Environmental Health Perspectives

Vol. 86, pp. 27-36, 1990

Inhalation Toxicology and Carcinogenicity

of 1,3-Butadiene in B6C3F1 Mice Following

65 Weeks of Exposure

by Ronald L. Melnick,* J. E. Huff,* Joseph H.

Roycroft,* Billy J. Chou,t and Rodney A. Millert

1,3-Butadiene, a large-production volume chemical used mainly in the manufacture of synthetic rubber,

was found to induce multiple-organ carcinogenicity in male and female B6C3F1 mice at exposure concentrations (625 and 1250 ppm) equivalent to and below the OSHA standard of 1000 ppm. Since this study was

terminated after 60 weeks of exposure because of reduced survival due to fatal tumors, and because

dose-response relationships for 1,3-butadiene-induced neoplastic and nonneoplastic lesions were not clearly

established, a second long-term inhalation study of 1,3-butadiene in B6C3F1 mice was conducted at lower

exposure concentrations, ranging from 6.25 to 625 ppm. Both the histopathological findings from animals

dying through week 65 and the results of evaluations of animals exposed for 40 and 65 weeks are presented in

this report. Exposure to 1,3-butadiene caused a regenerative anemia at concentrations of 62.5 ppm and

higher. Testicular atrophy was induced at 625 ppm, and ovarian atrophy was observed at 20 ppm and higher.

During the first 50 weeks of the study, lymphocytic lymphoma was the major cause of death of mice exposed

to 625 ppm 1,3-butadiene. Neoplasms of the heart, forestomach, lung, Harderian gland, mammary gland,

ovary, and liver were frequently observed in 1,3-butadiene-exposed mice that died between week 40 and week

65 of the study. Studies in which exposure to 1,3-butadiene was stopped after limited periods were also

included to assess the relationship between exposure levels and duration of exposures on the outcome of

1,3-butadiene-induced carcinogenicity. In these studies, lymphocytic lymphomas were induced in male mice

exposed to 625 ppm 1,3-butadiene for only 13 weeks. The incidence of lymphocytic lymphoma in male mice

exposed to 625 ppm 1,3-butadiene for 26 weeks was two times that in mice exposed to 625 ppm for 13 weeks.

However, when the exposure concentration was reduced by half to 312 ppm and the exposure duration

extended to 52 weeks, the incidence of lymphocytic lymphoma was reduced by 90%. Thus, the multiple of the

exposure concentration times the exposure duration did not predict the incidence of lymphocytic lymphoma

in mice. The early mortalities resulting from lymphocytic lymphomas in male mice exposed to 625 ppm

1,3-butadiene limited the expression of tumors at other sites. A clearer dose-response for 1,3-butadieneinduced neoplasia should be apparent from experiments in mice exposed to lower concentrations of this

chemical for 2 years.

Introduction

1,3-Butadiene is a colorless, flammable, noncorrosive

gas (bp: -4.4 °C, Vp: 1900 mmHg at 20 °C) used mainly

in the production of synthetic rubber and thermoplastic

resins (1). In production volume, 1,3-butadiene is one of

the top 25 organic chemicals manufactured in the United

States (2), with an annual production level of rubbergrade 1,3-butadiene in the U.S. of approximately 2.5

billion pounds (3). This gas has also been identified in

automobile exhaust, cigarette smoke, and in incineration products of fossil fuels (4). 1,3-Butadiene was

*National Institute of Environmental Health Sciences, Research

Triangle Park, NC 27709.

tBattelle Pacific Northwest Laboratories, Richland, WA 99352.

Address reprint requests to R. Melnick, NIEHS, P.O. Box 12233,

Research Triangle Park, NC 27709.

selected for evaluation of its toxicologic and carcinogenic

potential because of its large production volume, its

potential for human exposure in facilities that produce or

process this chemical, and the lack of long-term toxicology and carcinogenicity data for this compound (5). The

National Institute for Occupational Safety and Health

estimates that about 65,000 workers are potentially

exposed to 1,3-butadiene (6), and the U.S. Environmental Protection Agency estimates that between 5300 and

8200 workers are exposed to 1,3-butadiene in facilities

that either produce the monomer or process the monomer into polymers (7). Recent surveys conducted by

NIOSH at monomer and polymer manufacturing plants

indicate that most occupational exposures to 1,3-butadiene are less than 20 ppm; however, excursions in

certain jobs were as high as 374 ppm (8).

1,3-Butadiene has long been considered to have a low,
MELNICK ET AL.

noncumulative toxicity in animals and humans. The

LC50 for 1,3-butadiene was reported to be 270,000

mg/m3 (122,000 ppm) in mice exposed for 2 hr and

285,000 mg/m3 (129,000 ppm) in rats exposed for 4 hr (9).

Carpenter et al. (10) exposed rats, guinea pigs, rabbits,

and dogs to 0, 600, 2300, or 6700 ppm 1,3-butadiene 7.5

hr/day, 6 days/week, for 8 months. Slight growth retardation and cloudy swelling in hepatocytes were observed in the highest dose group; there were no apparent treatment-related pathologic changes in the adrenal

gland, heart, kidneys, lungs, skeletal muscle, pancreas,

spleen, testes, or ovares. Exposure of humans to 2000

ppm 1,3-butadiene or higher for 6 to 8 hr caused slight

irritation of the eyes (9).

