N rats and B6C3F1 mice after inhalation exposure to 1,2-epoxybutane

Toxicology, 50 (1988) 6 9 - 8 2 Elsevier Scientific Publishers Ireland Ltd.

R E S P I R A T O R Y T R A C T L E S I O N S IN F344/N RATS AND B6C3F 1 M I C E A F T E R I N H A L A T I O N E X P O S U R E TO 1,2-EPOXYBUTANE

J U N E K. DUNNICK ".', SCOT L. EUSTIS', WALTER W. PIEGORSCH a and RODNEY A. MILLER b

°National Institute of Environmental Health Sciences, National Toxicology Program, P.O. Box 12233, Research Triangle Park, NC 27709, and bBattelle, Pacific Northwest Laboratories, P.O. Box 999, Richland, WA 99352 (U.S.A) (Received July 14th, 1987) (Accepted October 7tb, 1987)

SUMMARY

1,2-Epoxybutane, a short-chain epoxide used as a stabilizer in chlorinated hydrocarbon solvents, was administered by inhalation exposure as a vapor 6 h/day, 5 day/week, for 24 months at exposure concentrations of 0, 200 or 400 ppm to F344/N rats and 0, 50, or 100 ppm to B6C3F 1 mice. Survival of all groups of rats was 50% or greater until week 98 but was reduced in exposed groups by the end of the study. Survival in male mice was comparable among groups. Survival in female mice was greater than 50% until week 86, but was then reduced in the high-exposure group of mice. Exposure-related inflammatory, degenerative, and proliferative lesions occurred in the nasal cavity of both rats and mice. Seven papillary adenomas Occurred in the nasal passages of high-exposure male rats and 2 in the nasal passages of highexposure female rats. Alveolar/bronchiolar adenoma or carcinoma {combined} occurred with increased incidence in exposed male rats relative to controls. No exposure-related neoplastic lesions were seen in mice. After inhalation exposure, 1,2-epoxybutane was carcinogenic in rodents as were other epoxides or related compounds including propylene oxide, 1,3-butadiene, and ethylene oxide. The site of carcinogenic activity was considered to be related to length of the carbon chain.

Key words: 1,2-Epoxybutane; Nasal lesions; F344/N rat; B6C3F 1 mouse; Inhalation exposure

*To whom all correspondence and reprint requests should be addressed. Printed and Published in Ireland

69

INTRODUCTION 1,2-Epoxybutane (synonym: 1,2-butylene oxide) is a short-chain epoxide that is used primarily as a stabilizer in chlorinated hydrocarbon solvents. This compound was selected for study as a representative of the short-chain epoxides, because it had a relatively high production of 8 000 000 pounds/ year in the U.S., and because there were no carcinogenicity data available [1]. Epoxides are known for their carcinogenicity in rodents [2,3]. Studies done by the National Toxicology Program (NTP) confirm previous findings which showed that 1,2-epoxybutane is a mutagen [4]. 1,2-Epoxybutane was mutagenic in Salmonella typhimurium strains, induced mutations in mouse lymphoma cells, and caused chromosomal aberrations and sister chromatid exchanges in Chinese hamster ovary cells (all effects were seen with and without metabolic activation). Sex-linked recessive mutations were seen in male Drosophila fed 1,2-epoxybutane [4]. This paper describes the respiratory tract lesions produced in F344/N rats and B6C3F 1 mice after inhalation exposure to 1,2-epoxybutane, and compares the carcinogenic activity of 1,2-epoxybutane with other epoxides and related compounds including propylene oxide, ethylene oxide, and 1,3-butadiene. All compounds were administered by the inhalation route 6 h/day, 5 days/ week to minic potential exposure in the work place. Chamber concentrations selected were based on the results of 13-week studies in which mice and rats received exposures from 50 to 800 ppm [4]. In these studies all mice exposed to 800 ppm died and mice exposed to 200 ppm and above had inflammation of the nasal cavity that was considered to be potentially life-threatening in exposures lasting up to 2 years. All rats survived the 13-week exposure to 800 ppm but developed inflammation of the nasal cavity at this concentration, but not at concentrations of 400 ppm or lower. Based on these results the high concentrations selected for the 2-year study were 100 ppm in mice and 400 ppm in rats [4]. MATERIALS AND METHODS

