Registration Dossier

Administrative data

Endpoint:
basic toxicokinetics
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: This study was conducted in accordance with GLP as well as comparable to guidelines and sufficient data is available for the interpretation study results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1983
Report Date:
1983

Materials and methods

Objective of study:
other: In order to develop an understanding of the biological processes which may influence butylene oxide toxicity, a series of in-vivo studies were conducted in rats.
Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 417 (Toxicokinetics)
GLP compliance:
yes
Remarks:
testing lab.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 1,2 Butylene Oxide
- Analytical purity: 98 % by gc/ms
- Lot/batch No.: #167
1-C14 labeled material- Specific activity= 5.6 mCi/mmol, Lot #NA 9181, purity >98% via gc/ms
Uniformly labeled material- Specific activity= 7.9 mCi/mmol, I.D. #906460, purity >98% via gc/ms

Radiolabelling:
yes
Remarks:
14C-butylene oxide (both uniformly labeled and specifically labeled at the C1 position

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male
Details on test animals and environmental conditions:
Male CDF Fischer 344 rats (Charles River Breeding Laborataries, Inc., Portage, MI) weighing 200-300 grams were employed in the present studies. This strain was chosen because it had been previously employed in the 90 day inhalation study of Miller et al. (1980). Animals were acclimated to the laboratory at least 7 days before being randomized by reference to a random number table and placed on test. Animals were maintained in rooms designed to maintain approximately 22C and 50% relative humidity with controlled lighting between 7 am and 7 pm each day. Certified Purina Lab Chow and municipal drinking water were provided ad libitum to the animals except during inhalation exposures.

Administration / exposure

Route of administration:
other: inhalation- vapor; oral-gavage
Vehicle:
other: inhalation-no vehicle; oral-corn oil
Details on exposure:
Balance Study (1-C14 Labeled BO).
Four male rats/group were acclimated to Roth metabolism cages for approximately 48 hours. The animals were then placed in a 30 liter dynamic inhalation chamber for exposure to 14C-BO (Butylene oxide) vapor. Flow through the exposure chamber was maintained at approximately 7 liters/min. Aliquots of the chamber atmosphere were analyzed for BO (by gas chromatography) or radioactivity (by bubbling through ACS scintillati.on fluid). Exposures were 6 hours in length and were initiated between 10':30 and 11:30 am. Specific radioactivity of the BO in the inhalation chamber after dilution with unlabeled BO was 3.65 x 10 exp -2 mCi/mmol (50 ppm exposure) or 2.49 X 10 exp -3 mCi/mmol (1000 ppm exposure). Concentrations.of BO in the chamber were determined by g.c. as 52.4 +/- 1.7 (50 ppm) and 992 +/- 32 (1000 ppm).

Balance Study [Uniformly labeled BO (UL-BO)].
This experiment was conducted as outlined above except that inhalation and gavage doses were compared. Three male rats were exposed to 50 ppm BO for 6 hr (inhalation) and two male rats were gavaged with 20 mg/kg BO in corn oil. Specific radioactivities of the administered BO were 1.59 x 10 exp-1 mCi/mmol (inhalation) and 1.98 x10 exp-2 mCi/mmol (gavage). The concentration of BO in the inhalation expousre was determined by g.c. to
be 50.2 +/- 2.2 ppm.

Steady State Uptake Study.
A plethesmograph/head-only exposure device, as described by Landry et al. (1982), was employed to measure steady state uptake rates in rats exposed to 50 or 1000 ppm of BO. Animals were placed in wire mesh restraining tubes to support the body of the rat. The rats head was exposed in an acrylic plastic chamber. The conical-shaped exposure chamber was designed to micimize dead volume and rebreathing of the test atmosphere. A rubber diaphragm was placed around the rat's neck to seal the exposure chamber from the plethysmograph chamber. Dual exhaust manifolds prevented the rat from blocking the chamber exhaust.
BO vapor of the appropriate concentration was pumped into the conical-shaped exposure chamber at the rate of 300 ml/min. (Experiments by Landry et al, 1982, indicate that this flow rate is adequate to supply the animal's respiratory needs.)