The National Toxicology Program (NTP) studied the

long-term effects ofinhalation exposure to 1,3-butadiene

in mice (5,11). Groups of 50 male and 50 female B6C3F1

mice were exposed 6 hr/day, 5 days/week to air containing 0 (chamber control), 625, or 1250 ppm 1,3-butadiene. This study, intended to last for 103 weeks, was

ended after 60 to 61 weeks because of cancer-related

mortality in both sexes at both exposure concentrations.

There were early induction and increased incidences of

malignant lymphomas, hemangiosarcomas of the heart,

alveolar-bronchiolar neoplasms, and squamous cell neoplasms of the forestomach in males and females; and

acinar cell carcinomas of the mammary gland, granulosa

cell neoplasms of the ovary, and hepatocellular neoplasms in females. The observation of high incidences of

hemangiosarcomas of the heart was a particularly unusual finding, since the historical rate for this uncommon

endothelial cell tumor is about 0.04% in untreated

B6C3F1 mice and has rarely been induced in B6C3F1

mice in long-term studies (12,13). In male mice, doseresponses for neoplasms of the heart, lung, and forestomach were not clearly demonstrated since both doses

produced similar responses, whereas in female mice, the

rate of malignant lymphomas was lower than that in

males, and the dose-responses for other neoplastic lesions were more clearly defined.

Nonneoplastic lesions associated with exposure of

B6C3F1 mice to 1,3-butadiene included epithelial hyperplasia of the forestomach, endothelial hyperplasia of the

heart, alveolar epithelial hyperplasia, hepatocellular

necrosis, testicular atrophy, ovarian atrophy, and nasal

cavity lesions (chronic inflammation, fibrosis, and osseous and cartilagenous metaplasia in the olfactory region) in high-dose males (5,11).

Stimulated by the unusual effects of 1,3-butadiene in

mice, the fact that the studies were terminated early,

the sometimes unclear dose-response relationships for

various lesions, and the importance of 1,3-butadiene as a

high-production volume chemical with potential human

exposure, additional toxicology/carcinogenicity studies

of various durations, including a 2-year exposure, were

initiated to better characterize dose-response relationships for 1,3-butadiene-induced nonneoplastic and neoplastic lesions in B6C3F1 mice. Data from the first 65

weeks (15 months) of that study (early death animals and

interim evaluations at 40 and 65 weeks) are presented.

The data from the 2-year exposures are not yet available

and will be reported separately.

Materials and Methods

Chemical and Exposure System

Liquified 1,3-butadiene of 99+% purity, containing

trace amounts of t-butyl catechol, a peroxide inhibitor,

was obtained from Phillips Chemical Co. (Borger, TX).

Concentrations of the dimer, 4-vinyl-1-cyclohexene, in

the cylinder headspace were determined by gas chromatographic analysis. Cylinders were not retained for

use if the dimer content was greater than 150 ppm.

1,3-Butadiene gas was metered to the exposure chambers via a distribution system and diluted in the fresh air

chamber inlets. Chamber concentrations of 1,3-butadiene were monitored continuously during the exposure periods with a Hewlett-Packard 5840 gas chromatograph equipped with a flame ionization detector

(oven temperature: 120°C; GC column: 12" x 1/8" nickel

column packed with 1% SP-1000 on 60/80 Carbopack B;

carrier gas: nitrogen, 20 mL/min). The daily mean concentrations of 1,3-butadiene, distributed uniformly in

the chambers, were within 2% of the target concentrations.

Animal Maintenance

Five-week-old male and female B6C3F1 mice, obtained from Frederick Cancer Research Facility (Frederick, MD), were quarantined and acclimatized for 10

days prior to the start of the study. Stainless steel

Hazelton 2000 chambers (2.3 m3) were used, and animals

were exposed in individual wire mesh cage units, each

having a floor area of 106 cm2. The chamber environment

was maintained at 75 + 3°F and 55 ± 15% relative

humidity, with a chamber air flow of 15 air changes/hr.

Fresh, softened tap water and NIH-07 diet were available ad libitum, except when the feed was removed

during the exposure periods and overnight prior to the

40- and 65-week necropsies.

Treatment Groups

Groups of 70 male and 70 female B6C3F1 mice were

exposed 6 hr/day, 5 days/week for periods up to 65 weeks

to 0, 6.25, 20, 62.5, or 200 ppm 1,3-butadiene; groups of

90 male and 90 female mice were similarly exposed to 625

ppm 1,3-butadiene. The additional animals in the

625-ppm exposure group were included out of concern

that high mortality rates observed previously (5,11) at

this exposure concentration might interfere with the

interim evaluations at weeks 40 and 65. Fifty animals

per group were included for evaluation of 1,3-butadieneinduced carcinogenicity after exposure for up to 103

weeks. The 625-ppm concentration corresponds to the

low exposure level in the previous inhalation study

(5,11), while 6.25 ppm and 20 ppm more closely represent occupational exposure levels to 1,3-butadiene (8).

28
INHALATION TOXICOLOGY OF 1,3-BUTADIENE IN MICE

Animals were observed twice daily for moribundity and

mortality, and body weights were measured weekly for

the first 13 weeks of the study and at 4-week intervals

thereafter.

Studies in which the exposure to 1,3-butadiene was

stopped after limited exposure periods (stop-exposure

studies, SE) were included to assess the relationship

between exposure level and duration of exposure on the

outcome of 1,3-butadiene-induced carcinogenicity.

