetuses for damages, especially malformations. The fetuses were sacrificed by an ether atmosphere and 50 % of the number of fetuses in each litter were examined for skeletal anomalies. The skeleton was double-stained with Alician blue for the examination of cartilage and with Alizarin red to reveal ossifications (according to). The remaining 50 % of the number of fetuses in each litter were examined for soft tissue anomalies. Body sections were made and examined according to.

All dams survived to sacrifice. At the high tested dose level of 1000 mg/kg bw, signs of toxicity in form of piloerection was noted in 5 of 20 dams. One animal showed pale eyes and ears on gestation days 7 to 12. Additionally, a slightly reduced body weight (up to 7 % below the control) and reduced food consumption (up to 36 % below the control value) and increased drinking water consumption were recorded. Necropsy revealed no test item-related pathological changes in any of the dams treated with 1000 mg/kg bw/day. Slightly increased absolute and relative liver weights and an increased activity of plasma aspartate aminotransferase were noted. A slight reduction by 9 % was noted for the gravid uterus weight caused by slightly lowered fetal weights. The carcass weight was 6 % below the control value.

The treatment of dams with 100 or 300 mg dibutyl ether/kg bw/day had no effects on behavior, external appearance, nature of the faeces, body weight, body weight change and body weight gain, food and drinking water consumption, uterus weight and net body weight change. Necropsy revealed no test item-related pathological changes in any of the dams treated with either 100 or 300 mg/kg bw/day. The marginal increase of absolute and relative weights of the liver at 100 and 300 mg/kg bw/day were regarded to be an inductive effect but not an adverse effect.

No test item-related influence on the prenatal fetal development was detected at either 100, 300 or 1000 mg/kg bw/day, with respect to the number of corpora lutea, implantation sites, resorptions, placental weights, live fetuses, the values calculated for the pre- and post-implantation losses and the sex distribution of fetuses. Laparotomy revealed one dead fetus each (both of them were runts) at 100 and 300 mg/kg bw/day. In total, five runts were noted at laparotomy: two runts each at 100 and 1000 mg/kg bw/day and one runt at 300 mg/kg bw/day. One twin implant (one male fetus and one late resorption) was detected in one dam of the intermediate dose-group. All findings of dead fetuses, runts and twin implants are regarded to be spontaneous and are within the normal range of variation.

At the high tested dose level of 1000 mg/kg bw, reduced fetal weights calculated for male and female fetuses and for all fetuses were slightly but statistically significantly below the control values. In addition, in this dose group skeletal examination according torevealed significantly increased fetal incidences for the following retardations: hyoid not ossified and absence of ossification in 5th metacarpalia. Significantly increased litter incidences were noted for the caudal vertebral bodies, only one body ossified. These incidences were above the range of the test facility background data and are regarded to be test item-related. However, the incidence of total skeletal retardations in the high dose was not increased. The examination of the fetal organs (according to) revealed statistically significantly increased fetal and litter incidences for dilatation of the 4th cerebral ventricle.

The mean fetal weights were not influenced by the administration of 100 or 300 mg/kg bw/day. Also no test item-related influence was noted for the incidence of skeletal retardations or soft tissue variations at the low and intermediate dose levels. At 100, 300 or 1000 mg/kg bw/day, no malformations were noted in the fetuses during external examination at laparotomy, skeletal examination (according to) or soft tissue examination (according to).

In conclusion, there were obvious signs of maternal toxicity at the dose level 1000 mg/kg bw/day, in form of piloerection, a reduction in body weight and food intake and an increase in water consumption, absolute and relative liver weights, and plasma ASAT activity. At this high tested and maternally toxic dose level of 1000 mg/kg bw/day the mean fetal weights were reduced, and in addition, statistically increased incidences were recorded for skeletal retardations (according to Dawson) in form of missing or incomplete ossification of the hyoid, caudal vertebral bodies and 5th metacarpalia and for soft tissue variations (according to Wilson) in form of dilatation of the 4th cerebral ventricle. Neither maternally toxic effects nor signs of fetal retardations appeared at the dose levels 300 and 100 mg/kg bw. Dibutyl ether possessed no teratogenic properties. There was no test item-related increase in the incidence of fetal malformations, external or skeletal variations, not even at the maternally toxic dose level. An increase in retardations and variations was only noted in the maternally toxic dose of 1000 mg/kg bw/day (Degussa AG, 2005e).

The no-observed-effect level (NOEL) for toxic effects on the dams and for fetal skeletal retar­dations and soft tissue variations was 300 mg/kg bw/day.

The no-observed-effect-level (NOEL) for teratogenic properties was 1000 mg/kg bw/day (highest dose tested).

Justification for classification or non-classification

No specific studies have been performed on the toxicity of dibutyl ether to reproduction. Organ weight determinations and gross and histopathological examinations in a 28-day study revealed no pathological change in reproductive organs (testes, epididymides, prostate, seminal vesicles, coagulating glands, ovaries, uterus, vagina and mammary glands) when dibutyl ether was administered by inhalation to male and female rats at concentration levels up to and including 1500 mg/m³. No histopathological effects or weight changes were found in testes and epididymides of rats treated orally with up to 200 mg/kg bw for four weeks in a study specifically designed to detect effects on male reproductive organs. In the latter study, clear testicular effects were produced in a concurrent group of animals treated with 1,6-dimethoxy hexane. This gives confidence that the study was sensitive enough to detect relevant adverse effects on the male reproductive organs, although the duration of treatment was relatively short (4 weeks). In the prenatal developmental toxicity study which is described below, there were no indications on adverse effects on female reproductive organs at doses up to and including 1000 mg/kg bw/day. The data set regarding female fertility is limited and therefore no firm conclusions can be drawn. However based on the minimal toxicological effects observed with this substance it is unlikely that this substance is toxic to reproduction.

In a GLP study performed in accordance with OECD TG 414 on rats with oral administration of dibutyl ether from the 6thto the 19thday of pregnancy, developmental effects (fetal weight reduction, skeletal retardations in form of missing or incomplete ossification of the hyoid, caudal vertebral bodies and 5thmetacarpalia, and soft tissue variations in form of dilatation of the 4thcerebral ventricle) were found at the maternally toxic dose level of 1000 mg/kg bw. There was no test item-related increase in the incidence of fetal malformations, or external or skeletal variations. Signs of maternal toxicity at 1000 mg/kg bw/day were piloerection, a reduction in body weight and food intake and an increase in water consumption, absolute and relative liver weights, and plasma aspartate aminotransferase activity. Necropsy revealed no test item-related pathological changes in reproductive organs. Under the conditions of this study the NOAEL for maternal toxicity and developmental toxicity was 300 mg/kg bw/day.

Although there is some small evidence of minor variations/retardations at the highest dose tested, these would not typically be sufficient to require classification for developmental toxicity. In addition, there was evidence of maternal toxicity at the highest dose that may have contributed to the delays in ossification observed.

In conclusion, no classification for reproductive or developmental toxicity is warrented. This conclusion was agreed by the OECD HPV programme.