Glutathione (GSH) Depletion.
Two experiments to estimate GSH depletion were conducted. In the first experiment, 6 male rats were exposed to 2000 ppm BO for 6 hr. Five additional rats of the same age were placed in an inhalation chamber but were not exposed to BO vapor (controls). All animals were deprived of food and water during the exposure period, and all exposures were initiated between 9:00 and 11:00 am. In the second experiment, 6 male rats were exposed to'1000 ppm BO for 6 hr, 6 male rats were exposed to 400 ppm BO, and 6 rats served as controls.
Duration and frequency of treatment / exposure:
Balance Study (1-C14 Labeled BO)- 6 hour exposure

Balance Study [Uniformly labeled BO (UL-BO)]- 6 hour exposure (inhalation) or single gavage dose

Steady State Uptake Study- up to 105 minutes
Doses / concentrations
Remarks:
Doses / Concentrations:
Balance Study (1-C14 Labeled BO)- target concentration of 50 or 1000 ppm, actual concentration 52.4 +/- 1.7 and 992 +/- 32 ppm

Balance Study [Uniformly labeled BO (UL-BO)]- target concentration 50 ppm (inhalation) or 20 mg/kg (gavage), actual concentration 50.2 +/- 2.2 ppm (inhalation)

Steady State Uptake Study- 50 or 1000 ppm
No. of animals per sex per dose:
Balance Study (1-C14 Labeled BO)- 4 male rats/group

Balance Study [Uniformly labeled BO (UL-BO)]- 3 male rats for inhalation and 2 male rats for oral gavage

Steady State Uptake Study- 3 male rats/concentration
Control animals:
other: Yes for glutathione depletion study
Details on study design:
Three types of experiments were conducted in these studies:
1. Balance Studies: 14C-BO was used to determine the major routes and rates of clearance following 6 hr exposure to either 50 or 1000 ppm BO.
2. Uptake Studies: Rats were placed in flow-through chambers and exposed to either 50 or 1000 ppm BO for several hours. Tidal volumes, respiratory frequencies, and concentrations of BO in the chamber supply and exit flows were measured, and the steady state uptake of BO was calculated from these values.
3. Glutathione Depletion: Non-protein sulfhydryl concentration of liver tissue was determined following exposure of rats to 400, 1000, or 2000 ppm BO (6 hr).
Details on dosing and sampling:
Balance Study (1-C14 Labeled BO)
Immediately following exposure, animals were placed in uniquely identified individual metabolism cages and maintained for 66 hr. Room air was drawn through the cages at 300-500 ml/min, passed over activated charcoal, and then bubbled through a CO2 trap. Details of the trapping procedure have been described elsewhere (Reitz et al., 1982). Samples of these traps as well as samples of urine and feces were collected at the following
times :

Charcoal - 0-6, 6-18, 18-36, 36-52, and 52-66 hr
CO2 - 0-18, 18-36, 36-52, and 52-66 hr
Urine - 0-24, and 24-66 hr
Feces - 0-66 hr
After 66 hr in the Roth cage, the animals were sacrificed and samples of liver, kidney, lungs, skin and carcass homogenate were obtained. Radioactivity in these fractions was determined as described elsewhere (Reitz et al., 1982).

Balance Study [Uniformly labeled BO (UL-BO)].
Animals receiving UL-BO by gavage were immediately placed in the Roth metabolism cages, while animals exposed to UL-BO vapor were maintained in the exposure chamber for 6 hours and then transferred to Roth cages. In this experiment, animals were maintained for 32 hr in the Roth cages before sacrifice.

Steady State Uptake Study
During exposure the tidal volume, respiratory rate, and concentrations of BO in the supply and exit lines from the chamber were monitored. Measurements were made after the animals had been in the chamber for approximately 45, 75, and 105 minutes. Preliminary experiments demonstrated that steady state was achieved within 30-45 minutes during BO exposure. The dose to the animal (material absorbed and not subsequently exhaled as unchanged BO) at steady state conditions was calculated from the difference in the concentrations of BO in the supply and exit streams. Three rats were studied at each concentration.

Glutathione (GSH) Depletion.
Immediately following exposure, animals were sacrificed and samples of liver and kidney tissue from each animal were analyzed for non-protein sulfhydryl (NPSH) content according to the method of Sedlak and Lindsay (1968).
Statistics:
Treatment.means were compared to appropriate control groups with a computer program which performs Bartlett's test for homogeneity of variance (Winer, 1971). If variances were homogeneous, a one way analysis of variance (ANOVA) was performed, followed by Dunnett's test if the ANOVA was significant.(p less than 0.1). If variances were non-homogeneous, treatment groups were compared .to controls using a nonparametric ANOVA followed by multiple comparisons as described by Hollander and Wolfe (1973).