Animals Male and female F344/N rats and B6C3F 1 mice were obtained from Charles River Breeding Laboratories, Portage, MI. At the start of the chronic study animals were 8--10 weeks of age. Animals were assigned to control, low, or high-exposure groups using a table of random numbers. Each exposure group contained 50 animals/sex per species at the start of the study; these animals were housed individually in stainless steel wire cages in Battelle-designed chambers (Hartford Systems/Lab Products, Inc., Aberdeen, MD) throughout the study. Tap water was provided ad libitum, and NIH 07 Open Formula (Zeigler Brothers, Gardners, PA) was provided ad libitum except during the exposure periods. Animals were maintained at a temperature of 22 _+ 2°C with a humidity of 43--69%. A 12-h fluorescent light cycle was used throughout the study, and room airflow during the non-

70

exposure perios was 20 air changes/h. All animals were checked twice daily for morbidity and mortality. Moribund animals were killed and necropsied. Clinical signs and body weights were recorded every 4 weeks.

Chemical and exposure system 1,2-Epoxybutane (Cas. No 106-88-7; synonym: 1,2-butylene oxide) was obtained from the Dow Chemical Company, Richmond, VA (lots MM10258 and RR810402). The chemical was vaporized at approximately 58°C to produce chamber concentrations of 0, 200, or 400 ppm (598-1178 mg/m a) for rats and 0, 50 or 100 ppm (147--294 mg/m 3) for mice. The liquid to be vaporized was contained in a 1.6-1 stainless steel reservoir housed in a vapor hood in the exposure room. The liquid was pumped from this reservoir to a stainless steel cylinder covered with a glass fiber wick from which the liquid was vaporized. An 80-W heater and a temperature-sensing element were incorporated within the cylinder. The heater maintained the vaporizer at approximately 58°C. To minimize material loss due to condensation on duct walls, each cylindrical Vaporizer was positioned in the fresh air duct leading directly into the exposure chamber. A photoionization detector and a gas chromatograph equipped with a flame ionization detector were used to monitor mean daily exposure concentrations. The vaporized chemical was greater than 98% pure [4]. Filtered room air was used in the control chamber. Pathology Complete necropsies were done on all animals, unless precluded by autolysis or cannibalism. Thus, the number of organs or tissues examined microscopically varied and did not necessarily equal the number of animals started on the study. Tissues were preserved in 10% neutral buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. The following tissues were examined microscopically: gross lesions and tissue masses, lymph nodes (mediastinal and tracheo-bronchial), salivary gland, mandibular lymph node, sternebrae including marrow, thyroid gland, parathyroids, small intestine, rectum, colon, mammary gland, prostate/testes/ epididymis or ovaries/uterus, lung and mainstem bronchi, nasal cavity and nasal turbinates, heart, esophagus, stomach, brain, thymus, trachea, pancreas, spleen, kidneys, adrenal glands, urinary bladder, pituitary gland, skin, clitoral or preputial gland (rats), and gallbladder (mice). Statistics Differences in survival were analyzed by life table methods [5]. Tumor incidence data were analyzed by survival-adjusted methods [6] and by Fisher's exact tests and Cochran-Armitage trend tests based on the overall proportion of tumor-bearing animals [7]. Non-neoplastic incidence data were analyzed by Fisher's exact test.

71

RESULTS

Body weights and survival Survival of all groups of rats was at least 50% until week 98 but survival after this time was reduced in exposed groups (final survival - male rats control (C), 30/50; low-exposure (LE); 18/50; high-exposure (HE) 23/50; female rats -- C, 32/50; LE, 21/50; HE, 22/50). Survival in male mice was comparable among groups (final survival -- C, 41/50; LE, 45/50; HE, 33/50). Survival in female mice was greater than 50% until week 86 and was then reduced in the high-exposure group (final survival - C, 29/50; LE, 25/50; HE 9/50). Suppurative inflammation of the ovary and uterus was found in female mice dying early and Klebsiella was cultured from some of these lesions. Initial and final body weights are summarized in Table I.