Groups of 50 male mice were exposed to one of the

following regimens: a) 625 ppm for 13 weeks (SE 13

WK), b) 200 ppm for 40 weeks (SE 40 WK), c) 625 ppm

for 26 weeks (SE 26 WK), or d) 312 ppm for 52 weeks (SE

52 WK). After the exposures were terminated, these

groups of animals were placed in control chambers for

the remainder ofthe 104-week study. The total exposure

to 1,3-butadiene (concentration multiplied by duration of

exposure, CT) was approximately equivalent for the

first and second treatments (8000 and 8125 ppm-weeks),

and it provided about half the total exposure given in the

third and fourth treatments (16,224 and 16,250 ppm

weeks).

Hematology

Blood samples were obtained at weeks 40 and 65 from

the supraorbital sinus of C02-anesthetized mice and

collected into tubes containing EDTA. Red blood cell

count (RBC), white blood cell count (WBC), hemoglobin

concentration (HGB), volume of packed red cells

(VPRC), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and platelet counts were

determined with an Ortho E LT-8/ds hematology

analyzer.

Histopathology

All moribund animals and those sacrificed at 40 and 65

weeks were killed by CO2 asphyxiation and then immediately necropsied. Tissue samples, preserved in 10%

neutral buffered formalin, were embedded in paraffin,

sectioned, and stained with hematoxylin and eosin. The

following tissues were examined microscopically: gross

lesions and tissue masses, bronchial, mediastinal, mandibular, and mesenteric lymph nodes; salivary gland;

sternebrae (including marrow); thyroid; parathyroid;

small intestine; large intestine; liver; gall bladder; prostate, testes, epididymis, and seminal vesicles; ovaries;

lungs and mainstem bronchi; nasal cavity and turbinates; heart; esophagus; stomach; uterus; brain; thymus; larynx; trachea; pancreas; spleen; kidneys; adrenals; urinary bladder; pituitary; and mammary gland.

Statistics

Mean body weights, organ weights, organ weight/

body weight ratios, and hematology results of treated

groups were compared to those of control groups using

Dunnett's t-test. The minimum level of probability accepted for significance was p<0.05.

Results

Survival, Body Weight, and Organ Weights

Exposure of B6C3F1 mice to 625 ppm 1,3-butadiene

caused a sharp decrease in survival of males and females

(Fig. 1). Increased mortality was apparent in exposed

males at about week 25 and in exposed females at about

week 30. Survival was reduced 50% by week 36 for males

and by week 46 for females. All mice exposed to 625 ppm

1,3-butadiene were dead by week 65. Exposure of mice

to 200 ppm 1,3-butadiene also caused a reduction in

survival; however, the rate of mortality at this exposure

level was much less than that at 625 ppm. At week 65 the

survival of mice exposed to 200 ppm 1,3-butadiene was

43 of 50 for males and 31 of 50 for females. In the control

and lower exposure concentration groups, survival was

generally greater than 90% at week 65.

Survival data from the stop-exposure groups (Fig. 2)

indicate that although mortality due to exposure to

1,3-butadiene was concentration related, the multiple of

exposure concentration times duration of exposure (CT)

did not predict the probability of survival. Mortalities

occurred at a greater rate for male mice exposed to 625

ppm 1,3-butadiene for 26 weeks, compared to 312 ppm

for 52 weeks (equivalent total exposure), and for male

mice exposed to 625 ppm for 13 weeks, compared to 200

ppm for 40 weeks.

Exposure of male or female mice to 6.25 to 625 ppm

1,3-butadiene for 65 weeks did not produce any significant adverse effects on mean body weight gain. The

initial mean body weight of male mice was 22.8 + 0.3 g

and of female mice was 18.5 + 0.2 g. At week 65, the

mean body weight of control male mice was 49.3 g, and

the mean body weights of the groups of male mice

exposed to 1,3-butadiene ranged from 46.0 to 47.4 g; the

mean body weight of control female mice was 43.5 g, and

the mean body weights of the groups of female mice

exposed to 1,3-butadiene ranged from 42.9 to 49.7 g.

At the interim evaluations at weeks 40 and 65, liver,

brain, kidney, spleen, lung, heart, thymus, and testis

weights were measured. The only significant concentration-related effect was a decrease in testis weight for

males exposed to 62.5 ppm and higher (Table 1). The

testis weight/body weight ratio was decreased in the 200

and 625 ppm exposure groups, compared to controls. A

similar dose-related decrease in testis weight was observed at the 65-week evaluation.

Hematology

Hematologic indices were measured in blood samples

taken from male and female mice at the 40- and 65-week

interim evaluations. For male mice at week 40, significant decreases were observed in red blood cell counts,

hemoglobin concentration, and packed red cell volume at

exposure concentrations of 62.5, 200, or 625 ppm 1,3-butadiene (Table 2). Similar decreases (data not shown)

were observed in female mice exposed to 625 ppm

1,3-butadiene and further demonstrate the dose-related

anemia caused by 1,3-butadiene in mice. These changes

29
MELNICK ET AL.

Males, 24

10o

0,9

00Oppm

08-

0.7

,200 ppm i

> 06

o 05

-rm

- 0.4-

0

a. -

0.2

0.1 -

30 40 50 60 70

WEEKS ON STUDY

FIGURE 1. Survival curves for male and female B6C3F1 mice exposed

to 200 or 625 ppm 1,3-butadiene for 65 weeks.

in male and female mice were not accompanied by increases in reticulocyte counts or in the frequency of

polychromatophilic cells in peripheral blood, and they

may indicate a partial or poorly regenerative response in

the bone marrow of 1,3-butadiene-exposed mice to reTable 1. Testis weights and testis weight/body weight ratios

(x 1000) for male mice exposed to 1,3-butadiene for 40 weeks.

