Developmental Toxicity of Thiodiglycol in Sprague-Dawley Rats

Abstract

Thiodiglycol (TG), a hydrolysis product of sulfur mustard (HD), is a potential contaminant of soil and water at certain military sites. To establish developmental toxicity criteria for TG, an oral developmental toxicity study was conducted in Sprague-Dawley rats. Neat thiodiglycol (99.9 %) was administered orally to mated female rats from gestation days (GDs) 5 through 19. The day of positive mating was considered day 0. A pilot study was conducted with TG at dose levels 250, 500, 1000, 2000, or 5000 mg/kg to select suitable doses for the main study. In the main study, three groups of rats (25/group) received TG by gavage at dose levels of 430, 1290, or 3870 mg/kg/day. A fourth group served as a sham control. On day 20 of gestation, all females were euthanized and a cesarean section performed. Litters were examined for soft tissue and skeletal alterations. Maternal toxicity was limited to dams receiving TG at 3870 mg/kg/day. At this dose, body weights and food consumption were reduced during certain periods of gestation. Fetuses derived from those dams exhibited a nonstatistically significant increased incidence of variations when compared to controls. Fetal body weights in the 3870 mg/kg/day group were significantly lower than controls. There was no increased incidence of anomalies when thiodiglycol-treated fetuses were compared to controls. It was concluded that TG did not produce terata. Developmental toxicity (decreased fetal weights and associated delays in development) occurred only at the maternally toxic dose of 3870 mg/kg. It appears that 1290 mg/kg/day could be considered no observed adverse effect level (NOAEL) for oral developmental toxicity. The lowest observed adverse effect level (LOAEL) was 3870 mg/kg for maternal toxicity.

Thiodiglycol (TG), a hydrolysis product of sulfur mustard, has been shown to be present in the ground and surface water near Old “O” Field at the Edgewood Area of Aberdeen Proving Ground, MD. The area had been a disposal site for, among other things, chemical warfare agents both pre- and post-World War II. Sulfur mustard (HD) undergoes hydrolysis to form various products such as thiodiglycol, 1,4-oxathiane, 1,4-dithiane, 2-vinylthionethanol, and mustard chlorohydrin in biological or environmental systems (D’Agostino and Provost 1988; Rosenblatt et al. 1996). Stockpiles of HD have been disposed through the hydrolysis process (National Research Council [NRC] 1996b). Toxicity data on TG is limited to only a few reports. The acute oral LD₅₀ values reported in male Wistar rats were 6610 mg/kg and in guinea pigs of mixed sexes 3960 mg/kg (Smyth, Seaton, and Fischer 1941). A subcutaneous LD₅₀ of 4 g/kg for rats and mice and an intravenous LD₅₀ of 3 g/kg for rabbits were reported (Anslow et al. 1948). The dermal LD₅₀ of 20 ml/kg TG was reported in rabbits (Union Carbide 1971). Thiodiglycol produced mild irritation to skin and moderate irritation to eyes of rabbits (Carpenter and Smyth 1946; Union Carbide 1971). It was also found not to be a sensitizer in guinea pigs (BASF AG 2005). Recently, Angerhofer, Michie, and Leach (1998 Recently, Angerhofer, Michie, and Leach (1999) conducted subacute (14-day) and subchronic (90-day) studies with TG in male and female rats and reported a no observed adverse effect level (NOAEL) for oral toxicity of 500 mg/kg/day. The U.S. Army conducted several toxicity studies, and developed provisional oral reference dose (RfD) for TG for the evaluation of environmental and health effect criteria (Reddy, Major, and Leach 2005). There were no reported studies for developmental toxicity or teratology on TG. Therefore, mammalian developmental toxicity was performed to acquire toxicity data and to determine a NOAEL for the derivation of a RfD that will be used to set remediation standards for health and environmental risk assessment.

MATERIAL AND METHODS

Chemicals

Thiodiglycol (2,2′-thiodiethanol; CAS no. 111-48-8, lot number 057 01 EQ) was procured from Aldrich Chemical, Milwaukee, Wisconsin. TG Samples for these studies were analyzed using gas chromatography with a flame ionization detector found to be 95% to 101% pure. The test substance was a clear, pale yellow liquid in appearance having a specific gravity of 1.221 at 25°C. Neat compound was used for these studies. No diluents were used with the test chemical.