Results and discussion

Toxicokinetic / pharmacokinetic studies

Details on absorption:
Steady-State Uptake of BO.
The net uptake of BO was calculated as mg BO/kg body wt. Animals exposed to 50 ppm BO absorbed material at the rate of 0.0433 mg/kg/min , while animals exposed to 1000 ppm BO absorbed material at the rate of 0.720 mg/kg/min. Since steady state is rapidly established during BO exposure, these uptake rates may be used to estimte the total uptake during a six hour exposure as 15.6 and 252 mg/kg respecti.vely.
The average chamber airflow rate during the experinent, the concentrations of BO in the supply and effluent streams, and the body
weights are reported as the mean value for all of the animals in a dose group. Net uptake, respiratory frequency, tidal volumes, and
percentage retention were calculated from individual animal data and then the calculated values were combined for tabluation.
The expired concentration (Cexp ) is calculated according to the following equation:
1-[(Cin-Cout ) x (Flow)/(Cin x Min Vol)
where Cin is the concentration of BO entering the chamber, Cout is the concentration of BO exiting the chamber, Flow is the flow rate in ml/min, and Min Vol is the product of respiratory frequency and tidal volume.
Animals exposed to 1000 ppm BO breathe more slowly and more shallowly than animals exposed to 50 ppm BO. These effects appear to be associated with a slightly higher relative retention of BO at 1000 ppm. The ratio of expired BO concentration to inspired BO concentration was 0.647 +/- 0.018 at 50 ppm, but dropped to 0.586 +/- 0.046 at 1000 ppm.
Details on excretion:
14C-Balance Studies.
Butylene oxide administered by either inhalation or gavage was rapidly metabolized and eliminated from the body. Major routes of elimination involved the formation of non-volatile urinary metabolites as well as expired carbon dioxide. Most of the material recovered in the urine (50 ppm- 44.4% and 1000 ppm- 53.4%), absorbed in the carbon dioxide trap (50 ppm- 33.6% and 1000 ppm- 26.8%), or absorbed on the charcoal trap (50 ppm- 4.86% and 1000 ppm- 5.4%) was excreted during the first 36 hr post exposure. Small amounts (less than 10%) of radioactivity were retained in the carcass homogenate after 32 or 66 hr. Minor amounts of radioactivity were recovered in the charcoal trap designed to recover unchanged 14C-BO.

When balance studies were conducted with C1-labeled BO (Cl-BO) and uniformly labeled 14C-BO (UL-BO) significant differences were noted in
the percentage of administered radioactivity recovered in the urine and carbon dioxide fractions. After exposure to Cl-BO, 40-46% of the administered label was recovered in the 0-24 hr urine (total 14C recovered in urine was 44-54%), but only 12% of the recovered radioactivity was recovered in the urine after administration of UL-BO. The carbon dioxide trap contained 27-33% of the recovered label following administration of C1-BO but about 60% of the label was trapped as carbon dioxide following administration of UL-BO. Only small changes in the disposition of BO were observed over the concentration range of 50-1000 ppm. Increasing the concentration of BO slightly increased the percentage of radioactivity which was recovered as urinary metabolites, while slightly decreasing the production of 14C-carbon dioxide. Urinary radioactivity increased from 44% to 53%. while 14C-carbon dioxide declined from 34% to 27% over this exposure range. No other remarkable changes were seen. A small number of animals were gavaged with a known amount of 14C-BO at approximately
the same dose received by animals inhaling 50 ppm BO for 6 hr. Total recovery of radioactivity in this experiment was 93.2 +/- 0.9% .
The routes of elimination of BO administered by gavage were very similar to the routes of elimination following inhalation exposure

Metabolite characterisation studies

Metabolites identified:
yes
Details on metabolites:
14C-Balance Studies.
The urinary metabolites obtained from animals exposed to 14C-BO were partially characterized by HPLC. Two major peaks (representing 35% and 53% of recovered radioactivity respectively) were seen in the urine obtained from animals exposed to either C1-BO or UL-BO. The remainder of the radioactivity (12%) eluted more slowly than the two major peaks, and was chromatographically diffuse. The chromatographic profiles of urine radioactivity from C1-BO and UL-BO exposed animals were identical within experimental error.

Any other information on results incl. tables

Glutathione Depletion.