Pathology- rats Inhalation exposure of male and female rats to 1,2-epoxybutane caused a spectrum of inflammatory lesions in the nose that were accompanied by degenerative and proliferative lesions of the mucosal epithelium and adjacent tissue (Table II). The inflammation varied from the accumulation of TABLE

I

MEAN BODY WEIGHTS AND SURVIVAL IN F344/N RATS AND B6C3F 1 MICE EXPOSED TO 1,2-EPOXYBUTANE FOR 2 YEARS"

Exposure

Initial body

Final body

(ppm)

weight (g)

weight (g)

Final survival

191 190 191

446 423 427

30/50 18/50 b 23/50

137 136 134

342 317 311

32/50 21/50 b 22/50

24 24 24

37 34 33

41/50 45/50 33/50

20 20 20

34 30 30

29/50 25/50 9/50 °

Male rats

0 200 400 Female r a t s

0 200 400 Male mice

0 50 100 Female mice

0 50 100

"Body weight is average of surviving animals. bp < 0.05 VS. control. cp < 0.01 vs. control.

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T A B L E II E X P O S U R E - R E L A T E D N O N - N E O P L A S T I C A N D N E O P L A S T I C LESIONS IN T H E N A S A L C A V I T Y A N D L U N G OF F344/N R A T S E X P O S E D TO 1,2-EPOXYBUTANE F O R 2 Y E A R S •

Lesions

Incidence Male rat

Female rat

0 ppm 200 ppm

400 ppm

0 ppm 200 ppm

400 ppm

9 2 10 0 8 0 4 0

36¢ 28c 37¢ 2 38¢ 0 22¢ 0

42¢ 36c 49° 11° 46¢ 5b 40¢ 7b

25 0 6 0 5 0 1 0

32 18¢ 26¢ 2 29~ 0 14e 0

43¢ 31¢ 45¢ 16e 4V 2 36¢ 2

0

18¢

12~

0

13c

8¢

Epithelial hyperplasia Adenoma Carcinoma

5 0 0

5 1 1

8 1 4

2 1 1

6 0 0

7b 1 0

Adenoma or carcinoma

0

2

5b

2

0

1

Nasal cavity Inflammation (unspecified) Serious inflammation Suppurative inflammation Hyperostosis Epithelial hyperplasia Adenomatous hyperplasia Squamous metaplasia Papillary adenoma

Olfactory sensory epithelium Atrophy

Lung {alveolarforonchiolar]

IBased on 50 animals examined in each group; lung tumor rates for males at 400 ppm based on 49 animals. bp < 0.05 VS. control. cp < 0.01 vs. control.

eosinophilic p r o t e i n a c e o u s m a t e r i a l in the l u m e n (serious inflammation) to i n f i l t r a t i o n of t h e m u c o s a b y n e u t r o p h i l s ( s u p p u r a t i v e i n f l a m m a t i o n ) a n d l y m p h o c y t i c a n d m a c r o p h a g e i n f i l t r a t e s ( u n s p e c i f i e d i n f l a m m a t i o n - - F i g . 1). E p i t h e l i a l h y p e r p l a s i a c o n s i s t e d of d i f f u s e c r o w d i n g of e p i t h e l i a l cells a n d i n c r e a s e d t h i c k n e s s of t h e r e s p i r a t o r y e p i t h e l i u m w i t h i n c r e a s e d n u m b e r s of g o b l e t cells (Figs. 2,3). F o c a l n o d u l a r p r o l i f e r a t i o n of r e s p i r a t o r y e p i t h e l i u m f o r m i n g g l a n d - l i k e s t r u c t u r e s w a s d i a g n o s e d as a d e n o m a t o u s h y p e r p l a s i a (Figs. 4,5). S q u a m o u s m e t a p l a s i a w a s a focal o r m u l t i f o c a l l e s i o n t h a t o c c u r r e d f r e q u e n t l y w i t h i n t h e r e s p i r a t o r y e p i t h e l i u m , e s p e c i a l l y in t h e a n t e r i o r s e c t i o n of t h e n a s a l c a v i t y (Fig. 1). A t r o p h y of t h e o l f a c t o r y s e n s o r y e p i t h e l i u m was o b s e r v e d at i n c r e a s e d incidence in exposed groups. H y p e r o s t o s i s of t h e n a s a l t u r b i n a t e b o n e s c o n s i s t i n g of p e r i o s t e a l cell proliferation and n e w bone formation, was o b s e r v e d at increased f r e q u e n c y in high-exposure male rats. Nasal papillary a d e n o m a s s e e n in exposed male and female r a t s w e r e