Animals

Male and female Sprague-Dawley rats were obtained from the Charles River Laboratories, Raleigh, North Carolina. The animals were 4 weeks of age upon arrival at this facility and approximately 10 weeks old at the start of the mating procedure. All rats were maintained at a set temperature (65°F to 78°F) and relative humidity (40% to 70%) and with a 12-h light/dark cycle. A certified rodent chow and tap water were available ad libitum. Rats were housed individually until mating in stainless steel hanging-wire cages. A number using cage cards and ear tags uniquely identified by the animals. The details were described in the report of Houpt, Crouse, and Angerhofer (2001). Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (NRC 1996a). These studies were conducted in accordance U.S. Environmental Protection Agency (EPA) health effects test guidelines in compliance with Good Laboratory Practice (GLP) (Title 40 Code of Federal Regulations [CFR], Part 160 GLP standards).

Methods

The test material was administered orally with syringe fitted with 2-inch, 16-gauge needle to the maternal animals from the time of embryonic implantation through the period of organogenesis. It was also desirable to produce some sign of maternal toxicity in rats receiving the highest daily dosage of TG. If the development of the fetus was unaffected where maternal toxicity was observed, TG would not be regarded as a developmental toxicant in this test system. A pilot study was first performed to establish acceptable dosage levels for the main developmental toxicity study.

Pilot Study

Rats were housed together, one female to one male, for mating purposes. The morning following cohabitation, cage pads were examined for the presence of sperm plugs. The finding of a plug(s) under a cage or vaginal evidence of copulation was construed to be a positive mating. That day was then designated as the female’s day 0 of gestation (GD 0). Each positively mated female was randomly assigned using a table of random numbers to one of six treatment groups (one negative control and five dosage levels of thiodiglycol). As positively mated females were removed from the males, they were replaced with virgin females. This procedure was continued until at least 36 females had been successfully mated and all groups were filled. Six or more (due to extra mated rats found on the final mating day) mated female rats were assigned to each of six treatment groups. Dosages were selected based upon the results of a 90-day oral toxicity study for TG in rats (Angerhofer, Mitchie, and Leach 1998, 1999). Rats received 250, 500, 1000, 2000, and 5000 mg/kg/day of neat TG via the oral route with a syringe fitted with 2-inch, 16-gauge gavage needle. A concurrent negative-control group was sham-treated without a vehicle. Mated female rats were dosed with the appropriate volumes beginning on their 5th day of gestation (GD 5). Dosing continued, once daily, at approximately the same time each day, through GD 19. Daily doses were calculated based upon the individual female’s most recently recorded body weight. All rats were observed grossly at least once a day. Individual body weights for all mated females were recorded on GDs 0, 5, 9, 13, 16, and 20. Food consumption was monitored for each test and control animal by weighing feed containers on those days when the mated females were weighed. Females which died before their scheduled sacrifice day were submitted for a complete gross necropsy.

On GD 20, surviving mated female rats were euthanized by CO₂ inhalation. Each gravid uterus was excised and weighed, after which counts and location of corpora lutea, implantations, resorptions, dead fetuses, and live fetuses were recorded. Fetuses were assigned temporary individual numbers starting at the upper right uterine horn and continuing around the cervix to the upper left horn. After opening the uterus, each viable fetus was removed, examined externally, sexed, and weighed. At this time, the litter was euthanized by CO₂ inhalation. The dam was examined grossly for any structural abnormalities or pathological changes which may have influenced the pregnancy.

The lowest dosage level of TG that significantly affected group maternal weight gain or produced other outward signs of group toxicity in the pilot study was used to determine the highest dosage level for the main developmental toxicity study.

Main Developmental Toxicity Study

In the main study the same mating procedure was followed with a new batch of animals as in the pilot study. Females were identified by cage cards, which conveyed species, animal number, dosage group, and mating group (A, B, C, etc., determined by the day of mating). Females were also identified by ear tag. This regimen was continued until all treatment groups had been filled with an appropriate number of mated females. At least 25 positively mated females were randomly assigned to each of the four groups.