Mean NPSH concentrations in the liver and kidney tissue of rats exposed to BO were depressed in a dose-related manner. NPSH in the liver and kidney tissues was significantly less than control values (p less than 0.05) following exposure to either 2000 ppm BO (approximately 65% depression) or 1000 ppm BO (approximately 35% depression). Exposure to 400 ppm BO decreased NPSH to a much lesser extent. NPSH was decreased 10% in liver tissue, and NPSH was decreased 11.3% in

kidney tissue. The decrease in NPSH in the kidney tissue was statistically significant (p less than 0.05), but the NPSH content of liver tissue from animals exposed to 400 ppm BO was not statistically different from that of control animals.

Steady state uptake of 1,2-butylene oxide (BO) during exposure to 50 or 1000 ppm. N=3 for all values reported.

50 PPM 1000 PPM

Chamber Flow 294 +/- 4.9 308 +/- 4.4

(ml/min)

Mean Conc BO 50.2 +/- 0.7 986 +/- 15

(Supply, ppm)

Mean Conc BO 37.5 +/- 1.0 788 +/- 27

(Exit, ppm)

Mean Body Wt 253 +/- 6.4 249 +/- 5.8

(grams)

Steady State Uptake 0.00433 +/- 0.0024 0.720 +/- 0.120

(mg/kg min)

Resp. Freq. 167 +/- 19 141 +/- 17

(Cycles/rnin)

Tidal Volume (ml) 1.27 +/- 0.15 1.06 +/- 0.08

Minute Vol. 210 +/- 2 149 +/- 7

(ml /min)

Ratio: 0.647 +/- .018 0.586 +/- 0.046

Expired/Inspired

Data from individual animals averaged after calculations have been performed.

Concentration expired calculated as outlined in methods section.

DISCUSSION

Basic disposition studies for 14C-labeled BO in rats have been conducted. These studies revealed that BO was readily absorbed following either inhalation or gavage. Following absorption, BO was extensively metabolized. Within 36 hr, 80-90% of the administered radioactivity could be recovered as either 14-carbon dioxide or urinary metabolites. Preliminary characterization of the urinary metabolites by HPLC suggested that there were two species of urinary metabolite formed following administration of 14C-BO.

Small amounts of residual radioactivity were detected in the liver (3% of dose) and carcass homogenates (8-9% of'dose) of animals 66 hr after exposure to 14C-BO . Since large amounts of the administered BO were metabolized to radioactive carbon dioxide, a portion of this residual radioactivity has undoubtedly been incorporated

into normal tissue components following labeling of the biosynthetic pools with radioactive C-1 fragments. It is not possible to determine whether any of the residual radioactivity has arisen from direct alkylation of biological macromolecules by BO with the techniques employed in the current study.

Further information about the disposition of BO may be obtained from comparison of the results obtained with C-1 labeled BO (Cl-BO) and uniformly-labeled BO (UL-BO) . None of the radioactivity recovered in the urine was volatile. Consequently, this radioactivity must represent either metabolic fragments of BO or conjugation products of BO (or a combination of these). Furthermore, the radioactivity recovered in the urine following administration of Cl-BO was chromatographcally indistinguishable from that recovered following administration of UL-BO (two major peaks with identical chromatographic behavior were seen in

each case, and the percentages of urinary radioacJivity recovered in each of these peaks was identical.

However, although the radioactive material recovered in the uri.ne behaved identically following C1-BO or UL-BO the percentage of the total dose administered to the animals which was recovered in the urine was dramatically different between Cl-BO and UL-BO. Almost half (44-53%) of the total radioactivity admbistered to the animals as C1-BO was recovered as urinary metabolites. In contrast, only 12% of the total radioactivity administered was recovered as urinary metabolites following administration of UL-BO. The decrease in the percentage of administered radioactivity recovered in the urine following UL-BO was accompanied by an increased recovery of label as radioactive carbon dioxide; 57.5% of the radioactivity administered as UL-BO was recovered as carbon dioxide, while 34-27% of administered radioactivity was recovered as carbon dioxide following exposure to Cl-BO.

The labeling ratio (ratio of the percentages of administered dose recovered in various fractions followig CL-BO and UL-BO) observed in, these experiments for urinary metabolites (C1-BO/UL-BO), is approximately 4. This is consistent with the hypothesis that the urinary metabolites contain only the label, associated with the

C1-atom of 1,2-butylene oxide (BO), and thus cannot be simple conjugates of BO (e.g. with glutathione, etc.) . This hypothesis is supported by the observation that the "extra" radioactivity recovered as carbon dioxide following UL-BO is approximately equal to that "missing" from the urinary metabolites.