73

Fig. 1. Nasal mucosa of a male rat exposed to 400 ppm of 1,2-epoxybutane for 2 years. The respiratory epithelium has been replaced by a stratified squamous epithelium (squamous metaplasia) and the underlying submucosa contains acute and chronic inflammatory cells. Hematoxylin and eosin, original magnification 50 × .

Fig. 2. Nasal mucosa of a male r a t exposed to 400 ppm of 1,2-epoxybutane for 2 years. There is hyperplasia of the respiratory epithelium with increased numbers of goblet cells and formation of irregular folds. Hematoxylin and eosiu, original magnification 40 × .

74

Fig. 3. Nasal mucosa of a male r a t exposed to 400 ppm of 1,2-epoxybutane for 2 years. There is regenerative hyperplasia of the epithelium with formation of intra-epithelial cysts and chronic inflammation in the submucosa. Hematoxylin and eosin, original magnification 50 x .

Fig. 4. Nasal mucosa of a male r a t exposed to 400 ppm of 1,2-epoxybutane for 2 years. The normal ciliated columnar respiratory epithelium is replaced by a non-ciliated cuboidal to columnar epithelium t h a t is arranged in irregular gland-like structures. Hematoxylin and eosin, original magnification 40 x .

75

Fig. 5. Nasal mucosa of a male rat exposed to 400 ppm of 1,2-epoxybutane for 2 years. The respiratory epithelium is replaced by a nodular proliferation of epithelial cells. The cells show moderate atypia and are less differentiated than the normal respiratory epithelium. Hematoxylin and eosin, original magnification 50 ×.

e x o p h y t i c p a p i l l a r y g r o w t h s of a c u b o i d a l to c o l u m n a r n o n - c i l i a t e d e p i t h e l i u m w h i c h w e r e a t t a c h e d to t h e u n d e r l y i n g m u c o s a b y t h i n s t a l k s or b r o a d b a s e s (Figs. 6,7). T h e r e w a s n o e v i d e n c e of local i n v a s i v e g r o w t h b y t h e s e adenomas.

Fig. 6. Nasal mucosa of a male rat exposed to 400 ppm of 1,2-epoxybutane for 2 years. A papillary adenoma arising from the nasal turbinate extends into the lumen of the nasal cavity. Hematoxylin and eosin, original magnification 13 x.

76

Fig. 7. Nasal mucosa of a male rat exposed to 400 ppm of 1,2-epoxybutane for 2 years. Higher magnification of the adenoma shown in Fig. 6. The epithelium is cuboidal to columnar and appears stratified in some areas. Gland-like invaginations of the epithelium form the body of the tumor. The point of origin of the tumor from the turbinate mucosa is apparent. Hematoxylin and eosin, original magnification 50 x .

Alveolar/bronchiolar carcinomas and alveolar/bronchiolar adenomas or carcinomas (combined) occurred with significant positive trends (P < 0.02) in male rats. The neoplasms seen in the lung and nasal cavity were primarily incidental findings and did not cause early death of the animals. Pathology

-- mice

Non-neoplastic lesions of the nasal cavity were seen in treated mice (Table III). Empyema and chronic inflammation were characterized by suppurative exudate in the nasal cavity and infiltration of the nasal mucosa with lymphocytes and macrophages. Erosion, respiratory epithelial regeneration and hyperplasia, and squamous metaplasia were observed at increased incidences in exposed mice. Cysts and hyperplasia of the nasal gland (Bowman's glands) suppurative and chronic inflammation and epithelial hyperplasia of the nasolacrimal duct, and atrophy of the olfactory sensory epithelium were also observed at increased incidences in exposed mice. A single squamous cell papilloma was seen in the incisive duct of one high-dose male mouse, but this finding was not statistically significant and could not clearly be related to chemical exposure. DISCUSSION