The dosage levels selected of neat TG chosen for the main study were 430, 1290, and 3870 mg/kg/day. The highest dosage was projected to be sufficient to produce some observable or measurable maternal toxicity while causing no more than 10% maternal deaths. The lowest dosage was designed to produce no signs of maternal or fetal toxicity. A concurrent negative control group was sham-treated without a vehicle using a 2-inch, 16-guage gavage needle.

Mated female rats were dosed via gavage with appropriate volumes beginning on the fifth day of gestation (GD 5). Dosing continued, once daily, at approximately the same time each day, through gestation day 19 (GD 19). Daily dose volumes were calculated based upon the individual female’s most recently recorded body weight.

As in the pilot study, all rats were observed grossly at least once daily. Body weights and food consumption were recorded on GDs 0, 5, 9, 13, 16, and 20. Females that died before the scheduled sacrifice day were submitted for a complete gross necropsy.

On GD 20, each surviving mated female rat was euthanized by CO₂ inhalation. The gravid uterus was excised, weighed, and examined for number and location of implantations, resorptions, dead fetuses, and living fetuses. Number of corpora lutea in each ovary was also recorded. Fetuses were assigned individual numbers starting at the upper right uterine horn and continuing around the cervix to the upper left horn. After opening the uterus, each viable fetus was removed, examined externally, sexed, and weighed. Following external examination, each fetus was placed in a numbered compartalized, plastic box in order to maintain identity as to the original uterine position. The female (dam) was examined grossly for structural abnormalities and pathological changes, especially those that might have influenced the pregnancy. After euthanizing by CO₂, even-numbered fetuses were decapitated Each head was placed in a numbered plastic vial, covered with Bouin’s fluid, capped, and allowed to fix for several weeks before examination. The even-numbered fetus and all decapitated odd-numbered fetuses were examined for soft tissue alterations (Staples 1974). The remaining fetuses in each litter were eviscerated, cleared, stained with alizarin red, and examined for skeletal changes (Staples and Schnell 1964). After staining, fetal skeletons were examined for malformations, completeness, and degree of ossification. Examination of fetal heads was accomplished by using Wilson’s sectioning technique, making a series of transverse sections viewed under a dissecting microscope to detect anomalies of the brain, nasal cavities, eyes, and palate (Wilson and Warkany 1965). Dosage groups were not made available to the examiners during any of these procedures.

Raw data were recorded on specialized forms or in a laboratory notebook; the following parameters, by test group, were considered when study results were analyzed and comparisons among test groups were made: (a) fertility index, (b) gestation index, (c) index of live fetuses, (d) resorption index, (e) index of variation, (f) index of anomaly, (g) maternal body weights, (h) maternal food consumption, (i) fetal body weights, (j) implantations per pregnancy, (k) fetuses per pregnancy, (l) dead fetuses per pregnancy, (m) resorptions per pregnancy, (n) variant fetuses per pregnancy, (o) anomalous fetuses per pregnancy, (p) fetal sex ratio (M/F).

Statistical Analysis

Maternal body weights, pregnancy parameters, litter parameters, fetal body weights, and food consumption were analyzed using a one-way analysis of variance (ANOVA) with an all pairwise multiple-comparison procedure (Dunn’s, Tukey, or Bonferroni’s). Malformations and variations among groups were compared using the chi-square statistic, with the litter selected as the unit of comparison. Females that were found not to be pregnant or died during the course of the study were not included in statistical analyses.

RESULTS

Pilot Study

Two mated females receiving 5000 mg/kg/day TG, died toward the end of the dosing period (gestation days 15 and 19). No cause of death could be determined, although one may have been an accidental dosing error. No other outward signs of toxicity were seen in any of the test animals. Body weight gains were unaffected by the treatment regimen. The mean food consumption for GDs 5 to 9 was significantly lower than controls for those females receiving TG at 5000 mg/kg/day. No consistent dose-related lesions were found at the necropsies of survivors and those animals that died during the course of the pilot study. Cesarean sections revealed no external anomalies in fetuses at any dosage level. Mean fetal weights at 5000 mg/kg/day of TG were significantly lower than the control and other treatment groups. Based upon the results of this study, the dosage levels selected for the main developmental toxicity study were 0, 430, 1290, and 3870 mg/kg/day, respectively.