Exposure

concentration, Testis weight, Testis weight/body weight,

ppm mg, ratioa

0 116 ± 6 2.89 ± 0.19

6.25 117 ± 9 2.93 ± 0.28

20 114 9 2.78 0.25

62.5 103 ± 12* 2.87 ± 0.36

200 102 ± 6* 2.54 ± 0.18*

625 60 ± 10* 1.60 + 0.07*

aResults are given as means ± SD; n = 10.

*Different compared to control, p < 0.05.

10 20 30 40 50 60 70

WEEKS ON STUDY

FIGURE 2. Survival curves for male B6C3F1 mice exposed to 1,3-

butadiene: stop-exposure groups. Exposure to 1,3-butadiene was

stopped after 13 (SE 13 wk), 26 (SE 26 wk), 40 (SE 40 wk), or 52

weeks (SE 52 wk). After these limited exposure periods, animals

were held in control chambers.

duced levels of circulating erythrocytes. Another

change caused by exposure of male and female mice to

1,3-butadiene was an increase in mean corpuscular volume and in mean corpuscular hemoglobin. A similar

profile of hematologic changes was observed in male and

female mice exposed to 625 ppm 1,3-butadiene for 65

weeks.

40-Week Evaluation

Gross and microscopic evaluations of early-death mice

at week 40 (i.e., those which died between the start of

the study and week 40), indicated that the predominant

cause of death in the 625-ppm exposure group was lymphocytic lymphoma (Table 3). The cumulative number of

deaths by week 40 at the lower exposure levels was too

Table 2. Hematologic changes in peripheral blood of male mice exposed to 1,3-butadiene by inhalation for 40 weeks.a

Exposure

concentration, RBC HGB, VPRC, MCV, MCH,

ppm x 106/p.L g/dL mL/dL fL pg

0 10.4 ± 0.3 16.5 ± 0.4 48.1 ± 1.5 46.3 ± 0.8 15.9 ± 0.2

6.25 10.3 ± 0.3 16.4 ± 0.5 47.8 ± 1.7 46.4 ± 1.0 16.0 ± 0.3

20 10.4 ± 0.4 16.7 ± 0.7 48.2 ± 2.2 46.3 ± 0.8 16.1 ± 0.4

62.5 9.9 ± 0.4* 15.9 ± 0.6* 45.9 ± 2.1* 46.7 ± 1.2 16.1 ± 0.3

200 9.6 ± 0.5* 15.6 ± 0.9* 45.4 + 2.7* 47.2 ± 1.0 16.2 ± 0.2*

625 7.6 ± 1.2* 13.5 ± 1.8* 39.9 5.3* 53.2 ± 2.9* 17.9 ± 0.8*

a n= 10.

*Different from control (0 ppm), p < 0.05.

-J

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0

-i

0.

30
INHALATION TOXICOLOGY OF 1,3-BUTADIENE IN MICE

Table 3. Incidence of microscopic lesions in early death mice

(through week-40) exposed to 625 ppm 1,3-butadiene.

SE SE

Malesa Femalesa week 13b week 26

Target: lesion n = 49 n = 21 n = 10 n = 20

Lymphocytic lymphoma 43 16 9 19

Heart:

Endothelial hyperplasia 20 0 0

Forestomach:

Epithelial hyperplasia 22 11 3 9

Squamous cell neoplasm 1 2 0 1

Lung:

Alveolar epithelial hyperplasia 3 0 10

Alveolar-bronchiolar neoplasm 21 0 1

Gonad:

Atrophy 35 15 2 4

aNumber of early death animals: males, n = 49; females, n = 21. There were

no deaths in either the male or female control groups.

bSE, stop-exposure groups (males).

few (3/group or less) to demonstrate any potential trend.

However, these deaths were generally due to lymphocytic lymphoma and included three mice at 200 ppm and

one mouse at 62.5 ppm. Male mice were more susceptible

to 1,3-butadiene-induced lymphoma than females; by

week 40, lymphocytic lymphoma was observed in 43 of

49 early-death males and 16 of 21 early-death females

exposed to 625 ppm 1,3-butadiene.

Lymphocytic lymphomas occurred in most earlydeath mice in the stop-exposure (SE) groups (Table 3).

Ten of 50 male mice exposed to 625 ppm 1,3-butadiene

for 13 weeks and 20 of 50 male mice exposed to 625 ppm

1,3-butadiene for 26 weeks died by week 40. Twentyeight of these deaths were due to lymphocytic lymphoma. The mortality rate and the incidence of lymphocytic lymphoma was markedly reduced in those groups

exposed to lower concentrations of 1,3-butadiene for a

longer duration (comparable total exposures); lymphocytic lymphoma was present in only two male mice

exposed to 200 ppm 1,3-butadiene for 40 weeks and one

male mouse exposed to 312 ppm.

The incidence of other tumor types, typically later

appearing than lymphocytic lymphoma, were too few to

clearly demonstrate an exposure-related effect. The low

incidences of squamous cell neoplasms of the forestomach and alveolar-bronchiolar neoplasms observed

by week 40 are noteworthy since they are uncommon,

especially in 46 to 47-week-old B63CF1, and because

they had been observed at increased incidences in the

previous inhalation study in mice (5,11). Epithelial hyperplasia ofthe forestomach was observed in over 50% of

the mice dying by week 40 that were exposed to 625 ppm

1,3-butadiene. Testicular and ovarian atrophy, lesions

observed at high rates in the previous study (5,11), were

prominent in this study as well.