Exposure of F344/N rats and B6C3F 1 mice to 1,2-epoxybutane caused

77

TABLE III EXPOSURE-RELATED NASAL NON-NEOPLASTIC E X P O S E D TO 1 , 2 - E P O X Y B U T A N E F O R 2 Y E A R S a Lesion

LESIONS

THE

B6C3F~

MICE

Incidence

Male m i c e

Number examined

IN

Female mice

0 ppm

50 p p m

100 p p m

0 ppm

50 p p m

100 p p m

49

49

50

50

50

48

0 0 0 0 0 1 0

32 b 33 ~ 7b 15 b 32 b 24 b 0

40 b 40 b 17 b 17 b 45 b 41 b 1

0 0 0 0 1 0 0

33 b 39 b 16 b 14 b 34 b 34 b 0

40 b 44 b 24 b 15 b 35 b 41 h 0

0 0

1 l0 b

6a 24 b

0 0

9b 23 b

7b 29 b

0 0 0 0

0 6a 3 12 b

0 2 4 21 b

0 1 1 1

2 3 5 18 b

5~ 4 6 21 b

0

13 b

32 b

0

25 h

35 b

Nasal cavity Empyema Chronic inflammation Erosion Regeneration Epithelial hyperplasia Squamous metaplasia S q u a m o u s cell p a p i l l o m a

Nasal gland Cyst Hyperplasia

Nasolacrimal duct Empyema Suppurative inflammation Chronic inflammation Epithelium hyperplasia

Olfactory sensory epithelium Atrophy

~P < 0.05 vs. c o n t r o l s . bp < 0.01 vs. c o n t r o l s .

inflammatory and degenerative lesions of the nasal cavity in both species, neoplastic lesions of both the nasal cavity and lung in male rats, and neoplastic lesions of the nasal cavity in female rats, but no exposure related neoplastic lesions in mice. The benign papillary adenomas seen in the nasal cavity of the rat are rare tumors and have been seen in only 0.1% of untreated control rats (2/1977 untreated male rats; 1/2021 untreated female rats) in all NTP studies. The incidence of alveolar/bronchiolar tumors in male rats is 2% for chamber controls at this laboratory (4/249) and 0.7% for untreated controls in all NTP studies (14/1973). There is currently no ACGIH threshold limit value for 1,2-epoxybutane although the manufacturer has established an 8-h time-weighted average (TWA) of 40 ppm [1]. The highest concentration used in the 2-year rat studies (400 ppm; 1,178 mg/m 3) is 10 times this TWA, and the highest concentration used in the mouse studies (100 ppm; 294 mg/m 3) is 2.5 times this TWA. A comparison of the carcinogenic activity of 1,2-epoxybutane with other 78