Main Developmental Toxicity Study

Maternal Parameters

Maternal body weights from GDs 16 to 20 and adjusted maternal weights on GD 20 at 3870 mg/kg/day were significantly less than the controls respectively (Tab. 1). Similar to maternal body weight, maternal food consumption was significantly lower during GDs 5 to 9, 9 to 13, and 16 to 20 at 3870 mg/kg/day compared to controls (Tab. 2). An analysis of food utilization (body weight gain/food consumed) revealed significantly lower food consumption during gestation during GDs 5 to 9 and 16 to 20 at 3,870 mg/kg/day (Tab. 3).

Litter and fetal parameters data are presented in Tab. 4, 5, and 6. Pregnancy occurred in 21 (78%), 26 (96%), 23 (82%), and 23 (85%) rats in four treatment groups, respectively. One female at 3870 mg/kg/day died prior to the schedule sacrifice. The cause of death was unknown. No effects on mean number of corpora lutea, implantations, early and late resorptions, and live fetuses were observed at any treatment group. However dams at 3870 mg/kg/day showed a statistically significant increase in the number of fetuses per dam (mean). A statistically significant decrease in average fetal body weights was observed at 3870 mg/kg/day. The average fetal body weights in the other treatment groups were similar to the control group.

Fetal evaluations (litters) were based on 274 (21), 346 (26), 334 (23), and 349 (23) in the control (vehicle), 430, 1290, and 3870 mg/kg/day treatment groups, respectively. The incidence of fetal variations at 3870 mg/kg/day was increased compared to the other dosage group and control group; however, there was no statistically significant difference when compared to controls. Additionally, the number of litters with variations at 3870 mg/kg/day (22) was similar to the control (20). The number of malformations fetuses observed in litters in all groups was comparable.

DISCUSSION

TG is a contaminant of soil and surface or ground water due to the decomposition of sulfur mustard near military disposal sites (Rosenblatt et al. 1996). Available toxicity data revealed it is not very toxic acutely (Smyth, Seaton, and Fischer 1941). Exposure to this material may become hazardous among military and civilian populations and ecological receptors such as plant and wildlife. This study was performed to develop toxicity data and to determine a NOAEL for the derivation of a RfD that will be used to set remediation standards for health and environmental risk assessment.

Analysis of individual fetal data has indicated a marked, but not statistically significant, increase in the rate of variation in fetuses originating from dams that had received 3,870 mg/kg/day TG. This finding must be viewed as having little biological relevance based upon several factors. It was described as a valid argument for the litter, not the fetus, to represent the basic independent variable in developmental toxicity studies (Palmer 1978). Using the litter as the basic unit for analysis, no significant differences were found in the rate of variation among treatment and control groups. Furthermore fetal variations at 3870 mg/kg/day were not a specific type but rather a variety of normal spontaneous occurring alterations . Moreover, no dose-related increase in malformed fetuses was observed in this study. The significantly reduced fetal body weights at 3870 mg/kg/day can be attributed to the concomitant maternal toxicity (reduced maternal body weight and food consumption) observed during the dosing period. This factor, along with no increase in pre-and postimplantation death, indicates that TG, even at maternally toxic levels, does not affect gestational parameters (except fetal body weights). There are no other reports or data on reproductive or developmental toxicity of TG in either in mammals or in vitro systems. It has been reported that other glycols such as ethylene glycol (EG) and propylene glycol (PG) when given in water did not produce reproductive toxicity in mice and rats (Gulati, Barnes, and Welch 1985, Lamb et al. 1985; LaKind et al. 1999). EG produced minimal maternal toxicity and teratogenic effects in CD-1 mouse by employing nose only exposure at 2500 mg/m³(Tyl et al. 1995a). However exposure of pregnant CD-1 mice to EG by occluded cutaneous application resulted in minimal or no observable maternal or developmental toxicity at 3,549 mg/kg/day during organogenesis (Tyl et al. 1995b).

In conclusion, the findings obtained from this study indicate that for the oral administration of TG to pregnant rats, both the maternal and developmental toxicity NOAELs were 1290 mg/kg/day. The lowest observed adverse effect level (LOAEL) was 3870 mg/kg/day.

It is recommended that another developmental toxicity study be performed with TG on a second species, namely the rabbit, to provide more information.