The incidence of selected histopathologic lesions in

groups of 10 male or 10 female mice killed at week 40 are

shown in Table 4. Lymphocytic lymphomas were observed in two males and one female exposed to 625 ppm

1,3-butadiene. The low incidence of lymphocytic lymphoma in these animals, compared to the early-death

mice (Table 3), reflects the rapid onset of a moribund

condition in lymphoma-bearing mice. Proliferative lesions of the forestomach (epithelial hyperplasia and

squamous cell papilloma) were present in both males and

females exposed to 625 ppm 1,3-butadiene. Epithelial

hyperplasia of the forestomach was observed in one

female mouse exposed to 200 ppm 1,3-butadiene. The

incidences of alveolar epithelial hyperplasia and alveolar-bronchiolar neoplasms were increased in male and

female mice exposed to 625 and 200 ppm 1,3-butadiene,

but not to 62.5 ppm 1,3-butadiene. Testicular atrophy, of

minimal to mild severity, was present in 6 of 10 males

exposed to 625 ppm 1,3-butadiene. Ovarian atrophy was

marked in female mice exposed to 200 or 625 ppm 1,3-butadiene, while ovaries of mice exposed to 62.5 ppm

appeared normal. The atrophic ovaries had no identifiable oocytes, follicles, or corpora lutea. A granulosa cell

neoplasm of the ovary was observed in one female mouse

in the 200-ppm dose group. Hemangiosarcomas of the

heart were not observed in animals sacrificed at the

40-week interim evaluation; however, endothelial hyperplasia, a proliferative vascular lesion of the heart,

was observed in one male and one female exposed to 625

ppm 1,3-butadiene for 40 weeks and two early death

male mice exposed to 625 ppm 1,3-butadiene.

65-Week Evaluation

Data from the 65-week interim sacrifice of male mice

(Table 5) show that exposure to 1,3-butadiene resulted

in dose-related increases in the incidences of proliferative lesions of the heart, forestomach, lung, and Harderian gland (a site not identified in the previous study).

Only 7 of the 90 male mice exposed to 625 ppm butadiene

survived until week 65. Proliferative responses were

induced in male mice at concentrations as low as 62.5 or

20 ppm after 65 weeks of exposure. Testicular atrophy

was observed only in the 625-ppm exposure group.

The interim sacrifice of female mice at week 65 (Table

6) also revealed 1,3-butadiene-induced proliferative

Table 4. Incidence of microscopic lesions in mice exposed to

1,3-butadiene for 40 weeks.a

Exposure concentration, ppm

Males Females

Target: lesion 0 200 625 0 200 625

Lymphocytic lymphoma 0 0 2 0 0 1

Forestomach:

Epithelial hyperplasia 0 0 6 0 1 6

Squamous cell papilloma 0 0 3 0 0 2

Lung.

Alveolar epithelial hyperplasia 0 3 3 0 1 0

Alveolar-bronchiolar neoplasm 1 2 4 3 1 0

Gonad:

Atrophy 0 0 6 0 9 8

an = 10, except for female mice exposed to 625 ppm 1,3-butadiene where n = 8.

31
MELNICK ET AL.

Table 5. Incidence of microscopic lesions in male mice exposed

to 1,3-butadiene for 65 weeks.a

Exposure concentration, ppm

Target: lesion 0 6.25 20 62.5 200 625

Lymphocytic lymphoma 0 0 0 0 0 2

Heart:

Endothelial hyperplasia 0 NEb 0 1 1 1

Hemangiosarcoma 0 NE 0 0 1 3

Forestomach:

Epithelial hyperplasia 0 0 0 1 0 2

Squamous cell neoplasm 0 0 0 1 3 4

Lung:

Alveolar epithelial hyperplasia 0 0 1 1 3 2

Alveolar-bronchiolar neoplasm 0 0 0 2 4 5

Harderian gland:

Adenoma 0 0 2 4 3 3

Liver:

Hepatocellular neoplasm 3 1 5 3 4 6

Testis:

Atrophy 0 NE NE NE 0 4

an = 10, except for mice exposed to 625 ppm 1,3-butadiene where

n = 7.

bNE, not examined microscopically.

Table 6. Incidence of microscopic lesions in female mice

exposed to 1,3-butadiene for 65 weeks.a

Exposure concentration, ppm

Target: lesion 0 6.25 20 62.5 200 625

Lymphocytic lymphoma 1 0 0 0 3 0

Heart:

Endothelial hyperplasia 0 NEb NE 0 4 0

Hemangiosarcoma 0 NE NE 0 1 2

Forestomach:

Epithelial hyperplasia 0 0 0 0 2 1

Squamous cell neoplasm 0 0 0 1 2 1

Lung:

Alveolar epithelial hyperplasia 0 0 2 1 4 0

Alveolar-bronchiolar neoplasm 0 0 0 3 4 2

Liver:

Hepatocellular neoplasm 1 1 1 1 3 1

Ovary:

Atrophy 0 0 2 9 8 2

an = 10, except for mice exposed to 625 ppm

n = 2.

bNE, not examined microscopically.