epoxides or chemicals metabolized to epoxides is summarized in Table IV. Propylene oxide at exposure concentrations of 200 or 400 ppm also caused nasal cavity lesions in F344/N rats and B6C3F 1 mice: in rats the lesions included inflammation, hyperplasia, and metaplasia of the nasal respiratory epithelium and papillary adenomas; in mice the lesions included inflammation and hemangiomas or hemangiosarcomas of the nasal cavity [8,9]. In contrast ethylene oxide and 1,3-butadiene, compounds which are metabolized to epoxides [10], did not cause nasal cavity tumors in mice or rats, but did cause tumors at other sites [11--14]. In mice ethylene oxide caused exposurerelated tumors of the lung, harderian gland, uterus, mammary gland, and hematopoietic system [11]. Snellings et al. [13] exposed F344/N rats to ethylene oxide at 10, 33, or 100 ppm and found tumors of the brain, hematopoietic system (leukemia), and peritoneal mesotheliomas. Lynch et al. observed similar tumors in male F344/N rats exposed to 50 and 100 ppm ethylene oxide [15]. Mice exposed to 625 or 1250 ppm butadiene had tumors of the lung, forestomach, heart, hematopoietic system (lymphoma), mammary gland, ovary, and liver [12]. F344/N rats exposed to butadiene at 1000 or 8000 ppm developed exposure-related tumors including Leydig cell adenomas, and tumors of the brain, pancreas, mammary gland, thyroid, uterus, and Zymbal's gland ([12; unpublished information from The International Institute of Synthetic Rubber Producers, Inc. New York]). 1,2-Epoxybutane, propylene oxide, and ethylene oxide are direct acting mutagens [4,8,11] while butadiene is not [12]. Ethylene oxide, an epoxide with a chain length of 2 carbons, was not carcinogenic to the nasal cavity while 1,2-epoxybutane, a 4-chain epoxide, and propylene oxide, a 3-chain epoxide, were carcinogenic to the nasal cavity. Increasing chain length of epoxides is related to increased nasal tumorigenicity. It must also be recognized, however, that the exposures used in the rat ethylene oxide study were lower than those used in the 1,2-epoxybutane and propylene oxide rat studies, and this difference in exposure concentrations may have contributed to the difference in nasal tumor response. Ethylene oxide and butadiene caused tumors at sites other than the site of maximum initial exposure, while the carcinogenicity of 1,2~poxybutane and propylene oxide were restricted to the site of maximum initial exposure (respiratory tract). The results suggest that ethylene oxide and butadiene (or metabolites) might be more potent mutagens than 1,2-epoxybutane or propylene oxide, with the ability to cause tumors in different organ systems. In vitro mutagenicity studies by Wade et ah and Voogd et al. [16,17] suggest that the mutagenicity of epoxides decreases with increasing length of the carbon chain. Under the conditions of these 2-year inhalation studies, there was clear evidence of carcinogenic activity of 1,2-epoxybutane for male F344/N rats as shown by an increased incidence of papillary adenomas of the nasal cavity and alveolar/bronchiolar carcinomas and alveolar/bronchiolar adenomas and carcinomas (combined); there was equivocal evidence of carcinogenic activity for female rats as shown by the presence of papillary adenomas of the nasal cavity; there was no evidence for carcinogenic activity in mice. Chronic 1,279

ov

F344/N rats Male Female B6C3F 1 mice Male Female F344/N r a t s Male Female B6C3F 1 mice Male Female F344/N rats Male Female

1,2-Epoxybutane b

Ethylene oxide d

H,C ~ C o H ~ C H z

Propylene oxide ~

H2C~CH~C2H 5

Strain/ species/ sex

Chemical

0, 10, 33, 100 0, 10, 33, 100

0, 200, 400 0, 200, 400

0, 200, 400 0, 200, 400

0, 50, 100 0, 50, 100

0, 200, 400 0, 200, 400

-

+ +

+ +

-

+ +/-

Nasal cavity

m

m

÷

Lung

+ +

m

m

m

EXPOSURE

(Brain, leukemia) (Brain, leukemia)

INHALATION

Other organ systems

B6C3F I M I C E A F T E R

Site or type of neoplasm

IN F344/N R A T S A N D

Exposure concentration (ppm)

COMPOUND-RELATED CARCINOGENIC RESPONSES EPOXYBUTANE AND RELATED COMPOUNDS i

T A B L E IV T O 1,2-

oo

= CH 2

Female

-

-

625, 1250

0, 625, 1250

0,

-

0, 1000, 8000

Female

B6C3F 1 mice Male

-

-

0, 1000, 8000

0, 50, 100 0, 50, 100

F344/N rats Male

Female

B6C3FIMalemice

+

+

-

-

+ +

+

+

+

+

+ +

(Heart, lymphomas, forestomach) (Heart, lymphomas, forestomach, ovary, m a m m a r y gland, liver)

(Leydig cell adenoma) (Mammary gland, thyroid, u t e r u s zymbal gland)

(Harderlan gland) (Harderian gland, lymphomas, u t e r u s m a m m a r y gland)

•Carcinogenic response: + , p r e s e n c e of compound-related neoplasms; + / - , equivocal evidence for compound-related neoplasms; - , no evidence for compound-related neoplasms. bNTP TR 329 [4]. ~NTP TR 267 [8]. dNTP TR 326 [11] - r a t s studied by o t h e r i n v e s t i g a t o r s [13]. •N T P TR 288 [12] - s t u d y t e r m i n a t e d early; - r a t s studied by International Institute of Synthetic R u b b e r Producers.