This labeling pattern is exactly what one would expect if BO initially formed a primary conjugate with some "acceptor" molecule, and this conjugate were then further degraded by side chain oxidation. However, the data presented here are also consistent with other pathways of metabolism, and futher experiments are required to clarify this point.

It is noteworthy that the metabolic fate of 14C-BO does not appear to be dose-dependent. Examination of the data in indicates that very similar percentages of radioactivity are recovered in the various fractions after exposure to either 50 ppm BO or 1000 ppm, BO. This implies that the major metabolic processes involved in BO disposition follow linear pharmacokinetics over the dose range studied (50-1000 ppm) .

Linearity of metabolic processes was also evident in the experiments which directly measured uptake of ion-radioactive BO. In these experiments, animals were forced to breath from a supply stream containing a known concentration of EO (either 50 or 1000 ppm BO). The breathing chamber was designed so that all gas exhaled by the animal was recovered in the exit stream. A specially designed plethysmograph was used to measure the respiratory frequency and minute volume during exposure according to the techniques described by Landry et al. (1982). Consequently, it is possible to determine the rate of uptake of BO before and during steady state inhalation exposure.

The rate of uptake of BO increased almost proportionately with exposure .concentration over the range studied. The small decrease in uptake observed (16.2 fold increase observed versus 20 fold expected) may have resulted from the fact that the higher concentration of BO appeared to be somewhat irritating to the animals, causing a significant reduction in both respiratory frequency and tidal volume. It is possible .to estimate the ratio of BO in inspired and expired air, (once steady state-has been achieved) as outlined earlier (Results section). Calculation of the concentration of BO in expired air for individual breaths involves the assumption that loss of BO occurs only in the,volume of air inspired by the animal. This appears to be a valid assumption, because when a glass plug was substituted for the head of a

living animal, the concentration of BO in input and exit stream differed by less than 2%. It is interesting to note that the decreased respiratory frequency and lower tidal volume observed at the higher concentration of BO appeared to result in a lower relative concentration of expired BO during exposure to 1000 ppm BO.

Glutathione (GSH) appears to play an important role in the detoxification of reactive materials in living animals. It has been previously reported that non-protein sulfhydryl groups (NPSH) (which approximate GSH levels in many tissues) were decreased 65-81% in rats and mice exposed to levels of ethylene oxide (EO) from 500-900 ppm for 4-5 hr (Tyler and McKelvey, 1980). Propylene oxide (PO) produced a similar effect on NPSH in experiments conducted by Nolan et al. (1980).

Exposure to 625 ppm PO for 6 hr depressed NPSH in the liver by 54%. BO also depresses NPSH during 6 hr exposures. However, it appears to be considerably less potent than either EO or PO in this respect. Exposure to 400 ppm BO (6 hr) failed to significantly depress liver NPSH, while exposure to 1000 ppm only caused a 35% depression in liver NPSH. Not until the concentration of BO was raised to 2000 ppm were depletions of GSH similar to those seen with 500-600 ppm of EO and

PO observed.

These results suggest that if conjugation of BO with GSH is an important detoxification mechanism in rodents, it is less likely to be overwhelmed by exposure to BO than exposure to EO or PO. Furthermore, if the results from a single exposure can be considered predictive of those seen during chronic exposures, the doses chosen by the NTP for their bioassay of BO in rats (400 and 200 ppm) would not be expected to seriously deplete endogenous reserves of GSH during exposure.

In conclusion, although BO is a member of a class of reactive chemicals (epoxides), it appears. to have significantly less biological activity than either EO or PO. This is reflected by the relative toxicities of these materials (EO greater than PO greater than 'BO; Fox et al., 1983) as well as their effects in depleting endogenous GSH.

Applicant's summary and conclusion

Conclusions:
Butylene oxide (1,2-epoxybutane, BO) is extensively metabolized and rapidly eliminated following either inhalation exposure or gavage in CDF Fischer 344 male rats. Acute exposures of CDF Fischer 344 males to concentrations of BO of 2000, 1000, and 400 ppm caused a dose-related depletion of non-protein sulfhydryl groups (NPSH) in liver and kidney tissue. Steady-state uptake rates of BO were determined to be 0.0433 mg/kg/min at 50 ppm and 0.720 mg/kg/min at 1000 ppm. These rates correspond to an estimated uptake of 15.6 mg/kg and 252 mglkg during a six hour exposure.Based on the results of this study it appears that the physical and biological processes involved in absorption, metabolism and elimination of BO are essentially linear throughout the exposure range of 50-1000 ppm.