1,3-butadiene where

changes in the heart, forestomach, and lung, especially

at 200 and 62.5 ppm 1,3-butadiene. Only two female mice

exposed to 625 ppm 1,3-butadiene survived until week

65. Ovarian atrophy was observed at exposure levels of

20 ppm and higher.

The cumulative incidence of microscopic lesions in

early-death mice through week 65 shows that lymphocytic lymphoma was the major cause of death at the 625

ppm exposure level, especially for male mice (Table 7).

Between week 40 and week 65, 24 male mice exposed to

625 ppm 1,3-butadiene died; 18 had lymphocytic lymphoma. Increased incidences of hemangiosarcomas of

the heart, forestomach neoplasms, alveolar-bronchiolar

neoplasms, Harderian gland adenomas, and hepatocellular neoplasms were also evident in early-death male mice

exposed to 625 ppm 1,3-butadiene. The number of early

deaths in male mice exposed to 62.5 or 200 ppm 1,3-

Table 7. Incidence of microscopic lesions in early-death mice

(through week 65) exposed to 1,3-butadiene.

Exposure concentration, ppm

Males Females

62.5 200 625 62.5 200 625

Target: lesion (4)a (7) (73) (6) (19) (80)

Lymphocytic lymphoma 1 2 61 1 6 36

Heart:

Endothelial hyperplasia 0 0 6 1 4 9

Hemangiosarcoma 0 0 3 0 3 24

Forestomach:

Epithelial hyperplasia 0 1 39 3 5 41

Squamous cell neoplasm 0 0 6 0 0 26

Lung:

Alveolar epithelial hyperplasia 0 0 9 1 4 10

Avelolar-bronchiolar neoplasm 1 3 3 1 5 22

Harderian gland:

Adenoma 0 1 4 0 5 6

Mammary gland:

Adenocarcinoma 1 3 11

Ovary:

Granulosa cell neoplasm 0 2 5

Liver:

Hepatocellular neoplasm 1 1 5 0 4 2

Gonad:

Atrophy 0 1 53 4 15 68

aNumbers in parentheses equal the number of early death animals

in each group. Only one control male mouse and one control female

mouse died during the first 65 weeks of the study; hepatocellular

adenoma was observed in the male mouse.

butadiene was too low to ascertain any potential carcinogenic effects. Fifty-nine female mice that were exposed

to 625 ppm died between week 40 and week 65; 20 had

lymphocytic lymphoma. The incidence of other primary

tumors was high in these 59 mice: 24 hemangiosarcomas

of the heart, 24 squamous cell neoplasms of the forestomach, 20 alveolar-bronchiolar neoplasms, 6 Harderian gland adenomas, 11 mammary gland adenocarcinomas, and 5 granulosa cell neoplasms of the ovary.

Elevated incidences of neoplasms at these sites were

also apparent in early-death female mice exposed to 200

ppm 1,3-butadiene. The development of neoplasms in

the heart, forestomach, and lung were supported by

high incidences of hyperplasias in these organs in both

males and females. Testicular atrophy was observed at a

high incidence in male mice exposed to 625 ppm, while

ovarian atrophy was observed in female mice exposed to

62.5, 200, or 625 ppm 1,3-butadiene.

The tumor incidence profiles in the stop-exposure

groups (Table 8) indicate that at comparable total exposures, the occurrence of lymphocytic lymphoma is more

likely with exposure to a higher concentration of 1,3-

butadiene for a short time, compared to exposure to a

lower concentration for an extended duration. This is

evident by comparing the incidence of lymphocytic lymphoma in the 625 ppm SE 13 WK group (15 lymphocytic

lymphomas) with that in the 200 ppm SE 40 WKgroup (3

lymphocytic lymphomas), and comparing the incidence

in the 625 ppm SE 26 WKgroup (30 lymphocytic lymphomas) with that in the 312 ppm SE 52 WK group (3

lymphocytic lymphomas). An obscuring effect of a high

32
INHALATION TOXICOLOGY OF 1,3-BUTADIENE IN MICE

incidence of lymphocytic lymphoma on 1,3-butadieneinduced neoplasia at other primary sites is evident by

comparing the tumor profile in the 312 ppm SE 52 WK

group with that in the 625 ppm SE 26 WK group. The

incidences of hemangiosarcomas of the heart, alveolarbronchiolar neoplasms, and hepatocellular neoplasms

were higher in the group with the lower incidence of

lymphocytic lymphoma (312 ppm SE 52 WK), indicating

that longer survival allows these other chemically induced tumors to manifest. Graphic presentation of the

incidences of lymphocytic lymphomas and hemangiosarcomas of the heart versus weeks on study for these two

stop-exposure groups (Fig. 3) shows that hemangiosarcomas of the heart are more likely to be observed when

the incidence of early lymphocytic lymphomas is low;

conversely, when the rate of early lymphocytic lymphoma is high, the incidence of 1,3-butadiene-induced

hemangiosarcoma of the heart is low.

Discussion

1,3-Butadiene has been shown to induce multiple organ carcinogenicity in rats and mice (5,11,14). In

B6C3F1 mice, exposure to 625 or 1250 ppm 1,3-

butadiene caused early induction and significantly increased incidences of malignant lymphomas, hemangiosarcomas of the heart, alveolar-bronchiolar neoplasms, squamous cell neoplasms of the forestomach,

acinar cell carcinomas of the mammary gland, granulosa

cell neoplasms of the ovary, and hepatocellular neoplasms (5,11); Sprague-Dawley rats exposed to 1000 or

8000 ppm 1,3-butadiene had increased incidences at

multiple sites: pancreatic exocrine adenomas, uterine

sarcomas, Zymbal gland carcinomas, mammary gland

neoplasms, thyroid follicular cell neoplasms, and Leydig

cell neoplasms of the testis (14). Morphological descripTable 8. Incidence of microscopic lesions in early-death male

mice: stop-exposure (SE) groups through week 65.