H2C = CH - CH

Butadiene e

H2CoCH2

epoxybutane exposure was associated with non-neoplastic lesions of the nasal cavity in rats and mice. ACKNOWLEDGEMENTS

We thank Drs. Joseph K. Haseman and Gary Boorman, NIEHS, and Dr. Trent Lewis, NIOSH, for their helpful comments and review of this manuscript. These studies were conducted at Battelle Pacific Northwest Laboratories. REFERENCES 1 2

3

4 5 6 7 8 9

10 11 12 13

14

15

16 17

82

U.S. Environmental Protection Agency, 1,2-Butylene oxide; response to the Interagency Testing Committee. Fed. Regist., 49 (1984) 504. Interagency Agency for Research on Cancer (IARC}, IARC monographs on the evaluation of carcinogenic risk of chemicals to humans. Cadmium, Nickel, Some Epoxides, Miscellaneous Industrial Chemicals and General Considerations on Volatile Anaesthetics, Lyon, France, 11 (1976) 115. Interagency Agency for Research on Cancer (IARC}, IARC monographs on the evaluation of carcinogenic risk of chemicals to humans. Allyl compounds, aldehydes, epoxides, and peroxides, Lyon, France, 36 (1985) 181. National Toxicology Program, Toxicology and carcinogenesis studies of 1,2-epoxybutane in F344/N rats and B6C3F 1 mice. NTP TR 329. NIH Publication No. 87-2585, 1986. D.R. Cox, Regression models and life tables. J. R. Stat. Soc., B 34 (19721 187. J.K. Haseman, Statistical issues in the design, analysis, and interpretation of animal carcinogenicity studies. Environ. Health Perspect., 58 (1984) 385. J. Gart, K. Chu and R. Tarone, Statistical issues in interpretation of chronic bioassay tests for carcinogenicity. J. Natl. Cancer Inst., 62 (1979) 957. National Toxicology Program (NTP}, Toxicology and carcinogenesis studies of propylene oxide in F344/N rats and B6C3F1 mice. NTP TR 267, NIH Publication No. 85-2527, 1985. R.A. Renne, W.E. Giddens, G.A. Boorman, R. Kovatch, J.E. Haseman and W.J. Clark, Nasal cavity neoplasia in F344/N rats and (C57BL/6 x C3H)F1 mice inhaling propylene oxide for up to two years. J. Natl. Cancer Inst., 77 (1986) 573. E. Malvoisin and M. Roberfroid, Hepatic microsomal metabolism of 1,3-butadiene. Xenobiotica, 12 (1982) 137. National Toxicology Program (NTP), Toxicology and carcinogenesis studies of ethylene oxide in B6C3F~ mice. NTP TR 326. NIH Publication No. 86-2582, 1986. National Toxicology Program {NTPI, Toxicology and carcinogenesis studies of 1,3-butadiene in B6C3F~ mice. NTP TR 288. NIH Publication No. 84-2544, 1984. W.M. Snellings, C.S. Weil and R.R. Maronpot, A two-year inhalation study of the carcinogenic potential of ethylene oxide in Fischer 344 rats. Toxicol Appl. Pharmacol., 75 (1984) 105. J.E. Huff, R.L. Melnick, H.A. Solleveld, J.K. Haseman, M. Powers and R.A. Miller, Multiple organ carcinogenicity of 1,3-butadiene in B6C3F 1 mice after 60 weeks of inhalation exposure. Science, 227 (1985) 548. D. Lynch, T. Lewis, W. Moorman, J. Burg D. Groth, A. Khan, L. Ockerman and B. Cockrell, Carcinogenic and toxicologic effects of inhaled ethylene oxide and propylene oxide in F344 rats. Toxicol. Pharmacol., 76 (1984} 69. D.R. Wade, S.C. Airy and J.E. Sinsheimer, Mutagenicity of aliphatic epoxides. Murat. Res., 58 (1978) 217. C.E. Voogd, J.J. van der Stel and J.J.A.A. Jacobs, The mutagenic action of aliphatic epoxides. Mutat. Res., 89 (19811 269.