Exposure concentration, ppma

200 625 312 625

SE 40 SE 13 SE 52 SE 26

Target: lesion (8000)a (8125) (16224) (16250)

Lymphocytic lymphoma 3 15 3 30

Heart:

Endothelial hyperplasia 0 1 1 5

Hemangiosarcoma 1 0 12 4

Forestomach:

Epithelial hyperplasia 2 5 12 13

Squamous cell neoplasm 0 3 3 9

Lung:

Alveolar eipthelial hyperplasia 1 0 3 4

Alveolar-bronchiolarneoplasm 13 9 5

Harderian gland:

Adenoma 1 1 3 2

Liver:

Hepatocellular neoplasm 2 0 8 4

Testis:

Atrophy 0 2 1 5

aNumber of early-death aninals in each group (n): 200 ppm, SE 40, n = 7; 625

ppm, SE 13, n = 20; 312 ppm, SE 52, n = 21; 625 ppm, SE 26, n = 36.

nTotal exposure expressed as ppm-weeks.

tions and illustrations of the neoplastic lesions induced

by 1,3-butadiene in B6C3F1 mice are presented in the

following paper (15). The large number of primary organ

sites of tumor induction by 1,3-butadiene was unusual

for chemicals studied by the NTP; only 1.9% of approximately 360 chemicals evaluated showed evidence of carcinogenicity at six or more primary sites.

Although the previous mouse study was ended after

only 60 weeks of exposure because of high rates of early

mortality (5,11), the doses used in that study are not

considered excessive for evaluation of carcinogenicity,

since survival was not reduced from toxic effects other

than from the induction of fetal neoplasms. Furthermore, those concentrations (625 and 1250 ppm) are

equivalent to and below the OSHA standard of 1000 ppm

for occupational exposure to 1,3-butadiene (16). Announcements have been made to lower this level considerably (17).

The results of the studies of 1,3-butadiene in mice are

unusual in that uncommon tumors, such as hemangiosarcoma of the heart and squamous cell neoplasms of the

forestomach, were induced after inhalation exposure. In

untreated B6C3F1 mice 110 to 112 weeks of age, the

historical rate for hemangiosarcomas of the heart is

about 0.04% (1 in 2500), while that of squamous cell

neoplasms of the forestomach is about 0.5% (1 in 200)

(12). In the present studies, Harderian gland adenomas

were observed in early-death mice (through week 65)

exposed to 200 or 625 ppm 1,3-butadiene and at the

65-week interim evaluation. These observations indicate

an additional site of tumor induction by 1,3-butadiene;

_ o Lymphoma, 625ppmSE 26wk

* Lymphoma, 312 ppm SE52wk

I o Hemongiosorcoma, 625 ppm SE 26wk 60 -

a Hemangiosarcoma, 312 ppm SE 52 wk

0

C,)

-J

a0a.

wllJ

z

L-) z

w

a

z

50

40

30

20

10

.10

0

30 40 70

WEEKS ON STUDY

FIGURE 3. Cumulative incidence of lymphocytic lymphomas or hemangiosarcomas of the heart versus weeks on study in two stopexposure groups for which the total exposures to 1,3-butadiene

were comparable (625 ppm SE 26 wk and 312 ppm SE 52 wk).

33
MELNICK ET AL.

the historical rate of Harderian gland neoplasms in untreated 110- to 112-week-old B6C3F1 mice is approximately 2% (12).

The present inhalation studies of 1,3-butadiene were

performed to better characterize dose-response relationships for butadiene-induced neoplastic and nonneoplastic lesions in B6C3F1 mice at various intervals and

after 2 years of exposure. The following conclusions are

based on the results of the 40-week and 65-week evaluations. 1,3-Butadiene is a gonadal toxicant in mice, inducing testicular atrophy in males at 625 ppm, and

ovarian atrophy in females at 20 ppm and higher. This

gas was also toxic to the bone marrow of mice, causing a

partially or poorly regenerative dose-dependent anemia

at exposure concentrations of 62.5 ppm and higher. The

increase in MCV and MCH at 625 ppm 1,3-butadiene

indicates that although 1,3-butadiene caused suppression of hematopoiesis in the bone marrow, young larger

cells may have been released into the blood from extramedullary sites. It has been reported that exposure of

B6C3F1 mice to 1250 ppm 1,3-butadiene produces a

macrocytic-megaloblastic anemia (18) and promotes extramedullary hematopoiesis in the spleen (19).

For male or female mice exposed to 625 ppm 1,3-

butadiene, lymphocytic lymphoma was the major cause

of death in the first 50 weeks of the study. Prior to week

65, the lowest exposure concentration at which lymphocytic lymphomas were observed was 62.5 ppm; lymphocytic lymphoma was observed in one male mouse exposed to this concentration of 1,3-butadiene for 29

weeks. Lymphocytic lymphomas were also induced in

male mice exposed to 625 ppm 1,3-butadiene for only 13

weeks (i.e., stop-exposure study). The incidence of lymphocytic lymphoma was two times higher in male mice

exposed to 625 ppm 1,3-butadiene for 26 weeks, compared to male mice exposed to 625 ppm for 13 weeks.

When the exposure concentration was reduced to 312

ppm and the exposure duration was extended to 52

weeks (312 ppm SE 52 WK), the incidence of lymphocytic lymphoma was 10% of the incidence in the group of

mice that received the same total exposure to 1,3-

butadiene (625 ppm SE 26 WK). Thus, the multiple of

exposure concentration times exposure duration does

not predict the incidence of lymphocytic lymphoma in

mice. At equivalent total exposures, the likelihood for

induction of lymphocytic lymphoma is greater with exposure to a higher concentration of 1.3-butadiene for a

shorter time than for exposure to a lower concentration

of 1,3-butadiene over an extended duration.

The early mortalities due to lymphocytic lymphomas,

especially in male mice exposed to 625 ppm 1,3-

butadiene, appear to limit the expression of neoplasms

at other sites. Increased incidences of hemangiosarcomas of the heart, squamous cell neoplasms of the

forestomach, alveolar-bronchiolar neoplasms, Harderian gland adenomas, adenocarcinomas of the mammary

gland, granulosa cell neoplasms of the ovary, and hepatocellular neoplasms were frequently observed in

1,3-butadiene-exposed mice that died between weeks 45

and 65 of the study. Since early lymphocytic lymphomas

were not as prevalent at concentrations of 6.25 to 200

ppm, it is likely that a clearer dose response for

1,3-butadiene-induced neoplasia at these sites will be

obtained over this concentration range. The lack of a

clear dose response for some of the neoplastic and nonneoplastic lesions observed in the previous inhalation

study of 1,3-butadiene in B6C3F1 mice (5,11) was likely

due to reduced survival caused by early development of

malignant lymphomas. Proliferative, nonneoplastic lesions of the forestomach, heart, and lung which were

observed in early-death mice and at the 40-week and

65-week evaluations may represent treatment related

preneoplastic changes at these target sites.

The carcinogenicity studies of 1,3-butadiene in rats

and in mice demonstrate a species difference with respect to sites of tumor induction and magnitude of response. Irons and coworkers (20,21) have suggested that

activation of an endogenous ecotropic retrovirus may

play a critical role in butadiene-induced thymic

lymphomalleukemia in B6C3F1 mice. However, the finding that exposure to 1250 ppm 1,3-butadiene for up to 1

year also caused a 14% incidence of thymic lymphoma/

leukemia in NIH Swiss mice, a strain that does not

express the endogenous ecotropic retrovirus recovered

from tissues of B6C3F1 mice. Further, Swiss mice have

a background rate of zero for this neoplasm, indicating

that 1,3-butadiene can induce thymic lymphoma independently of this activated retrovirus.

Butadiene monoxide (1,2-epoxybutene-3) has been

identified as the primary metabolite of 1,3-butadiene

biotransformation by rat liver microsomal monooxygenase (22). This intermediate may be further metabolized by liver monooxygenase or epoxide hydrolase to

diepoxybutane or 3,4-epoxy-1,2-butanediol, respectively (23). Butadiene monoxide and diepoxybutane

have been shown to be mutagenic to Salmonella typhimurium strains, which are sensitive to base pair

substitution mutagens (24,25), and to induce local neoplasms when applied to the skin ofmice or when administered to rats or mice by subcutaneous injection (26,27).

Since 1,3-butadiene is mutagenic to Salmonella typhimurium only when incubated with liver S-9 activation systems (28), epoxide intermediates may be the

ultimate carcinogenic forms of this chemical. Butadiene

monoxide has been identified in the expired air of

Sprague-Dawley rats exposed to 1,3-butadiene (29).

Although the rate of butadiene metabolism is greater in

B6C3F1 mice than Sprague-Dawley rats at equivalent

exposure concentrations (30-32), the magnitude of this

difference at carcinogenic exposure levels in these species does not seem to be sufficient to account for the

species difference in carcinogenicity of 1,3-butadiene.

The present studies indicate that 200 ppm butadiene is

carcinogenic to the heart, lung, Harderian gland, mammary gland, and ovary of B6C3F1 mice; however, other

than for the mammary gland, neoplasms were not induced at these sites in Sprague-Dawley rats exposed to

1000 or 8000 ppm 1,3-butadiene. The metabolism of

34
INHALATION TOXICOLOGY OF 1,3-BUTADIENE IN MICE 35

1,3-butadiene is greater in Sprague-Dawley rats exposed to 1000 ppm 1,3-butadiene than in B6C3F1 mice

exposed to 200 ppm 1,3-butadiene (30,31). Perhaps

steady-state tissue concentrations of these intermediates at target sites are sufficiently different to account

for the species difference in carcinogenicity of 1,3-butadiene.

The inhalation carcinogenicity studies of 1,3-butadiene in rats and mice have raised concerns of risk for

humans exposed to this chemical. Results of epidemiology studies indicate an association between occupational exposure to 1,3-butadiene and the development

of lymphatic and hematopoietic neoplasms (33-36).

These findings are consistent with the results of the

inhalation studies of 1,3-butadiene in mice.

NOTE ADDED IN PROOF: Data from the 2-year exposure studies clearly showed that the incidence of

alveolar-bronchiolar neoplasms in female B6C3F1, mice

was increased at all exposure concentrations ofof 1,3-butadiene, including 6.25 ppm, compared to controls (R.L.

Melnick et al., Cancer Res., in press).

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